EDWIN ROGER PARRA CUENTAS
Projetos de Pesquisa
Unidades Organizacionais
10 resultados
Resultados de Busca
Agora exibindo 1 - 10 de 10
conferenceObject Increased decorin and type V collagen in SSc pulmonary fibrosis(2012) TEODORO, W.; VELOSA, A. P.; MARCELINO, A.; MARTIN, P.; CARRASCO, S.; GOLDENSTEIN-SCHAINBERG, C.; PARRA, E.; YOSHINARI, N.; CAPELOZZI, V.Objective: To evaluate COL V and decorin expression in pulmonary tissue and to characterize biochemical profile of COLV from lung fibroblasts culture from SSc patients. Method: We evaluated COL V and decorin expression and tridimensional reconstruction (3D) of 6 patients with SSc without pulmonary hypertension that underwent surgical lung biopsy and as control was obtained lung fragments from 6 normal individuals who died from trauma. COL V amount in lung sections was evaluated with immunofluorescence. To biochemical characterization of COL V from lung fibroblasts culture was used quantitative immunoblot. Results: It was found that the structure of COLV fibers was distorted and strongly thickened in lung tissue from SSc patients compared with thin fibers pattern in the healthy controls. Decorin was distributed around COL V fibrils in the bronchovascular interstitium and vascular walls. Histomorphometric analysis of SSc lung demonstrated increased expression of both COL V and decorin when compared to the control (p<0.01). The semiquantitative imunoblot detected an increased high molecular weight COLV fraction in patients when compared to the control. Conclusion: The over expression and unusual organization of COLV fibers with biochemical changes associated to increased decorin indicates that matrix signalization pathway is involved in COLV fibrillogenesis process in SSc pulmonary fibrosis.conferenceObject Fibrogenesis failure of type V collagen observed in pulmonary and cutaneous fibroblast culture reinforce the pathogenic participation of this collagen in the pathway of systemic sclerosis(2012) TEODORO, W. R.; MORAIS, J.; MARTIN, P.; VELOSA, A. P. P.; CARRASCO, S.; SOUZA, R. B. C.; KATAYAMA, M. L.; GOLDEINSTEIN-SCHAINBERG, C.; PARRA, E. R.; CAPELOZZI, V. L.; YOSHINARI, N. H.Introduction: Unusual type V collagen (COLV) accumulation was demonstrated in systemic sclerosis (SSc) by our group. In this regard, this study analyzed tridimensional reconstruction (3D), biochemical and molecular profile of COLVα1 and COLVα2 chains in pulmonary and cutaneous fibroblasts culture from patients with SSc. Materials and Methods: Pulmonary and cutaneous fibroblasts for culture were obtained from 7 patients with SSc and from six controls respectively. COLV 3D reconstruction was performed by confocal microscopy. COLVα1 and COLVα2 gene expression was performed by RT-PCR and COLV protein expression by immunoblotting. Results: COL V 3D reconstruction showed distorted and strongly thickened fibers with irregular bundles resulting in a dense network in lung and skin fibroblast cultures from SSc patients compared to the thin fibers from fibroblast controls. Collagen quantification showed significant increased COLV fiber expression in SSc cutaneous and pulmonary fibroblasts (P<0.01) compared with the respective controls. In the same way, molecular evaluation demonstrated an increased significance (P=0.05) of COLVα1 and COLVα2 mRNA expression in cutaneous and pulmonary fibroblasts from SSc patients to that of control groups. The immunoblotting analysis demonstrated the increased weight of the molecular COLV chains. Conclusion: COLV overexpression and an unusual organization of these fibers including molecular and biochemical changes, suggest an interference process of the COLV fibrillogenesis in patients with SSc, reinforcing the participation of this collagen in SSc pathogenesis and open new therapeutic perspectives for these patients.article 39 Citação(ões) na Scopus Experimental diabetes modulates collagen remodelling of joints in rats(2012) ATAYDE, Sandra A.; YOSHINARI, Natalino H.; NASCIMENTO, Dafne P.; CATANOZI, Sergio; ANDRADE, Priscila C.; VELOSA, Ana Paula P.; PARRA, Edwin R.; PASSARELLI, Marisa; NAKANDAKARE, Edna R.; CAPELOZZI, Vera L.; TEODORO, Walcy R.The aim of this study was to evaluate extracellular matrix components in articular cartilage, ligaments and synovia in an experimental model of diabetes. Young Wistar rats were divided into a streptozotocin-induced (STZ; 35 mg/kg) diabetic group (DG; n=15) and a control group (CG; n=15). Weight, blood glucose and plasma anti-carboxymethyllysine were measured 70 days after STZ infusions. Knee joints, patellar ligaments, and lateral and medial collateral ligaments were isolated and stained with hematoxylineosin and Picrosirius. The total collagen content was determined by morphometry. Immunofluorescence was employed to evaluate types I, III, and V collagen in ligaments and synovial tissues and types II and XI collagen in cartilage. Results: Higher blood glucose levels and plasma anti-carboxymethyllysine were observed in DG rats when compared to those in CG rats. The final weight was significantly lower in the DG rats than in the CG rats. Histomorphometric evaluation depicted a small quantity of collagen fibers in ligaments and articular cartilage in DG rats, as well as increased collagen in synovial tissue. There was a decrease in cartilage proteoglycans in DG rats when compared with CG rats. Immunofluorescence staining revealed an increase of collagen III and V in ligaments, collagen XI in cartilage, and collagen I in synovial tissue of DG rats compared with CG rats. Conclusion: The ligaments, cartilage and synovia are highly affected following STZ-induced diabetes in rats, due the remodeling of collagen types in these tissues. This process may promote the degradation of the extracellular matrix, thus compromising joint function. Our data may help to better understand the pathogenesis of joint involvement related to diabetes.conferenceObject DYNAMIC COLLAGEN V REMODELING IS RELATED TO SKIN THICKENING IN SSc(2012) MARTIN, P.; TEODORO, W. R.; VELOSA, A. P.; CARRASCO, S.; MORAIS, J. de; CHRISTMANN, R. B.; PARRAS, E. R.; CAPELOZZI, V. L.; YOSHINARI, N. H.Background. Normal physiological properties of skin, one of the primary organs affected in SSc, depends on collagen Types I (COL I), III (COLIII) and V (COLV) assembly forming heterotypic fibres. COLV regulates fibril diameter and loss of this function could result in tissue fibrosis. In this way, our aim was to evaluate the histological and molecular profiles of COLI, COLIII and COLV in SSc skin and its correlation with skin thickening and disease activity. Methods. Skin biopsies of 18 patients (5 at early and 13 at late disease stage) and 10 healthy controls were studied. Assessment of skin thickening was performed using the modified Rodnan skin score (MRSS) and disease activity was calculated by Valentini Disease Activity Index. Quantification of COLI, COLIII and COLV was evaluated by histomorphometry in dermis and quantitative RT–PCR in dermal fibroblast culture. Results. A higher expression of abnormal COLV was observed in dermis of patients with early disease when compared with control group and late disease. The COLIII content was also higher in early SSc when compared with healthy controls and late SSc. On the other hand, the amount of COLI was higher in late disease when compared with control and early SSc. A positive correlation between COLV and MRSS (r = 0.42, P = 0.04) as well as disease activity (r = 0.45, P = 0.03) was observed, but there was no correlation between COLI and COLIII expression and these parameters. COLV α-1 and COLV α-2, as well as COLI α-1 and COLIII α-1 mRNA expression were higher in SSc when compared with control group. Conclusion. We found increased COLIII and COLV deposition in early SSc and increased COLI expression in late SSc indicating that collagen remodelling in SSc is a dynamic process. The fact that abnormal COLV expression decreases in later disease stages could explain why skin thickening sometimes improves spontaneously with time. Besides, COLV is correlated to MRSS and disease activity. These findings include COLV as an important regulator of cutaneous thickness in SSc and may add this protein as a new target for future treatments.conferenceObject Collagen V-induced nasal tolerance increase FOXp3 in systemic sclerosis model(2012) TEODORO, W.; VELOSA, A. P.; CRUZ, I. Brindo da; SANTOS FILHO, A.; FERNEZLIAN, S.; PARRA, E.; YOSHINARI, N.; CAPELOZZI, V.Objective: To evaluate FOXp3 expression in bronchus-associated lymphoid tissue (BALT) and correlate with the inflammatory process and collagen content in the lung tissue in an experimental model of scleroderma (SSc) after type V collagen (COL V)-induced nasal tolerance. Method: Female New Zealand rabbits (N=12) were immunized with 1 mg/ml of COL V in Freund’s adjuvant (IM). After 150 days, six immunized animals were nasally tolerated with COL V (25νg/day), during 60 days (IM-TOL). Animals (N=6) only tolerated served as control (CT). FOXP3 expression in BALT and inflammatory cells in pulmonary interstitium were evaluated by point counting method. Types I, III and V collagen gene expression were evaluated by Real-time PCR. Results: IM-TOL when compared to IM presented decreased lymphocytes, macrophages and monocytes and types I (p=0,002) and V (p=0,009) collagen mRNA expression in pulmonary tissue. T lymphocytes FOXp3 were expressed in 100 % of IM-TOL and 33,3 % of CT (p=0,03). Additionally, BALT was higher expressed in IM-TOL in relation to CT. Conclusion: COL V-induced nasal tolerance in SSc model induces FOXp3 regulatory T cells in BALT which can trigger an immune regulatory mechanism resulting in decreased inflammation and collagen expression. It suggests that COL V tolerance could be a promising therapeutic for human scleroderma treatment.conferenceObject COLLAGEN V-INDUCED NASAL TOLERANCE PROMOTES DECREASE IN TOPO I PROTEIN SYNTHESIS AND PULMONARY FIBROSIS OF SSc MODEL(2012) VELOSA, A. P.; TEODORO, W. R.; CALLADO, M. R.; FILHO, A. S.; FERNEZLIAN, S. M.; KATAYAMA, M. L.; PARRA, E. R.; CAPELOZZI, V. L.; YOSHINARI, N. H.Background. Autoantibodies against topo I (anti-Scl-70) are found to be associated with increased mortality and correlate with the extent of pulmonary fibrosis in SSc. To evaluate anti-Scl-70 antibodies and topo I expression in lung and to correlate with pulmonary fibrosis in experimental SSc after collagen V (COL V)-induced nasal tolerance. Methods. Female New Zealand rabbits (n = 12) were immunized with 1 mg/ml of COL V in Freund's adjuvant (IM). After 150 days, six immunized animals were tolerated by nasal administration of type COL V (25 μg/day) (IM-TOL), daily during 60 days. Anti-Scl-70 antibodies were evaluated by ELISA. Immunohistochemistry, histomorphometry and RT–PCR evaluated pulmonary topo I expression, types I, III and V collagen and TGF-β expression in pulmonary parenchyma. Results. A significant decrease in topo I expression by pulmonary endothelial cells was found comparing IM-TOL vs IM [29.86 (10.48) vs 76.11 (20.91), P = 0.019]. No difference was found for the anti-Scl-70 frequency after tolerance. Type V collagen content around the small vessels [0.371 (0.118) vs 0.874 (0.282), P < 0.001] and bronchioles [0.294 (0.139) vs 0.646 (0.172), P < 0.001], beyond mRNA expression to 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 decreased in IM-TOL, when compared with IM TGF-β expression decreased in endothelial [10.77 (4.3) vs 43.5 (5.7), P < 0,0001] and smooth muscle cells [9.93 (3.77) vs 53.68 (4.06), P < 0,0001] from pulmonary vessels, epithelial cells [6.03 (1.47) vs 13.65 (1.39), P < 0,0001] and interstitial fibroblasts [11.55 (1.88) vs 20.13 (1.60), P < 0,0001] in IM-TOL compared with IM. Conclusions. The results showed that a direct link between nasal type V collagen tolerance and a decline in topo I expression may reduce pulmonary fibrosis, suggesting that strategies aimed at preventing the increase of the type V collagen synthesis, or the local responses to increased topo I expression, may have a greater impact in SSc.conferenceObject TRIDIMENSIONAL RECONSTRUCTION, BIOCHEMICAL AND MOLECULAR PROFILE OF COLLAGEN V IN SKIN AND LUNG FIBROBLASTS CULTURE FROM SSc INDICATE A FAILING IN FIBRILOGENESIS(2012) TEODORO, W.; MORAIS, J.; MARTIN, P.; VELOSA, A. P. P.; CARRASCO, S.; SOUZA, R. B. C.; KATAYAMA, M. L.; GOLDEINSTEIN-SCHAINBERG, C.; PARRA, E. R.; CAPELOZZI, V. L.; YOSHINARI, N. H.Background. The type V collagen (COL V) mutations are involved in collagen vascular diseases, such as SSc, in which an unusual accumulation of this collagen was demonstrated (Pathol Res Pract 2004; 200:681). In this context, our purpose was to analyse the 3D reconstruction, biochemical and molecular profile of COL Vα1 and α2 chains in skin and lung fibroblasts culture from patients with SSc. Methods. Lung biopsies of seven patients, skin of six patients and respective matched controls were obtained from SSc according ACR. For fibroblast culture skin and lung evaluations were used the following score: intense expression (1–4), fibroblast number/field (1, 2) and collagen fibres architecture (1–3). The total evaluations were: mild (3–5), moderate (6, 7) and severe (8, 9). COLV 3D reconstruction was performed by confocal microscopy, COL Vα1 and Vα2 gene expression in fibroblasts of skin and lung was performed in PCR–RT and COLV protein expression by immunoblotting. Results. The structure of COL V fibre in 3D reconstruction showed distorted and strongly thickened fibres in skin and lung fibroblasts with irregular bundles of COL V distributed in parallel and perpendicular arrangements resulting in a dense network in SSc patients compared with thin fibres pattern from the healthy controls. Collagen quantification showed increase of COL V fibres expression in SSc cutaneous fibroblast [82.5 (9.5)% vs 47.5 (9.5)%, P = 0.002] and lung fibroblast 38.87 (2.99)% vs 20.33 (7.50)%, P = 0,002) compared with respective controls. The molecular evaluation demonstrated an increased of COL Vα1 and α2 mRNA expression in SSc fibroblast skin when compared with control [1.375 (0.373) au vs 0.0047 (0.0013) au, P = 0.05). Similar results were observed in lung [1.61 (0.654) vs 0.99 (0.51) au; P = 0.05).The proportion COL Vα1/COL Vα2 mRNA in fibroblast lung and skin was higher in SSc than in controls being the chains ratio 1 : 2. COL V chains from skin and lung fibroblasts presented alteration of molecular weight of the quoted chain. Conclusion. The overexpression and the unusual organization of COLV fibres, besides the biochemical changes, suggest an interference with the fibrillogenesis process in skin and pulmonary fibrosis from SSc patients, reinforcing the participation of this collagen in pathogenesis of SSc and open new therapeutic perspectives for these patients.- Abnormal collagen V deposition in dermis correlates with skin thickening and disease activity in systemic sclerosis(2012) MARTIN, Patricia; TEODORO, Walcy R.; VELOSA, Ana Paula P.; MORAIS, Jymenez de; CARRASCO, Solange; CHRISTMANN, Romy B.; GOLDENSTEIN-SCHAINBERG, Claudia; PARRA, Edwin R.; KATAYAMA, Maria Lucia; SOTTO, Mirian N.; CAPELOZZI, Vera L.; YOSHINARI, Natalino H.Objective: The physiological and mechanical properties of the skin, the primary tissue affected by systemic sclerosis, depend on the assembly of collagen types I, Ill 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.
conferenceObject Stem cells of adipose rabbit tissue are stimulate into chondrocyte-like phenotype by collagen V in vitro(2012) CRUZ, I. Brindo da; GOLDENSTEIN-SCHAINBERG, C.; FULLER, R.; VELOSA, A. P. P.; CARRASCO, S.; PARRA, E. R.; CAPELOZZI, V. L.; YOSHINARI, N. H.; TEODORO, W. R.Introduction: Among a variety of biological functions, including an anti-inflammatory effect, collagen V (COLV) regulates the diameter of collagen fibers with an important role in the development of functional tissues. Therefore the aim of this study was to evaluate, in rabbits, the influence of COLV in the induction of differentiation of adipose tissue-derived stem cells to a chondrocyte-like cell phenotype. Materials and Methods: New Zealand Rabbits were used as source of adipose tissues for the isolation of mesenchymal stem cells (MSCs). Preliminary characterization of mesenchymal lineage and differentiation into chondrocyte-like phenotype was confirmed by immunofluorescence analysis using antibodies to collagens I, II (polyclonals), III and CD34 (monoclonals). After 2 and 3 weeks in culture with and without COLV, the cell aggregates were fixed for 2 h in 4% formaldehyde, dehydrated with ethanol, washed with xylene and embedded in paraffin. Different sections were cut and stained with Toluidine blue, Alcian blue and Picrosirius red respectively. Results: Proteoglicans and collagen fibers were observed by assessment of the different stains, confirming the collagen expression. Remarkably, compared to control cultures, in the presence of COLV timulation, MSCs were capable to increase production of collagen I and II, confirming its chondrocyte-like cell phenotype. Conclusion: We conclude that COLV may facilitate the differentiation of rabbit adipose tissue-derived stem cells into a chondrocyte-like phenotype and this result can be considered to be candidate for therapies.conferenceObject COLLAGEN V AND DECORIN INTERACTION ARE INVOLVED IN PULMONARY FIBROSIS OF SSc(2012) TEODORO, W.; MARCELINO, A.; CRUZ, I. C. Brindo da; VELOSA, A. P. P.; MARTIN, P.; CARRASCO, S.; GOLDENSTEIN-SCHAINBERG, C.; PARRA, E. R.; CAPELOZZI, V. L.; YOSHINARI, N. H.Aims. Type V collagen (COL V) is involved in SSc pathogenesis since immunization of health rabbits with this protein induces an experimental model reproducing the main pathogenic manifestation of this disease. We have demonstrated an increased amount of unusual COL V fibrils deposition in lung of SSc patients indicating an important role for this protein in fibrosis. Formation of fibrotic tissue can be induced both by cytokines and cell–matrix interaction involving signalization mechanism. COL V and decorin participate of this mechanism interfering with fibrilogenesis. The aim was to evaluate COL V and decorin expression in pulmonary tissue and to characterize biochemical profile of COLV from lung fibroblasts culture from SSc patients. Methods. We evaluated COL V and decorin expression as well as 3D reconstruction using IF in lung specimens from six patients with SSc without pulmonary hypertension and six normal individuals died from trauma. The amount of COL V in lung sections was evaluated with software Image Pro-Plus 6.0 in Olympus-BX51. Quantitative immunoblotting was used to characterize COL V biochemistry from lung fibroblasts culture. Results. It was found that the structure of COL V fibres was distorted and strongly thickened in lung tissue from SSc patients compared with thin fibres pattern in the healthy controls. Decorin was distributed around COL V fibrils in the bronchovascular interstitium and vascular walls. Histomorphometric analysis of SSc lung demonstrated increased expression of both COL V and decorin when compared with the control (68.52 + 7.36% vs 5.01 + 2.12%, P < 0.01 and 22.99 + 0.59% vs 32.93 + 3.81%, P = 0.01, respectively). The semiquantitative immunoblotting detected an increased high molecular weight COL V fraction in patients when compared with the controls (P = 0.02). Conclusions. The overexpression and unusual organization of COL V fibres with biochemical changes associated to increased decorin indicates that matrix signalization pathway is involved in COL V fibrillogenesis process in SSc pulmonary fibrosis.