Role of nitric oxide in the remodelling of extracellular matrix in myxomatous mitral valve degeneration of dogs and pigs

Myxomatous mitral valve degeneration (MMVD) or endocardiosis is a heart valve disease that occurs in many mammalian species, especially in humans, dogs and pigs. Nitric oxide (NO) plays an important role in the MMVD development. NO can be indirectly evaluated by the nitric-oxide synthase (NOS) expression and by the histochemical reaction of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d). The aim of this study was to evaluate NOS activity, by NADPH-d reaction, in the anterior leaflet of dogs with regular mitral valves and in those with MMVD, as well as in young swine and old females, comparing the reaction level with the degree of endocardiosis disease and also the histological alterations. Twelve mitral valves of dogs and 22 of swine were used for the research. All the valves were macroscopically analyzed for the occurrence or not of endocardiosis. They were fixed in a 4% paraformaldehyde, exposed to NADPH-d reaction, routinely processed and microscopically evaluated for the detection of mucopolysaccharides (MPS) deposition, collagen degeneration, fibrosis and level of endocardiosis. In dogs, relation was observed between higher intensity of the NADPH-d reaction, higher endocardiosis degree, MPS deposition as well as the collagen degeneration. No alteration in color was observed in pigs ́ valves during NADPH-d reaction. In conclusion, NO works in canine mitral valve remodeling extracellular matrix and plays an important role in endocardiosis disease. In swine, the lack of reaction reinforces the absence of macroscopical endocardiosis lesions, suggesting restrict NO action or major differences in the structures of swine valves.

Elevated hyaluronan and extracellular matrix metalloproteinase inducer levels in women with preeclampsia

Purpose: Preeclampsia (PE) is a specific syndrome of pregnancy clinically identified by hypertension and proteinuria from the 20th week of gestation associated with a systemic inflammatory response and oxidative stress. While pro-inflammatory cytokines have been extensively studied in PE, other factors in the circulation that also influence the magnitude of inflammation have received much less attention. The present study compared serum concentrations of five immune-regulatory compounds in normotensive pregnant women and in women with gestational hypertension (GH) or PE. Methods: Sixty women with PE, 53 with GH and 40 normotensive women paired by gestational age were evaluated. Sera were evaluated for concentrations of extracellular matrix metalloproteinase inducer (EMMPRIN), hyaluronan, gelsolin, visfatin and histone 2B by ELISA. Differences between groups were analyzed by nonparametric tests, with a significance level of 5 %. Results: Increased levels of EMMPRIN and hyaluronan were present in preeclamptic women as compared to the GH and normotensive groups. There was no difference between groups in gelsolin, visfatin or histone 2B. Conclusion: Increased release of EMMPRIN and hyaluronan may contribute to an elevated pro-inflammatory response and tissue damage in women with PE. © 2013 Springer-Verlag Berlin Heidelberg.

Urethral striated muscle and extracellular matrix morphological characteristics among mildly diabetic pregnant rats: translational approach

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Extracellular matrix remodeling of the testes through the male reproductive cycle in Teleostei fish

During the fish reproductive cycle, testes undergo morphological changes related to germinal epithelium and remodeling of extracellular matrix components (ECM). ECM is degraded mainly by action of matrix metalloproteinases (MMPs). Due to the natural renewal of ECM in fish testes, we choose Pimelodus maculatus to study remodeling of ECM throughout reproductive cycle, using picrosirius (to identify type I, II, III collagen) and reticulin (type III collagen), and to immunolocalize MT1-MMP (membrane type 1-matrix metalloproteinase) and MMP-2 in testis cells. Testes were classified in four reproductive phases: regenerating, development, spawning capable and regressing. Picrosirius and reticulin demonstrated a differential distribution of total collagen fibers during the reproductive cycle. Immunohistochemistry showed MT1-MMP only in acidophilic granulocyte cells mainly inside blood vessels, in connective tissue of capsule close to the germinal compartment, and also infiltrated in interstitial connective tissue. MMP-2 was detected in fibroblast and endothelial cells of interstitial and capsule blood vessels, in epithelial cells of capsule, and in acidophilic granulocyte cells at same description for MT1-MMP. The fish testes ECM were remodeled throughout reproductive cycle in according to morphophysiological alterations. During reproductive season (spawning capable), the interstitium increased in total collagen fibers (type I, II, III). After spermiation period (regression and regenerating), the amount of collagen fibers decreased in response to action of MMPs on collagen degradation and other interstitial components (not assessed in this study). MMPs seem to be indispensable components for natural cyclic events of ECM remodeling of fish testes and for guarantee tissue homeostasis throughout reproductive cycle.

Prostate hyperplasia caused by long-term obesity is characterized by high deposition of extracellular matrix and increased content of MMP-9 and VEGF

Recent studies have shown a positive association of cancer and obesity, but the morphological and molecular mechanisms involved in this relationship are still unknown. This study analysed the impact of long-term obesity on rat prostate, focusing on stromal changes. Male adult Wistar rats were treated with high-fat diet to induce obesity, while the control group received a balanced diet. After 30 weeks of feeding, the ventral prostate was analysed by immunohistochemistry for cell proliferation, smooth muscle α-actin, vimentin, chondroitin sulphate and metalloproteinases (MMP-2 and 9). The content of androgen receptor (AR), oestrogen receptors (ERs) and vascular endothelial growth factor (VEGF) was measured by Western blotting, and activity of catalase and Glutathione-S-Transferase (GST) were quantified by enzymatic assay. Long-term obesity decreased testosterone plasma levels by 70% and resulted in stromal prostate hyperplasia, as evidenced by increased collagen fibres. Such stromal hyperplasia was associated with increased number of blood vessels and raised VEGF content, and increased expression of chondroitin sulphate, vimentin, α-actin and MMP-9. In spite of the high cell density in prostate, the proliferative activity was lower in the prostates of obese rats, indicating that hyperplasia was established during the early phases in this obesity model. AR levels increased significantly, whereas the ERα decreased in this group. Moreover, the levels of catalase and GST were changed considerably. These findings indicate that long-term obesity, besides disturbing the antioxidant control, causes intense stromal remodelling and release of factors that create an environment that can promote proliferative disorders in the gland, culminating with diffuse hyperplasia.

Alterations in extracellular matrix composition in the aging larynx

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Streptococcus mutans-derived extracellular matrix in cariogenic oral biofilms

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Alterations of protein expression in conditions of copper-deprivation for Paracoccidioides lutzii in the presence of extracellular matrix components

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Changes in cells's secretory organelles and extracellular matrix during endochondral ossification in the mandibular condyle of the growing rat

The mandibular condyle from 20-day-old rats was examined in the electron microscope with particular attention to intracellular secretory granules and extracellular matrix. Moreover, type II collagen was localized by an immunoperoxidase method. The condyle has been divided into five layers: (1) the most superficial, articular layer, (2) polymorphic cell layer, (3) flattened cell layer, (4) upper hypertrophic, and (5) lower hypertrophic cell layers. In the articular layer, the cells seldom divide, but in the polymorphic layer and upper part of the flattened cell layer, mitosis gives rise to new cells. In these layers, cells produce two types of secretory granules, usually in distinct stacks of the Golgi apparatus; type a, cylindrical granules, in which 300-nm-long threads are packed in bundles which appear lucent after formaldehyde fixation; and type b, spherical granules loaded with short, dotted filaments. The matrix is composed of thick banded lucent fibrils in a loose feltwork of short, dotted filaments. The cells arising from mitosis undergo endochondral differentiation, which begins in the lower part of the flattened cell layer and is completed in the upper hypertrophic cell layer; it is followed by gradual cell degeneration in the lower hypertrophic cell layer. The cells produce two main types of secretory granules: type b as above; and type c, ovoid granules containing 300-nm-long threads associated with short, dotted filaments. A possibly different secretory granule, type d, dense and cigar-shaped, is also produced. The matrix is composed of thin banded fibrils in a dense feltwork. In the matrix of the superficial layers, the lucency of the fibrils indicated that they were composed of collagen I, whereas the lucency of the cylindrical secretory granules suggested that they transported collagen I precursors to the matrix. Moreover, the use of ruthenium red indicated that the feltwork was composed of proteoglycan; the dotted filaments packed in spherical granules were similar to, and presumably the source of, the matrix feltwork. The superficial layers did not contain collagen II and were collectively referred to as perichondrium. In the deep layers, the ovoid secretory granules displayed collagen II antigenicity and were likely to transport precursors of this collagen to the matrix, where it appeared in the thin banded fibrils. That these granules also carried proteoglycan to the matrix was suggested by their content of short dotted filaments. Thus the deep layers contained collagen II and proteoglycan as in cartilage; they were collectively referred to as the hyaline cartilage region.

Extracellular Matrix Composition of the Cricopharyngeus Muscle

The aim of this study was to analyze the presence and distribution of total collagen, type I and type III collagen, elastic fibers, fibronectin, and versican in the endomysium of cricopharyngeus muscles from adults of various ages. The study was a cross-sectional analysis of human cricopharyngeus muscles. Twenty-seven muscles obtained from autopsies of men and women ranging in age from 28 to 92 years were analyzed with the Picrosirius method, oxidized Weigert resorcin-fuchsin, immunohistochemistry, and image analysis. Collagen had the highest density among the analyzed components. Elastic fibers surrounded each muscle cell; they were aligned longitudinally by their long axis and associated with traversing fibers, thereby forming a fiber network with embedded muscle cells. The fibronectin and versican contents varied widely among the specimens. We found no statistically significant differences between the proportion of extracellular matrix (ECM) components and factors such as gender and race. We conclude that the higher proportion of type I and type III collagen is compatible with the cricopharyngeus muscle's sphincteric behavior, and the arrangement of the elastic fibers may also contribute to the muscle's elasticity. We found no statistically significant correlation between the ECM components and age.

OmpL1 Is an Extracellular Matrix- and Plasminogen-Interacting Protein of Leptospira spp.

Leptospirosis is a zoonosis with multisystem involvement caused by pathogenic strains of the genus Leptospira. OmpL1 is an outer membrane protein of Leptospira spp. that is expressed during infection. In this work, we investigated novel features of this protein. We describe that OmpL1 is a novel leptospiral extracellular matrix (ECM)-binding protein and a plasminogen (PLG) receptor. The recombinant protein was expressed in Escherichia coli BL21(DE3) Star/pLysS as inclusion bodies, refolded, and purified by metal-chelating chromatography. The protein presented a typical beta-strand secondary structure, as evaluated by circular dichroism spectroscopy. The recombinant protein reacted with antibodies in serum samples from convalescent leptospirosis patients with a high specificity compared to serum samples from individuals with unrelated diseases. These data strengthen the usefulness of OmpL1 as a diagnostic marker of leptospirosis. The characterization of the immunogenicity of recombinant OmpL1 in inoculated BALB/c mice showed that the protein has the capacity to elicit humoral and cellular immune responses, as denoted by high antibody titers and the proliferation of lymphocytes. We demonstrate that OmpL1 has the ability to mediate attachment to laminin and plasma fibronectin, with KD (equilibrium dissociation constant) values of 2,099.93 +/- 871.03 nM and 1,239.23 +/- 506.85 nM, respectively. OmpL1 is also a PLG receptor, with a KD of 368.63 +/- 121.23 nM, capable of generating enzymatically active plasmin. This is the first report that shows and characterizes OmpL1 as an ECM-interacting and a PLG-binding protein of Leptospira spp. that may play a role in bacterial pathogenesis when expressed during infection.

Extracellular matrix in airway smooth muscle is associated with dynamics of airway function in asthma

Background: Altered deposition of extracellular matrix (ECM) in the airway smooth muscle (ASM) layer as observed in asthma may influence ASM mechanical properties. We hypothesized that ECM in ASM is associated with airway function in asthma. First, we investigated the difference in ECM expression in ASM between asthma and controls. Second, we examined whether ECM expression is associated with bronchoconstriction and bronchodilation in vivo. Methods: Our cross-sectional study comprised 19 atopic mild asthma patients, 15 atopic and 12 nonatopic healthy subjects. Spirometry, methacholine responsiveness, deep-breath-induced bronchodilation (Delta R-rs) and bronchoscopy with endobronchial biopsies were performed. Positive staining of elastin, collagen I, III and IV, decorin, versican, fibronectin, laminin and tenascin in ASM was quantified as fractional area and mean density. Data were analysed using Pearson's or Spearman's correlation coefficient. Results: Extracellular matrix expression in ASM was not different between asthma and controls. In asthmatics, fractional area and mean density of collagen I and III were correlated with methacholine dose-response slope and DRrs, respectively (r = 0.71, P < 0.01; r = 0.60, P = 0.02). Furthermore, ASM collagen III and laminin in asthma were correlated with FEV1 reversibility (r = -0.65, P = 0.01; r = -0.54, P = 0.04). Conclusion: In asthma, ECM in ASM is related to the dynamics of airway function in the absence of differences in ECM expression between asthma and controls. This indicates that the ASM layer in its full composition is a major structural component in determining variable airways obstruction in asthma.

Immunophenotyping and extracellular matrix remodeling in pulmonary and extrapulmonary sarcoidosis

Objective: To investigate the significance of cellular immune markers, as well as that of collagen and elastic components of the extracellular matrix, within granulomatous structures in biopsies of patients with pulmonary or extrapulmonary sarcoidosis. Methods: We carried out qualitative and quantitative evaluations of inflammatory cells, collagen fibers, and elastic fibers in granulomatous structures in surgical biopsies of 40 patients with pulmonary and extrapulmonary sarcoidosis using histomorphometry, immunohistochemistry, picrosirius red staining, and Weigert's resorcin-fuchsin staining. Results: The extrapulmonary tissue biopsies presented significantly higher densities of lymphocytes, macrophages, and neutrophils than did the lung tissue biopsies. Pulmonary granulomas showed a significantly higher number of collagen fibers and a lower density of elastic fibers than did extrapulmonary granulomas. The amount of macrophages in the lung samples correlated with FVC (p < 0.05), whereas the amount of CD3+, CD4+, and CD8+ lymphocytes correlated with the FEV1/FVC ratio and VC. There were inverse correlations between TLC and the CD1a+ cell count (p < 0.05), as well as between DLCO and collagen/elastic fiber density (r = -0.90; p = 0.04). Conclusions: Immunophenotyping and remodeling both showed differences between pulmonary and extrapulmonary sarcoidosis in terms of the characteristics of the biopsy samples. These differences correlated with the clinical and spirometric data obtained for the patients, suggesting that two different pathways are involved in the mechanism of antigen clearance, which was more effective in the lungs and lymph nodes.

Influence of a hypercholesterolemic diet on the collagen composition of the bladder wall extracellular matrix in rats

Purpose: To investigate the effects of hypercholesterolemic diet on the collagen composition of urinary bladder wall. Materials and methods: Forty-five female 4-week-old Wistar rats were divided into three groups: 1) control group fed a normal diet (ND); 2) model of bladder outlet obstruction (BOO) group fed a ND; and 3) group fed a HCD (1.25% cholesterol). Total serum cholesterol, LDL cholesterol and body weight were assessed at baseline. Four weeks later, group 2 underwent a surgical procedure resulting in a partial BOO, while groups 1 and 3 underwent a sham similar surgical procedure. Six weeks later, all animals had their bladders removed; serum cholesterol and LDL cholesterol levels and body weights were measured. Morphological and morphometric analysis was performed by Picrosirius staining and collagen types I and III were identified by immunofluorescence. Statistical analysis was completed and significance was considered when p<0.05. Results: Rats fed an HCD exhibited a significant increase in LDL cholesterol levels (p<0.001) and body weight (p=0.017), when compared to the groups fed a ND during the ten-week study period. Moreover, the HCD induced morphological alterations of the bladder wall collagen, regarding thin collagen fibers and the amounts of type III collagen when compared to the control group (p=0.002 and p=0.016, respectively), resembling the process promoted in the BOO model. Conclusions: A hyper-cholesterolemic diet in Wistar rats promoted morphological changes of the bladder types of collagen, as well as increases in body weight and LDL cholesterol.

Extracellular matrix composition in COPD

Extracellular matrix (ECM) composition has an important role in determining airway structure. We postulated that ECM lung composition of chronic obstructive pulmonary disease (COPD) patients differs from that observed in smoking and nonsmoking subjects without airflow obstruction. We determined the fractional areas of elastic fibres, type-I, -III and -IV collagen, versican, decorin, biglycan, lumican, fibronectin and tenascin in different compartments of the large and small airways and lung parenchyma in 26 COPD patients, 26 smokers without COPD and 16 nonsmoking control subjects. The fractional area of elastic fibres was higher in non-obstructed smokers than in COPD and nonsmoking controls, in all lung compartments. Type-I collagen fractional area was lower in the large and small airways of COPD patients and in the small airways of non-obstructed smokers than in nonsmokers. Compared with nonsmokers, COPD patients had lower versican fractional area in the parenchyma, higher fibronectin fractional area in small airways and higher tenascin fractional area in large and small airways compartments. In COPD patients, significant correlations were found between elastic fibres and fibronectin and lung function parameters. Alterations of the major ECM components are widespread in all lung compartments of patients with COPD and may contribute to persistent airflow obstruction.