Fibronectin glycation increases IGF-I induced proliferation of human aortic smooth muscle cells

The advanced glycation end products, namely AGEs, contribute to long-termed complications of diabetes mellitus, including macroangiopathy, where smooth muscle cells (SMC) proliferation stimulated by platelet-derived growth factor (PDGF) isoforms and insulin-like growth factor-I (IGF-I) plays an important role. The objective of the present study was to investigate the effect of an AGE-modified extracellular matrix protein on IGF-I induced SMC proliferation and on the IGF-I-IGF binding protein 4 (IGFBP-4) axis under basal conditions and after stimulation with PDGF-BB. IGF-I resulted in significantly higher thymidine incorporation in SMC seeded on AGE-modified fibronectin (AGE-FN) in comparison to cells seeded on fibronectin (FN). This augmented proliferation could not be accounted for by increased expression of IGF-IR, by decreased secretion of IGFBP-4, a binding protein that inhibits IGF-I mitogenic effects or by increased IGF-IR autophosphorylation. PDGF-BB did not modulate IGF-IR and IGFBP-4 mRNA expression in any of the substrata, however, this growth factor elicited opposite effects on the IGFBP-4 content in the conditioned media, increasing it in cells plated on FN and diminishing it in cells plated on AGE-FN. These findings suggest that one mechanism by which AGE-modified proteins is involved in the pathogenesis of diabetes-associated atherosclerosis might be by increasing SMC susceptibility to IGF-I mitogenic effects.

Adhesion of Trypanosoma cruzi Trypomastigotes to Fibronectin or Laminin Modifies Tubulin and Paraflagellar Rod Protein Phosphorylation

Background: The unicellular parasite Trypanosoma cruzi is the causative agent of Chagas disease in humans. Adherence of the infective stage to elements of the extracellular matrix (ECM), as laminin and fibronectin, is an essential step in host cell invasion. Although members of the gp85/TS, as Tc85, were identified as laminin and fibronectin ligands, the signaling events triggered on the parasite upon binding to these molecules are largely unexplored. Methodology/Principal Findings: Viable infective parasites were incubated with laminin, fibronectin or bovine serum albumin for different periods of time and the proteins were separated by bidimensional gels. The phosphoproteins were envisaged by specific staining and the spots showing phosphorylation levels significantly different from the control were excised and identified by MS/MS. The results of interest were confirmed by immunoblotting or immunoprecipitation and the localization of proteins in the parasite was determined by immunofluorescence. Using a host cell-free system, our data indicate that the phosphorylation contents of T. cruzi proteins encompassing different cellular functions are modified upon incubation of the parasite with fibronectin or laminin. Conclusions/Significance: Herein it is shown, for the first time, that paraflagellar rod proteins and alpha-tubulin, major structural elements of the parasite cytoskeleton, are predominantly dephosphorylated during the process, probably involving the ERK1/2 pathway. It is well established that T. cruzi binds to ECM elements during the cell infection process. The fact that laminin and fibronectin induce predominantly dephosphorylation of the main cytoskeletal proteins of the parasite suggests a possible correlation between cytoskeletal modifications and the ability of the parasite to internalize into host cells.

Fibronectin expression is decreased in metastatic renal cell carcinoma following endostatin gene therapy

Tumor cells induce the disruption of homeostasis between cellular and extracellular compartments to favor tumor progression. The expression of fibronectin (FN), a matrix glycoprotein, is increased in several carcinoma cell types, including renal cell carcinoma (RCC). RCC are highly vascularized tumors and are often amenable to antiangiogenic therapy. Endostatin (ES) is a fragment of collagen XVIII that possesses antiangiogenic activity. In this study, we examined the modulation of FN gene expression by ES gene therapy in a murine metastatic renal cell carcinoma (mRCC) model. Balb/C mice bearing Renca cells were treated with NIH/3T3-LXSN cells or NIH/3T3-LendSN cells. At the end of the experiment, the ES serum levels were measured, and the FN gene expression was assessed using real-time PCR. The tissue FN was evaluated by western blotting and by immunofluorescence analysis. The ES serum levels in treated mice were higher than those in the control group (P < 0.05). ES treatment led to significant decreases at the FN mRNA (P < 0.001) and protein levels (P < 0.01). Here, we demonstrate the ES antitumor effect that is mediated by down-regulation of FN expression in mRCC. (C) 2012 Elsevier Masson SAS. All rights reserved.

Calcium Binding to Leptospira Outer Membrane Antigen LipL32 Is Not Necessary for Its Interaction with Plasma Fibronectin, Collagen Type IV, and Plasminogen

LipL32 is the most abundant outer membrane protein from pathogenic Leptospira and has been shown to bind extracellular matrix (ECM) proteins as well as Ca2+. Recent crystal structures have been obtained for the protein in the apo-and Ca2+-bound forms. In this work, we produced three LipL32 mutants (D163-168A, Q67A, and S247A) and evaluated their ability to interact with Ca2+ and with ECM glycoproteins and human plasminogen. The D163-168A mutant modifies aspartate residues involved in Ca2+ binding, whereas the other two modify residues in a cavity on the other side of the protein structure. Loss of calcium binding in the D163-D168A mutant was confirmed using intrinsic tryptophan fluorescence, circular dichroism, and thermal denaturation whereas the Q67A and S247A mutants presented the same Ca2+ affinity as the wild-type protein. We then evaluated if Ca2+ binding to LipL32 would be crucial for its interaction with collagen type IV and plasma proteins fibronectin and plasminogen. Surprisingly, the wild-type protein and all three mutants, including the D163-168A variant, bound to these ECM proteins with very similar affinities, both in the presence and absence of Ca2+ ions. In conclusion, calcium binding to LipL32 may be important to stabilize the protein, but is not necessary to mediate interaction with host extracellular matrix proteins.

Antifibrotic Effect of Tamoxifen in a Model of Progressive Renal Disease

Tamoxifen, a selective estrogen receptor modulator, has antifibrotic properties; however, whether it can attenuate renal fibrosis is unknown. In this study, we tested the effects of tamoxifen in a model of hypertensive nephrosclerosis (chronic inhibition of nitric oxide synthesis with L-NAME). After 30 days, treated rats had significantly lower levels of albuminuria as well as lower histologic scores for glomerulosclerosis and interstitial fibrosis than untreated controls. Tamoxifen was renoprotective despite having no effect on the sustained, severe hypertension induced by L-NAME. Tamoxifen prevented the accumulation of extracellular matrix by decreasing the expression of collagen I, collagen III, and fibronectin mRNA and protein. These renoprotective effects associated with inhibition of TGF-beta 1 and plasminogen activator inhibitor-1, and with a significant reduction in a-smooth muscle actin-positive cells in the renal interstitium. Furthermore, tamoxifen abrogated IL-1 beta- and angiotensin-II-induced proliferation of fibroblasts from both kidney explants and from the NRK-49F cell line. Tamoxifen also inhibited the expression of extracellular matrix components and the production and release of TGF-beta 1 into the supernatant of these cells. In summary, tamoxifen exhibits antifibrotic effects in the L-NAME model of hypertensive nephrosclerosis, likely through the inhibition of TGF-beta 1, suggesting that it may have therapeutic use in CKD treatment.

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.

Lepstospira interrogans shotgun phage display identified LigB as a heparin-binding protein

LigB is an adhesin from pathogenic Leptospira that is able to bind to extracellular matrix and is considered a virulence factor. A shotgun phage display genomic library was constructed and used for panning against Heparan Sulfate Proteoglycan (HSPG). A phage clone encoding part of LigB protein was selected in panning experiments and showed specific binding to heparin. To validate the selected clone, fragments of LigB were produced as recombinant proteins and showed affinity to heparin and to mammalian cells. Heparin was also able to reduce the binding of rLB-Ct to mammalian cells. Our data suggests that the glycosaminoglycan moiety of the HSPG is responsible for its binding and could mediate the attachment of the recombinant protein rLB-Ct. Thus, heparin may act as a receptor for Leptospira to colonize and to invade the host tissue. (C) 2012 Elsevier Inc. All rights reserved.

Virulence attributes and genetic variability of oral Candida albicans and Candida tropicalis isolates

The wide spectrum of candidiasis and its clinical importance encourage the research with the purpose of clarifying the mechanisms of pathogenicity and identification of virulence factors of Candida sp. Therefore, the aim of this study was to verify the adhesion capacity, protease activity and genotypic diversity of oral C. albicans and C. tropicalis isolates. The adhesion ability to the extracellular matrix glycoproteins laminin and fibronectin was evaluated using the ELISA technique. The research of proteases was carried out in agar plate containing bovine albumin and through a quantitative method in buffer solution containing haemoglobin. Intra and interspecies polymorphisms was verified through random amplified polymorphic DNA (RAPD) technique. All C. albicans and C. tropicalis isolates binded to immobilised laminin and fibronectin. Ca33 and Ct13 isolates had relative adhesion index significantly higher than the other isolates for both glycoproteins (P < 0.001). Protease activity was observed in all isolates of C. albicans using either the semi-quantitative or quantitative assay. The protease activity of C. tropicalis was better detected through the quantitative assay. The genotypic diversity by RAPD revealed a heterogeneous population in both species. Nevertheless, C. tropicalis presented higher genetic variability than C. albicans strains.

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.

Lsa30, a novel adhesin of Leptospira interrogans binds human plasminogen and the complement regulator C4bp

Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Surface proteins have the potential to promote several activities, including adhesion. This work aimed to study the leptospiral coding sequence (CDS) LIC11087, genome annotated as hypothetical outer membrane protein. The LIC11087 gene was cloned and expressed in Escherichia coil BL21 (DE3) strain by using the expression vector pAE. The recombinant protein tagged with N-terminal 6XHis was purified by metal-charged chromatography and characterized by circular dichroism (CD) spectroscopy. The recombinant protein has the ability to mediate attachment to the extracellular matrix (ECM) components, laminin and plasma fibronectin, and was named Lsa30 (Leptospiral surface adhesin of 30 kDa). Lsa30 binds to laminin and to plasma fibronectin in a dose-dependent and saturable manner, with dissociation equilibrium constants (K-D) of 292 +/- 24 nM and 157 +/- 35 nM, respectively. Moreover, the Lsa30 is a plasminogen (PLC) receptor, capable of generating plasmin, in the presence of activator. This protein may interfere with the complement cascade by interacting with C4bp regulator. The Lsa30 is probably a new surface protein of Leptospira as revealed by immunofluorescence assays with living organisms and the reactivity with antibodies present in serum samples of experimentally infected hamsters. Thus, Lsa30 is a novel versatile protein that may play a role in mediating adhesion and may help pathogenic Leptospira to overcome tissue barriers and to escape the immune system. (C) 2012 Elsevier Ltd. All rights reserved.

Features of two proteins of Leptospira interrogans with potential role in host-pathogen interactions

Background: Leptospirosis is considered a re-emerging infectious disease caused by pathogenic spirochaetes of the genus Leptospira. Pathogenic leptospires have the ability to survive and disseminate to multiple organs after penetrating the host. Leptospires were shown to express surface proteins that interact with the extracellular matrix (ECM) and to plasminogen (PLG). This study examined the interaction of two putative leptospiral proteins with laminin, collagen Type I, collagen Type IV, cellular fibronectin, plasma fibronectin, PLG, factor H and C4bp. Results: We show that two leptospiral proteins encoded by LIC11834 and LIC12253 genes interact with laminin in a dose - dependent and saturable mode, with dissociation equilibrium constants (K-D) of 367.5 and 415.4 nM, respectively. These proteins were named Lsa33 and Lsa25 (Leptospiral surface adhesin) for LIC11834 and LIC12253, respectively. Metaperiodate - treated laminin reduced Lsa25 - laminin interaction, suggesting that sugar moieties of this ligand participate in this interaction. The Lsa33 is also PLG - binding receptor, with a K-D of 23.53 nM, capable of generating plasmin in the presence of an activator. Although in a weak manner, both proteins interact with C4bp, a regulator of complement classical route. In silico analysis together with proteinase K and immunoflorescence data suggest that these proteins might be surface exposed. Moreover, the recombinant proteins partially inhibited leptospiral adherence to immobilized laminin and PLG. Conclusions: We believe that these multifunctional proteins have the potential to participate in the interaction of leptospires to hosts by mediating adhesion and by helping the bacteria to escape the immune system and to overcome tissue barriers. To our knowledge, Lsa33 is the first leptospiral protein described to date with the capability of binding laminin, PLG and C4bp in vitro.

Batroxase, a new metalloproteinase from B. atrox snake venom with strong fibrinolytic activity

The structures and functional activities of metalloproteinases from snake venoms have been widely studied because of the importance of these molecules in envenomation. Batroxase, which is a metalloproteinase isolated from Bothrops atrox (Para) snake venom, was obtained by gel filtration and anion exchange chromatography. The enzyme is a single protein chain composed of 202 amino acid residues with a molecular mass of 22.9 kDa, as determined by mass spectrometry analysis, showing an isoelectric point of 7.5. The primary sequence analysis indicates that the proteinase contains a zinc ligand motif (HELGHNLGISH) and a sequence C164I165M166 motif that is associated with a "Met-turn" structure. The protein lacks N-glycosylation sites and contains seven half cystine residues, six of which are conserved as pairs to form disulfide bridges. The three-dimensional structure of Batroxase was modeled based on the crystal structure of BmooMP alpha-I from Bothrops moojeni. The model revealed that the zinc binding site has a high structural similarity to the binding site of other metalloproteinases. Batroxase presented weak hemorrhagic activity, with a MHD of 10 mu g, and was able to hydrolyze extracellular matrix components, such as type IV collagen and fibronectin. The toxin cleaves both a and beta-chains of the fibrinogen molecule, and it can be inhibited by EDTA. EGTA and beta-mercaptoethanol. Batroxase was able to dissolve fibrin clots independently of plasminogen activation. These results demonstrate that Batroxase is a zinc-dependent hemorrhagic metalloproteinase with fibrin(ogen)olytic and thrombolytic activity. Published by Elsevier Ltd.

Leptospiral Immunoglobulin-like Proteins Interact With Human Complement Regulators Factor H, FHL-1, FHR-1, and C4BP

Leptospira, the causative agent of leptospirosis, interacts with several host molecules, including extracellular matrix components, coagulation cascade proteins, and human complement regulators. Here we demonstrate that acquisition of factor H (FH) on the Leptospira surface is crucial for bacterial survival in the serum and that these spirochetes, besides interacting with FH, FH related-1, and C4b binding protein (C4BP), also acquire FH like-1 from human serum. We also demonstrate that binding to these complement regulators is mediated by leptospiral immunoglobulin-like (Lig) proteins, previously shown to interact with fibronectin, laminin, collagen, elastin, tropoelastin, and fibrinogen. Factor H binds to Lig proteins via short consensus repeat domains 5 and 20. Competition assays suggest that FH and C4BP have distinct binding sites on Lig proteins. Moreover, FH and C4BP bound to immobilized Ligs display cofactor activity, mediating C3b and C4b degradation by factor I. In conclusion, Lig proteins are multifunctional molecules, contributing to leptospiral adhesion and immune evasion.

Intestinal Lymph-Borne Factors Induce Lung Release of Inflammatory Mediators and Expression of Adhesion Molecules After an Intestinal Ischemic Insult

Background. Intestinal ischemia and reperfusion (I/R) is a documented cause of acute lung injury (ALI) and systemic inflammation. We previously reported that obstruction of thoracic lymphatic flow during intestinal I/R blunts pulmonary neutrophil recruitment and microvascular injury and decreases the systemic levels of tumor necrosis factor. Here, we consider the existence of a gut-lung axis promoting the induction of systemic inflammation, whereby drained intestinal lymph stimulates lung expression of adhesion molecules and matrix components and generation of inflammatory mediators. Material and Methods. Upon administration of anesthesia, male Wistar rats were subjected to occlusion of the superior mesenteric artery for 45 min, followed by 2 h of intestinal reperfusion (I/R); groups of rats were subjected to I/R with or without thoracic lymphatic duct ligation immediately before the procedure. The non-manipulated rats were used to investigate basal parameters. Results. Obstruction of thoracic lymphatic flow before intestinal I/R decreased the ability of cultured lung tissue explants to release IL-1 beta, IL-10, and VEGF. In contrast, lymphatic obstruction normalized the elevated lung expression of PECAM-1 caused by intestinal I/R. On the other hand, lung E-selectin expression was significantly reduced, whereas fibronectin expression and collagen synthesis were not affected. Lymph levels of LTB4 and TXB2 were found to be significantly increased. Conclusions. These data suggest that lymph factors drained from the intestine during ischemic trauma stimulate the lung to generate inflammatory mediators and alter the expression of adhesion molecules. Disturbances in lung homeostasis mediated by lymph might contribute to the spread of inflammatory processes, thereby accounting for the systemic inflammation induced by intestinal I/R. (C) 2012 Elsevier Inc. All rights reserved.