Análise da influência de proteínas da matriz extracelular e fibrinogênio na formação de biofilme por cepas de Enterococcus faecalis isoladas de infecção endodôntica primária / Analysis of influence of extracellular matrix proteins and fibrinogen in biofilm formation by Enterococcus faecalis strains isolated from primary endodontic infection

Enterococcus faecalis é um patógeno oportunista com peculiar potencial para a manutenção da infecção perirradicular endodôntica após o preparo químico-mecânico do sistema de canais radiculares. Adicionalmente, possui aptidão para desenvolver-se em biofilme e apresenta em sua parede celular adesinas compatíveis com substratos colagênicos, como a composição da matriz extracelular da dentina e dos túbulos dentinários. Esse estudo propôs-se a caracterizar geneticamente 23 amostras de E faecalis isoladas de infecções endodônticas primárias através da técnica da reação em cadeia da polimerase (PCR, do inglês Polymerase Chain Reaction) e investigar a influência de COL I (colágeno tipo I), FN (fibronectina) e fibrinogênio (FBG) na formação de biofilme em superfície abiótica. Assim, após a sensibilização de ¾ dos poços de placas de poliestireno estéreis com 50 µl da solução de proteína de matriz (COL I, FN e FBG) na concentração de 1mg/ml, transferiu-se 50µl de suspensão bacteriana (1,5 x 108 bact/mL) correspondente a cada amostra, de modo a preencher tanto os poços sensibilizados como os não sensibilizados. A quantificação da formação de biofilme foi realizada por meio de leitura por densidade óptica, cujos resultados revelaram que houve formação de biofilme por todas as em superfície abiótica, porém com diferentes graus de intensidade. Todas as cepas foram identificadas geneticamente como Enterococcus faecalis e a presença do gene gelE foi dominante. Contudo, nenhuma apresentou amplificação para os genes esp e agg, e, apesar de 73,9% das amostras amplificarem para o gene ace, apenas 2 cepas (P7 e P75) isoladas de infecções endodônticas primárias tiveram aumento de formação de biofilme na presença de COL I (P<0,05). Embora a presença de FBG não forneça subsídio estatisticamente significante para a formação de biofilme, COL I e FN influenciaram na redução da formação do biofilme para a maior parte das amostras. É possível que a capacidade de formação de biofilme inerente ao E. faecalis e a afinidade para FN e COL I através da expressão gênica de ace contribuam substancialmente para a manutenção desse micro-organismo no ambiente radicular mesmo após o tratamento endodôntico minucioso.

Enterococcus faecalis is an opportunistic pathogen with peculiar potential to maintain the periradicular endodontic infection even after chemical-mechanical preparation of the root canal system. In addition, it has the ability to develop into biofilms and presents in your cell wall adhesins compatible with collagenous substrates, as the composition of the extracellular matrix of the dentine and dentinal tubules. This study aims to characterize genetically 23 samples of E. faecalis isolated from primary endodontic infections by Polymerase Chain Reaction (PCR) technique and investigate the influence of collagen type I (COL I), fibronectin (FN) and fibrinogen (FBG) in biofilm formation on abiotic surface. Thus, after the sensitization of ¾ the wells of sterile microtiter plates with 50 ul of matrix protein solution (COL I and FN FBG) at a concentration of 1mg / ml, was transferred 50mL of bacterial suspension (1.5 x 108 bact / ml) corresponding to each sample in order to fill both wells sensitized and non-sensitized. Quantification of biofilm formation was performed by optical density, so the results showed that there were biofilm formation by all strains on abiotic surface, but with different degrees of intensity. All strains were genetically identified as Enterococcus faecalis and the presence of gelE gene was prevalent. However, none showed amplification for the esp and agg gene, and, while 73.9% of the samples for amplifying ace gene, only 2 strains (P7 and P75) isolated from primary endodontic infections they had increased biofilm formation in the presence of COL I (P <0.05). Although the presence of FBG no provides significant support for the biofilm formation, COL I and FN were relevant influence in the reduction of biofilm formation for most of the samples. It is possible that the biofilm-forming ability inherent in E. faecalis and affinity for FN and COL I through ace gene expression contribute substantially to maintain of this microorganism in the root environment even after thorough endodontic treatment.

Heparan sulfate proteoglycans: structure, protein interactions and cell signaling

Heparan sulfate proteoglycans are ubiquitously found at the cell surface and extracellular matrix in all the animal species. This review will focus on the structural characteristics of the heparan sulfate proteoglycans related to protein interactions leading to cell signaling. The heparan sulfate chains due to their vast structural diversity are able to bind and interact with a wide variety of proteins, such as growth factors, chemokines, morphogens, extracellular matrix components, enzymes, among others. There is a specificity directing the interactions of heparan sulfates and target proteins, regarding both the fine structure of the polysaccharide chain as well precise protein motifs. Heparan sulfates play a role in cellular signaling either as receptor or co-receptor for different ligands, and the activation of downstream pathways is related to phosphorylation of different cytosolic proteins either directly or involving cytoskeleton interactions leading to gene regulation. The role of the heparan sulfate proteoglycans in cellular signaling and endocytic uptake pathways is also discussed.

Proteoglicanos de heparam sulfato são encontrados tanto superfície celular quanto na matriz extracelular em todas as espécies animais. Esta revisão tem enfoque nas características estruturais dos proteoglicanos de heparam sulfato e nas interações destes proteoglicanos com proteínas que levam à sinalização celular. As cadeias de heparam sulfato, devido a sua variedade estrutural, são capazes de se ligar e interagir com ampla gama de proteínas, como fatores de crescimento, quimiocinas, morfógenos, componentes da matriz extracelular, enzimas, entreoutros. Existe uma especificidade estrutural que direciona as interações dos heparam sulfatos e proteínas alvo. Esta especificidade está relacionada com a estrutura da cadeia do polissacarídeo e os motivos conservados da cadeia polipeptídica das proteínas envolvidas nesta interação. Os heparam sulfatos possuem papel na sinalização celular como receptores ou coreceptores para diferentes ligantes. Esta ligação dispara vias de sinalização celular levam à fosforilação de diversas proteínas citosólicas ou com ou sem interações diretas com o citoesqueleto, culminando na regulação gênica. O papel dos proteoglicanos de heparam sulfato na sinalização celular e vias de captação endocítica também são discutidas nesta revisão.

Blancos terapéuticos potenciales para revertir la cirrosis hepática / Therapeutical targets for revert liver fibrosis

Liver fibrosis is the common response to chronic liver injury, ultimately leading to cirrhosis and its complications: portal hypertension, liver failure, hepatic encephalopathy, and hepatocellular carcinoma and others. Efficient and well-tolerated antifibrotic drugs are still lacking, and current treatment of hepatic fibrosis is limited to withdrawal of the noxious agent. Efforts over the past decade have mainly focused on fibrogenic cells generating the scarring response, although promising data on inhibition of parenchymal injury or reduction of liver inflammation have also been obtained. A large number of approaches have been validated in culture studies and in animal models, and several clinical trials are underway or anticipated for a growing number of molecules. This review highlight recent advances in the molecular mechanisms of liver fibrosis and discusses mechanistically based strategies that have recently emerged.

The extracellular matrix of the lung and its role in edema formation

The extracellular matrix is composed of a three-dimensional fiber mesh filled with different macromolecules such as: collagen (mainly type I and III), elastin, glycosaminoglycans, and proteoglycans. In the lung, the extracellular matrix has several functions which provide: 1) mechanical tensile and compressive strength and elasticity, 2) low mechanical tissue compliance contributing to the maintenance of normal interstitial fluid dynamics, 3) low resistive pathway for an effective gas exchange, d) control of cell behavior by the binding of growth factors, chemokines, cytokines and the interaction with cell-surface receptors, and e) tissue repair and remodeling. Fragmentation and disorganization of extracellular matrix components comprises the protective role of the extracellular matrix, leading to interstitial and eventually severe lung edema. Thus, once conditions of increased microvascular filtration are established, matrix remodeling proceeds fairly rapidly due to the activation of proteases. Conversely, a massive matrix deposition of collagen fiber decreases interstitial compliance and therefore makes the tissue safety factor stronger. As a result, changes in lung extracellular matrix significantly affect edema formation and distribution in the lung.

A matriz extracelular é um aglomerado tridimensional demacromoléculas composta por: fibras colágenas (principalmente, tipos I e III), elastina, glicosaminoglicanos e proteoglicanos. No pulmão, a matriz extracelular tem várias funções, tais como: 1) promover estresse tensil e elasticidade tecidual, 2) contribuir para a manutenção da dinâmica de fluidos no interstício, 3) propiciar efetiva troca gasosa, 4) controlar a função celular através de sua ligação com fatores de crescimento, quimiocinas, citocinas e interação com receptores de superfície, e 5) remodelamento e reparo tecidual. A fragmentação e a desorganização da matriz extracelular pode acarretar edema intersticial e, eventualmente, edema alveolar grave. Logo, quando há aumento da filtração microvascular ocorre rápido remodelamento da matriz por ativação de proteases. Destarte, a deposição de fibras colágenas reduz a complacência intersticial limitando o edema. Em conclusão, modificações na matriz extracelular podem afetar a formação e distribuição do edema no pulmão.

The extracellular matrix provides directional cues for neuronal migration during cerebellar development

Normal central nervous system development relies on accurate intrinsic cellular programs as well as on extrinsic informative cues provided by extracellular molecules. Migration of neuronal progenitors from defined proliferative zones to their final location is a key event during embryonic and postnatal development. Extracellular matrix components play important roles in these processes, and interactions between neurons and extracellular matrix are fundamental for the normal development of the central nervous system. Guidance cues are provided by extracellular factors that orient neuronal migration. During cerebellar development, the extracellular matrix molecules laminin and fibronectin give support to neuronal precursor migration, while other molecules such as reelin, tenascin, and netrin orient their migration. Reelin and tenascin are extracellular matrix components that attract or repel neuronal precursors and axons during development through interaction with membrane receptors, and netrin associates with laminin and heparan sulfate proteoglycans, and binds to the extracellular matrix receptor integrins present on the neuronal surface. Altogether, the dynamic changes in the composition and distribution of extracellular matrix components provide external cues that direct neurons leaving their birthplaces to reach their correct final location. Understanding the molecular mechanisms that orient neurons to reach precisely their final location during development is fundamental to understand how neuronal misplacement leads to neurological diseases and eventually to find ways to treat them.

Specific structural features of syndecans and heparan sulfate chains are needed for cell signaling

The syndecans, heparan sulfate proteoglycans, are abundant molecules associated with the cell surface and extracellular matrix and consist of a protein core to which heparan sulfate chains are covalently attached. Each of the syndecan core proteins has a short cytoplasmic domain that binds cytosolic regulatory factors. The syndecans also contain highly conserved transmembrane domains and extracellular domains for which important activities are becoming known. These protein domains locate the syndecan on cell surface sites during development and tumor formation where they interact with other receptors to regulate signaling and cytoskeletal organization. The functions of cell surface heparan sulfate proteoglycan have been centered on the role of heparan sulfate chains, located on the outer side of the cell surface, in the binding of a wide array of ligands, including extracellular matrix proteins and soluble growth factors. More recently, the core proteins of the syndecan family transmembrane proteoglycans have also been shown to be involved in cell signaling through interaction with integrins and tyrosine kinase receptors.

Regulation of pituitary hormones and cell proliferation by components of the extracellular matrix

The extracellular matrix is a three-dimensional network of proteins, glycosaminoglycans and other macromolecules. It has a structural support function as well as a role in cell adhesion, migration, proliferation, differentiation, and survival. The extracellular matrix conveys signals through membrane receptors called integrins and plays an important role in pituitary physiology and tumorigenesis. There is a differential expression of extracellular matrix components and integrins during the pituitary development in the embryo and during tumorigenesis in the adult. Different extracellular matrix components regulate adrenocorticotropin at the level of the proopiomelanocortin gene transcription. The extracellular matrix also controls the proliferation of adrenocorticotropin-secreting tumor cells. On the other hand, laminin regulates the production of prolactin. Laminin has a dynamic pattern of expression during prolactinoma development with lower levels in the early pituitary hyperplasia and a strong reduction in fully grown prolactinomas. Therefore, the expression of extracellular matrix components plays a role in pituitary tumorigenesis. On the other hand, the remodeling of the extracellular matrix affects pituitary cell proliferation. Matrix metalloproteinase activity is very high in all types of human pituitary adenomas. Matrix metalloproteinase secreted by pituitary cells can release growth factors from the extracellular matrix that, in turn, control pituitary cell proliferation and hormone secretion. In summary, the differential expression of extracellular matrix components, integrins and matrix metalloproteinase contributes to the control of pituitary hormone production and cell proliferation during tumorigenesis.

Extracellular matrix molecules play diverse roles in the growth and guidance of central nervous system axons

Axon growth and guidance represent complex biological processes in which probably intervene diverse sets of molecular cues that allow for the appropriate wiring of the central nervous system (CNS). The extracellular matrix (ECM) represents a major contributor of molecular signals either diffusible or membrane-bound that may regulate different stages of neural development. Some of the brain ECM molecules form tridimensional structures (tunnels and boundaries) that appear during time- and space-regulated events, possibly playing relevant roles in the control of axon elongation and pathfinding. This short review focuses mainly on the recognized roles played by proteoglycans, laminin, fibronectin and tenascin in axonal development during ontogenesis

Adhesion of the human pathogen Sporothrix schenckii to several extracellular matrix proteins

The pathogenic fungus Sporothrix schenckii is the causative agent of sporotrichosis. This subcutaneous mycosis may disseminate in immunocompromised individuals and also affect several internal organs and tissues, most commonly the bone, joints and lung. Since adhesion is the first step involved with the dissemination of pathogens in the host, we have studied the interaction between S. schenckii and several extracellular matrix (ECM) proteins. The binding of two morphological phases of S. schenckii, yeast cells and conidia, to immobilized type II collagen, laminin, fibronectin, fibrinogen and thrombospondin was investigated. Poly (2-hydroxyethyl methacrylate) (poly-HEMA) was used as the negative control. Cell adhesion was assessed by ELISA with a rabbit anti-S. schenckii antiserum. The results indicate that both morphological phases of this fungus can bind significantly to type II collagen, fibronectin and laminin in comparison to the binding observed with BSA (used as blocking agent). The adhesion rate observed with the ECM proteins (type II collagen, fibronectin and laminin) was statistically significant (P<0.05) when compared to the adhesion obtained with BSA. No significant binding of conidia was observed to either fibrinogen or thrombospondin, but yeast cells did bind to the fibrinogen. Our results indicate that S. schenckii can bind to fibronectin, laminin and type II collagen and also show differences in binding capacity according to the morphological form of the fungus

Integrinas: mediadores de interacción entre células y matriz extracelular / Integrins: interaction mediators between cells and extracellular matrix

Se presenta una revisión actualizada sobre integrinas, glicoproteínas transmembrana, que unen selectivamente proteínas constitutivas de la matriz extracelular y que sincrónicamente se relacionan con filamentos del citoesqueleto, modulando de esta forma la morfología y fisiología celular. Se describe también, la expresión de estos receptores en la transformación de células epiteliales mamarias

Thrombospondin: relationship to protease and growth factor/cytokine activity

1. The finding in the last two years of different proteins presenting structural homology with platelet thrombospondin (TSP-1) has permitted to establish the existence of a set of related genes referred to as thrombospondin family. While much work remains to be done concerning the characterization of the newly described members of the family, careful studies carried out on TSP-1 have been implicating this high molecular weight molecule (420-450 KDa) in a variety of aspects of celular physiology. 2. The present text discusses the implications of the matrix-bound and fluid TSP-1 forms for cell adhesion and protease activity generation. Their relationships with growth factors in matrices are also discussed