Network organization of interstitial connective tissue cells in the human endolymphatic duct.

The human endolymphatic duct (ED) and sac of the inner ear have been suggested to control endolymph volume and pressure. However, the physiological mechanisms for these processes remain obscure. We investigated the organization of the periductal interstitial connective tissue cells and extracellular matrix (ECM) in four freshly fixed human EDs by transmission electron microscopy and by immunohistochemistry. The unique surgical material allowed a greatly improved structural and epitopic preservation of tissue. Periductal connective tissue cells formed frequent intercellular contacts and focally occurring electron-dense contacts to ECM structures, creating a complex tissue network. The connective tissue cells also formed contacts with the basal lamina of the ED epithelium and the bone matrix, connecting the ED with the surrounding bone of the vestibular aqueduct. The interstitial connective tissue cells were non-endothelial and non-smooth muscle fibroblastoid cells. We suggest that the ED tissue network forms a functional mechanical entity that takes part in the control of inner ear fluid pressure and endolymph resorption.     

Laminin-111 derived peptides AG73 and C16 regulate invadopodia activity of a human adenoid cystic carcinoma cell line

Adenoid cystic carcinoma is a frequently occurring malignant salivary gland neoplasm with high level of recurrence and distant metastasis long time after treatment. Metastatic tumor cells that actively migrate and invade surrounding tissues rely on invadopodia to degrade extracellular matrix (ECM) barriers. Invadopodia are actin-rich membrane protrusions that localize enzymes required for ECM degradation. Breakdown of ECM macromolecules releases fragments and bioactive peptides. We have already demonstrated that laminin-111 and its derived peptides regulate migration, invasion and protease activity of adenocarcinoma cells. Here we addressed the role of laminin-111 peptides AG73 and C16 in invadopodia activity of cells (CAC2) derived from human adenoid cystic carcinoma. CAC2 cells were treated by AG73 and C16, and subjected to fluorescent gelatin substrate degradation assay. In this assay invadopodia activity areas appear as black dots in a fluorescent background. Both peptides significantly increased invadopodia formation and activity compared to controls. We analyzed putative receptors and signaling pathways related to peptide effects. β1 integrin silencing by siRNA decreased AG73- and C16-induced invadopodia. Furthermore inhibition of Rac1 and ERK signaling pathways decreased both C16- and AG73-related invadopodia activities. We propose that laminin-111 peptides AG73 and C16 increase invadopodia activity in CAC2 cells through β1 integrin. Rac1 and ERK1/2 signaling pathways would transduce signals generated by both peptides.     

Mechanoregulation of gene expression in fibroblasts

Mechanical loads placed on connective tissues alter gene expression in fibroblasts through mechanotransduction mechanisms by which cells convert mechanical signals into cellular biological events, such as gene expression of extracellular matrix components (e.g., collagen). This mechanical regulation of ECM gene expression affords maintenance of connective tissue homeostasis. However, mechanical loads can also interfere with homeostatic cellular gene expression and consequently cause the pathogenesis of connective tissue diseases such as tendinopathy and osteoarthritis. Therefore, the regulation of gene expression by mechanical loads is closely related to connective tissue physiology and pathology. This article reviews the effects of various mechanical loading conditions on gene regulation in fibroblasts and discusses several mechanotransduction mechanisms. Future research directions in mechanoregulation of gene expression are also suggested.     

Cellular signaling by peptides of the endothelin gene family

Endothelins (ET) are a family of regulatory peptides synthesized by selected endothelial and epithelial cells that act in a paracrine fashion on nearby smooth muscle or connective tissue cells. We review the pathways of transmembrane signaling triggered by binding of endothelin peptides to receptors on the plasma membrane. Although our understanding of many components is unclear, endothelin peptides appear to evoke a phosphoinositide-linked signaling system that bears a striking resemblance to signaling pathways activated by other regulatory peptides. Expression of endothelin receptors and specific pathways stimulated by activated receptors are controlled in a cell- and tissue-specific manner, which perhaps explains the diverse biological actions of endothelin in different tissues. Complex negative feedback pathways regulate endothelin-induced signaling at the receptor and second messenger levels. Moreover, by regulating the activity of sequence-specific DNA binding proteins, short-term signals by ET can be extended to long-term effects involving gene expression. Regulation of gene expression by ET could account for complex events such as mitogenesis and vascular and tissue remodeling in disease.     

The antitumor properties of the alpha3(IV)-(185-203) peptide from the NC1 domain of type IV collagen (tumstatin) are conformation-dependent

Tumor progression may be controlled by various fragments derived from noncollagenous 1 (NC1) C-terminal domains of type IV collagen. We demonstrated previously that a peptide sequence from the NC1 domain of the alpha3(IV) collagen chain inhibits the in vitro expression of matrix metalloproteinases in human melanoma cells through RGD-independent binding to alpha(v)beta(3) integrin. In the present paper, we demonstrate that in a mouse melanoma model, the NC1 alpha3(IV)-(185-203) peptide inhibits in vivo tumor growth in a conformation-dependent manner. The decrease of tumor growth is the result of an inhibition of cell proliferation and a decrease of cell invasive properties by down-regulation of proteolytic cascades, mainly matrix metalloproteinases and the plasminogen activation system. A shorter peptide comprising the seven N-terminal residues 185-191 (CNYYSNS) shares the same inhibitory profile. The three-dimensional structures of the CNYYSNS and NC1 alpha3(IV)-(185-203) peptides show a beta-turn at the YSNS (188-191) sequence level, which is crucial for biological activity. As well, the homologous MNYYSNS heptapeptide keeps the beta-turn and the inhibitory activity. In contrast, the DNYYSNS heptapeptide, which does not form the beta-turn at the YSNS level, is devoid of inhibitory activity. Structural studies indicate a strong structure-function relationship of the peptides and point to the YSNS turn as necessary for biological activity. These peptides could act as potent and specific antitumor antagonists of alpha(v)beta(3) integrin in melanoma progression.     

Fine Structural Observations on the Turbellarians Stenostomum sp. and Microstomum lineare with Special Reference to the Extracellular Matrix and Connective Tissue Systems

Employing transmission electron microscopy, observations were made on epidermis, muscle cells and connective tissue systems, with special emphasis on extracellular matrix components (ECM), in two rather primitive turbellarians: Stenostomum sp. (Catenulida) and Microstomum lineare (Macrostomida). In Stenostomum the only ECM components found are basal laminae, predominantly situated subepidermally. In Microstomum ECM is well developed and connective tissue filaments abundant in conspicuous extracellular spaces. It is uncertain whether basal laminae exist. The finding of basal lamina structures as the only ECM component present in Stenostomum makes it now possible to establish a complete ECM and connective tissue hierarchy in turbellarians, ranging from a purely cellular type with no ECM present to systems dominated by ECM and very similar to loose connective tissue in vertebrates. Comparative aspects of ECM and connective tissue systems in turbellarians are discussed in addition to the difficulties and ambiguities regarding definition and nomenclature of basal matrices as basal laminae and subepidermal membranes.     

Laying the groundwork for growth: Cell–cell and cell–ECM interactions in cardiovascular development

Cardiac development is reliant upon the spatial and temporal regulation of both genetic and chemical signals. Central to the communication of these signals are direct interactions between cells and their surrounding environment. The extracellular matrix (ECM) plays an integral role in cell communication and tissue growth throughout development by providing both structural support and chemical signaling factors. The present review discusses elements of cell–cell and cell–ECM interactions involved in cardiogenesis, and how disruption of these interactions can result in numerous heart defects. Examining the relationships between cells and their immediate environment has implications for novel and existing therapeutic strategies to combating congenital disorders. Birth Defects Research (Part C) 90:1–7, 2010. © 2010 Wiley‐Liss, Inc.     

The ultrastructure of the morula cells of Eupentacta quinquesemita (Echinodermata: Holothuroidea) and their role in the maintenance of the extracellular matrix

The ultrastructure of the morula cells of Eupentacta quinquesemita and the distribution of these cells in the dermal connective tissue are described. Morula cells are abundant in the dermis and appear to function in the maintenance of the extracellular matrix (ECM) as a source of ground substance material. The synthetic activity of these cells is described in detail. Morula cells are filled with large secretory vesicles containing three electrondense materials which are derived from rough endoplasmic reticulum and Golgi activity. The synthetic product of these cells contains glycosaminoglycans and is secreted into the ECM by degranulation. The ultrastructural and histochemical similarity of the degranulation product to the ECM ground substance suggests that they are comprised of the same material. Morula cells appear to function primarily in connective tissues where ground substance predominates. The cells often contain secretory vesicles at various stages of formation, all of which eventually mature and degranulate. The synthetic pathway of the morula cells appears to result ultimately in the complete disruption and death of the cells. The function of morula cells in the holothuroid ECM is discussed, and the synthetic activity of the cells is compared with that of other secretory cells.     

Methods in studying ECM degradation

Almost all tissues in our body contain specific cells associated with the tissue itself, and an extracellular matrix (ECM) that consists of a variety of proteins of which the bulk is formed by different types of collagens, glycoproteins and proteoglycans. The ECM plays a pivotal role in numerous processes not only related to the mechanical properties of a tissue, but also in modulating cellular activity. For a proper functioning of a tissue remodeling of the ECM is essential. Some connective tissues are characterized by a very rapid turnover (e.g. periodontal ligament) whereas others hardly show signs of turnover (e.g. cartilage). In all situations degradation of the ECM constituents occur. Under certain conditions, especially during a pathological situation, a high level of degradation may take place. In other situations matrix synthesis and deposition outstrips breakdown, leading to a fibrosis. In order to obtain information on the level of degradation of the different ECM components, various methods have been employed. A number of these methods will be discussed in this article.     

Functional role of periostin in development and wound repair: implications for connective tissue disease

Integrity of the extracellular matrix (ECM) is essential for maintaining the normal structure and function of connective tissues. ECM is secreted locally by cells and organized into a complex meshwork providing physical support to cells, tissues, and organs. Initially thought to act only as a scaffold, the ECM is now known to provide a myriad of signals to cells regulating all aspects of their phenotype from morphology to differentiation. Matricellular proteins are a class of ECM related molecules defined through their ability to modulate cell-matrix interactions. Matricellular proteins are expressed at high levels during development, but typically only appear in postnatal tissue in wound repair or disease, where their levels increase substantially. Members of the CCN family, tenascin-C, osteopontin, secreted protein acidic rich in cysteine (SPARC), bone sialoprotein, thrombospondins, and galectins have all been classed as matricellular proteins. Periostin, a 90 kDa secreted homophilic cell adhesion protein, was recently added to matricellular class of proteins based on its expression pattern and function during development as well as in wound repair. Periostin is expressed in connective tissues including the periodontal ligament, tendons, skin and bone, and is also prominent in neoplastic tissues, cardiovascular disease, as well as in connective tissue wound repair. This review will focus on the functional role of periostin in tissue physiology. Fundamentally, it appears that periostin influences cell behaviour as well as collagen fibrillogenesis, and therefore exerts control over the structural and functional properties of connective tissues in both health and disease. Periostin is a novel matricellular protein with close homology to Drosophila fasciclin 1. In this review, the functional role of periostin is discussed in the context of connective tissue physiology, in development, disease, and wound repair.     

A bioreactor test system to mimic the biological and mechanical environment of oral soft tissues and to evaluate substitutes for connective tissue grafts

Gingival cells of the oral connective tissue are exposed to complex mechanical forces during mastication, speech, tooth movement and orthodontic treatments. Especially during wound healing following surgical procedures, internal and external forces may occur, creating pressure upon the newly formed tissue. This clinical situation has to be considered when developing biomaterials to augment soft tissue in the oral cavity. In order to pre‐evaluate a collagen sponge intended to serve as a substitute for autogenous connective tissue grafts (CTGs), a dynamic bioreactor system was developed. Pressure and shear forces can be applied in this bioreactor in addition to a constant medium perfusion to cell‐material constructs. Three‐dimensional volume changes and stiffness of the matrices were analyzed. In addition, cell responses such as cell vitality and extracellular matrix (ECM) production were investigated. The number of metabolic active cells constantly increased under fully dynamic culture conditions. The sponges remained elastic even after mechanical forces were applied for 14 days. Analysis of collagen type I and fibronectin revealed a statistically significant accumulation of these ECM molecules (P < 0.05–0.001) when compared to static cultures. An increased expression of tenascin‐c, indicating tissue remodeling processes, was observed under dynamic conditions only. The results indicate that the tested in vitro cell culture system was able to mimic both the biological and mechanical environments of the clinical situation in a healing wound. Biotechnol. Bioeng. 2010;107: 1029–1039. © 2010 Wiley Periodicals, Inc.     

Ultrastructure of the capillaries of nerve tissue transplants growing in the anterior chamber of the eye

Blood capillaries have been studied electron microscopically in the areas of grafts (rat embryonal hippocamp and septal cerebral parts transplanted to mature rats) containing mainly nervous, glial or connective tissue cells. Certain differences in the capillary wall structure have been revealed. In areas with a great concentration of nervous cells, the blood capillaries are characterized by a dense arrangement of cellular elements in their walls, a continuous layer of the glial end-feet, this is specific for the CNS capillaries providing the blood--brain barrier. In peripheral area of the grafts, where glial elements predominate, the capillaries have loose arrangement of the mural cellular elements, great endotheliocyte activity, thick connective tissue tunic, lack of a dense glial surrounding. These characteristics make dubious the statement whether these capillaries possess the blood--brain barrier function. In places where connective tissue cells make aggregates, the capillaries do not possess the barrier properties because of perforations and fenestrae in endothelium and interruptions of the basal membrane, absence of pericapillary glial elements. All types of the capillaries demonstrate certain signs of a high functional activity. Formation of the capillary structure depends on the surrounding tissue.     

Nanoscale engineering of extracellular matrix-mimetic bioadhesive surfaces and implants for tissue engineering

Background

The goal of tissue engineering is to restore tissue function using biomimetic scaffolds which direct desired cell fates such as attachment, proliferation and differentiation. Cell behavior in vivo is determined by a complex interaction of cells with extracellular biosignals, many of which exist on a nanoscale. Therefore, recent efforts in tissue engineering biomaterial development have focused on incorporating extracellular matrix- (ECM) derived peptides or proteins into biomaterials in order to mimic natural ECM. Concurrent advances in nanotechnology have also made it possible to manipulate protein and peptide presentation on surfaces on a nanoscale level.

Scope of Review

This review discusses protein and peptide nanopatterning techniques and examples of how nanoscale engineering of bioadhesive materials may enhance outcomes for regenerative medicine.

Major Conclusions

Synergy between ECM-mimetic tissue engineering and nanotechnology fields can be found in three major strategies: (1) Mimicking nanoscale orientation of ECM peptide domains to maintain native bioactivity, (2) Presenting adhesive peptides at unnaturally high densities, and (3) Engineering multivalent ECM-derived peptide constructs.

General Significance

Combining bioadhesion and nanopatterning technologies to allow nanoscale control of adhesive motifs on the cell–material interface may result in exciting advances in tissue engineering.This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.     

Role of TGF-beta1 in the development of pancreatic fibrosis in Otsuka Long-Evans Tokushima Fatty rats

Recently established Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of naturally occurring obesity diabetes, exhibit progressive accumulation of connective tissue in the pancreas. The present study was designed to determine the pathogenic role of transforming growth factor-beta1 (TGF-beta1) in the development of pancreatic fibrosis in OLETF rats by investigating the serial changes in the expression of TGF-beta1 and extracellular matrix (ECM) in the pancreas. Progressive proliferation of connective tissue arose from the interstitial region surrounding islets at 20 wk of age and extended to the exocrine pancreas adjacent to the islets. TGF-beta1 mRNA levels in the pancreas increased at 20 wk of age and reached a peak value at 30 wk of age. Fibronectin (FN) and procollagen types I and III mRNAs peaked at 20 wk of age and remained at higher levels than those in the nondiabetic counterparts Long-Evans Tokushima Otsuka rats until 50 wk of age. Immunoreactivities for TGF-beta1 and FN were found in islets of OLETF rats at 20 wk of age and were seen in acinar and interstitial cells at 50 wk of age. Moreover, alpha-smooth muscle actin was located at interstitial region surrounding the islets. Proliferation of the connective tissue in the pancreas of OLETF rats closely correlated with expression of TGF-beta1 and ECM. Our results suggest that the development of pancreatic fibrosis in OLETF rats extends from endocrine to exocrine pancreas and that TGF-beta1 is involved in pancreatic fibrosis of OLETF rats.     

Terminal differentiation of nuchal ligament fibroblasts: characterization of synthetic properties and responsiveness to external stimuli

The temporal expression of elastogenesis is unique among connective tissues in that elastin production occurs primarily during late fetal and early neonatal periods and is essentially fully repressed once fiber assembly is completed. To test whether elastin synthesis in adult nuchal ligament fibroblasts is permanently repressed or whether the cells retain the ability to reinitiate production upon proper stimulation, we examined in adult ligament cells various parameters known to be involved in the regulation of elastin production. Elastin synthetic capacity, as determined by the levels of steady-state tropoelastin mRNA, of adult tissue was significantly decreased relative to fetal tissue. Likewise, fibroblasts grown from explants of adult ligament had about a fourfold decrease in elastin production and elastin-specific mRNA levels. On the other hand, adult cells were similar to fetal ligament cells in that they were sensitive to glucocorticoid stimulation and demonstrated chemotactic responsiveness to elastin peptides. Since our previous studies have shown that the extracellular matrix (ECM) plays an important role in influencing elastin phenotypic expression, fetal and adult fibroblasts were grown on slices of nonviable adult ligament to test if repression of elastin production was directed by factors in ECM of adult tissues. No change in elastin synthesis was detected with either cell type grown on adult ligament, whereas both fetal and adult cells demonstrated increased elastin production in response to contact with fetal ligament. These results suggest that adult ligament ECM does not provide a metabolic signal to shut off the elastin gene and that adult cells remain responsive to external stimuli that may reinitiate high levels of elastin synthesis.     

Interaction between cell and extracellular matrix in heart disease: multiple roles of tenascin-C in tissue remodeling

The heart remodels myocardial tissue in physiological and pathological response. The cell-extracellular matrix (ECM) interaction provides not only structural and mechanical support but also important biological signaling during tissue remodeling. Among various ECM molecules, tenascin-C (TNC) is well known as a regulator of multiple cellular functions during embryogenesis, wound healing or cancer progression. In the heart, TNC appears in several important steps of embryonic development such as the initial differentiation of cardiomyocytes or coronary vasculo/angiogenesis, but it is not detected in a normal adult myocardium. However, TNC is found to re-express after myocardial injury and may regulate cellular behavior during tissue remodeling by modulating the attachment of cardiomyocytes to connective tissue, by enhancing migration and differentiation of myofibroblasts, and by inducing matrix metallo-proteinases. TNC also interacts with other ECM molecules and may modulate progression of fibrosis. Furthermore, transient and site specific expression of TNC closely associated with myocardial injury and inflammation suggests not only its key roles during tissue remodeling but also that TNC can be a marker for myocardial disease activity.