Convective tissue movements play a major role in avian endocardial morphogenesis

Endocardial cells play a critical role in cardiac development and function, forming the innermost layer of the early (tubular) heart, separated from the myocardium by extracellular matrix (ECM). However, knowledge is limited regarding the interactions of cardiac progenitors and surrounding ECM during dramatic tissue rearrangements and concomitant cellular repositioning events that underlie endocardial morphogenesis. By analyzing the movements of immunolabeled ECM components (fibronectin, fibrillin-2) and TIE1 positive endocardial progenitors in time-lapse recordings of quail embryonic development, we demonstrate that the transformation of the primary heart field within the anterior lateral plate mesoderm (LPM) into a tubular heart involves the precise co-movement of primordial endocardial cells with the surrounding ECM. Thus, the ECM of the tubular heart contains filaments that were associated with the anterior LPM at earlier developmental stages. Moreover, endocardial cells exhibit surprisingly little directed active motility, that is, sustained directed movements relative to the surrounding ECM microenvironment. These findings point to the importance of large-scale tissue movements that convect cells to the appropriate positions during cardiac organogenesis.     

Extracellular matrix dynamics in heart failure: A prospect for gene therapy

In chronic congestive heart failure, an illness affecting more than 4 million Americans, there is impairment of myocardial extracellular matrix (ECM) remodeling. Failing human ventricular myocardium contains activated matrix metalloproteinases (MMPs), which are involved in adverse ECM remodeling. Our studies support the concept that impaired ECM remodeling and MMP activation are, in part, responsible for the cardiac structural deformation and heart failure. There is no known program that has declared its aim the investigation of the role of ECM gene therapy in heart failure. The development of transgenic technology, and emerging techniques for in vivo gene transfer, suggest a strategy for improving cardiac function by overexpressing or downregulation of the ECM components such as MMPs, tissue inhibitor of metalloproteinases (TIMPs), transforming growth factor-β1 (TGF-β), decorin, and collagen in cardiomyopathy and heart failure. J. Cell. Biochem. 68:403–410, 1998. © 1998 Wiley-Liss, Inc.     

Regulation of myocardial matrix metalloproteinase expression and activity by cardiac fibroblasts

Cardiac fibroblasts (CF) play a key role in orchestrating the structural remodeling of the myocardium in response to injury or stress, in part through direct regulation of extracellular matrix (ECM) turnover. The matrix metalloproteinases (MMPs) are a family of over 25 zinc-dependent proteases that together have the capacity to degrade all the protein components of the ECM. Fibroblasts are a major source of several MMPs in the heart, thereby representing a viable therapeutic target for regulating ECM turnover in cardiac pathologies characterized by adverse remodeling, such as myocardial infarction, cardiomyopathy, hypertension and heart failure. This review summarizes current knowledge on the identity and regulation of MMPs expressed by CF and discusses future directions for reducing adverse myocardial remodeling by modulating the expression and/or activity of CF-derived MMPs.     

MicroRNA-18 and microRNA-19 regulate CTGF and TSP-1 expression in age-related heart failure

To understand the process of cardiac aging, it is of crucial importance to gain insight into the age‐related changes in gene expression in the senescent failing heart. Age‐related cardiac remodeling is known to be accompanied by changes in extracellular matrix (ECM) gene and protein levels. Small noncoding microRNAs regulate gene expression in cardiac development and disease and have been implicated in the aging process and in the regulation of ECM proteins. However, their role in age‐related cardiac remodeling and heart failure is unknown. In this study, we investigated the aging‐associated microRNA cluster 17–92, which targets the ECM proteins connective tissue growth factor (CTGF) and thrombospondin‐1 (TSP‐1). We employed aged mice with a failure‐resistant (C57Bl6) and failure‐prone (C57Bl6 × 129Sv) genetic background and extrapolated our findings to human age‐associated heart failure. In aging‐associated heart failure, we linked an aging‐induced increase in the ECM proteins CTGF and TSP‐1 to a decreased expression of their targeting microRNAs 18a, 19a, and 19b, all members of the miR‐17–92 cluster. Failure‐resistant mice showed an opposite expression pattern for both the ECM proteins and the microRNAs. We showed that these expression changes are specific for cardiomyocytes and are absent in cardiac fibroblasts. In cardiomyocytes, modulation of miR‐18/19 changes the levels of ECM proteins CTGF and TSP‐1 and collagens type 1 and 3. Together, our data support a role for cardiomyocyte‐derived miR‐18/19 during cardiac aging, in the fine‐tuning of cardiac ECM protein levels. During aging, decreased miR‐18/19 and increased CTGF and TSP‐1 levels identify the failure‐prone heart.     

Interactions between the extracellular matrix and the cell surface determine tooth morphogenesis and the cellular differentiation of the dental mesenchyme

A series of reciprocal interactions between epithelial and mesenchymal tissues control the morphogenesis and cell differentiation in the developing tooth. The molecular mechanisms operating in these interactions are, however, unknown at present. Structural components of the extracellular matrix (ECM) affect cellular behavior in the embryo and appear to be involved also in these regulatory processes. The ECM molecules exert their effects on cells through binding to specific matrix receptors on the cell surface. This review article summarizes our findings on the distribution patterns during tooth development of the ECM glycoproteins, fibronectin and tenascin, and of the cell surface proteoglycan, syndecan, which functions as a receptor for interstitial matrix. Based on the observed changes in these distribution patterns and on experimental evidence, roles for these molecules in epithelial-mesenchymal interactions during tooth development are suggested. Fibronectin and tenascin are enriched in the dental basement membrane at the time of odontoblast differentiation. These matrix glycoproteins may be involved in the cell-matrix interaction which controls differentiation of the dental mesenchymal cells into odontoblasts. Tenascin and syndecan are accumulated in the dental mesenchyme during bud stage of development. We have shown in tissue recombination experiments that the presumptive dental epithelium induces the expression of tenascin and syndecan in mesenchyme. We suggest that these molecules are involved in cell-matrix interactions, which regulate mesenchymal cell condensation during the earliest stages of tooth morphogenesis.     

Matrix metalloproteinases and their function in myocardium

A significant number of myocardial diseases are accompanied by increased synthesis and degradation of the extracellular matrix (ECM) as well as by changed maturation and incorporation of ECM components. Important groups of enzymes responsible for both normal and pathological processes in ECM remodeling are matrix metaloproteinases (MMPs). These enzymes share a relatively conserved structure with a number of identifiable modules linked to their specific functions. The most important function of MMPs is the ability to cleave various ECM components; including such rigid molecules as fibrillar collagen molecules. The amount and activity of MMPs in cardiac tissue are regulated by a range of activating and inhibiting processes. Although MMPs play multifarious roles in many myocardial diseases, here we have focused on their function in ischemic cardiac tissue, dilated cardiomyopathy and hypertrophied cardiac tissue. The inhibition of MMPs by means of synthetic inhibitors seems to be a promising strategy in cardiac disease treatment. Their effects on diseased cardiac tissue have been successfully tested in several experimental studies.     

Epithelial-mesenchymal interactions in tooth morphogenesis: the roles of extracellular matrix, growth factors, and cell surface receptors.

Morphogenesis and cell differentiation in the developing tooth are controlled by a series of reciprocal interactions between the epithelial and mesenchymal tissues. The exact molecular mechanisms operating in these interactions are unknown at present, but both structural components of the extracellular matrix (ECM) and diffusible growth factors have been suggested to be involved. In this review article we summarize our findings on the distribution patterns of three ECM molecules and two cell surface receptors during tooth morphogenesis through bud, cap, and bell stages of development. The examined molecules include fibronectin, type III collagen, and tenascin, which all represent components of the mesenchymal ECM, the cell surface proteoglycan, syndecan, which functions as a receptor for interstitial matrix, and the cell surface receptor for epidermal growth factor. Based on the observed changes in distribution patterns and on experimental evidence, roles are suggested for these molecules in epithelial-mesenchymal interactions during tooth development. Fibronectin is suggested to be involved in the cell-matrix interaction that controls odontoblast differentiation. Epidermal growth factor and its receptors are suggested to be involved in a paracrine fashion in the epithelial-mesenchymal interactions regulating morphogenesis of bud- and cap-stage teeth. Tenascin and syndecan are accumulated in the dental mesenchyme during the bud stage of development, and it is suggested that they represent a couple of a cell surface receptor and its matrix ligand and that they are involved in mesenchymal cell condensation during the earliest stages of tooth morphogenesis.     

Ontogeny of the basal lamina in the sea urchin embryo

The patterns of expression for several extracellular matrix components during development of the sea urchin embryo are described. An immunofluorescence assay was employed on paraffin-sectioned material using (i) polyclonal antibodies against known vertebrate extracellular matrix components: laminin, fibronectin, heparan sulfate proteoglycan, collagen types I, III, and IV; and (ii) monoclonal antibodies generated against sea urchin embryonic components. Most extracellular matrix components studied were found localized within the unfertilized egg in granules (0.5-2.0 micron) distinct from the cortical granules. Fertilization initiated trafficking of the extracellular matrix (ECM) components from within the egg granules to the basal lamina of the developing embryo. The various ECM components arrived within the developing basal lamina at different times, and not all components were unique to the basal lamina. Two ECM components were not found within the egg. These molecules appeared de novo at the mesenchyme blastula stage, and remained specific to the mesoderm through development. The reactivity of antibodies to vertebrate ECM antigens with components of the sea urchin embryo suggests the presence of immunologically similar ECM molecules between the phyla.     

Long form of latent TGF-beta binding protein 1 (Ltbp1L) is essential for cardiac outflow tract septation and remodeling

Latent TGF-beta binding protein 1 (LTBP1) is a member of the LTBP/fibrillin family of extracellular proteins. Due to the usage of different promoters, LTBP1 exists in two major forms, long (L) and short (S), each expressed in a temporally and spatially unique fashion. Both LTBP1 molecules covalently interact with latent TGF-beta and regulate its function, presumably via interaction with the extracellular matrix (ECM). To explore the in vivo role of Ltbp1 in mouse development, at the time when only the L isoform is expressed, we mutated the Ltbp1L locus by gene targeting. Ltbp1L-null animals die shortly after birth from defects in heart development, consisting of the improper septation of the cardiac outflow tract (OFT) and remodeling of the associated vessels. These cardiac anomalies present as persistent truncus arteriosus (PTA) and interrupted aortic arch (IAA), which are associated with the faulty function of cardiac neural crest cells (CNCCs). The lack of Ltbp1L in the ECM of the septating OFT and associated vessels results in altered gene expression and function of CNCCs and decreased Tgf-beta activity in the OFT. This phenotype reveals a crucial role for Ltbp1L and matrix as extracellular regulators of Tgf-beta activity in heart organogenesis.     

The developmental roles of the extracellular matrix: beyond structure to regulation

Cells in multicellular organisms are surrounded by a complex three-dimensional macromolecular extracellular matrix (ECM). This matrix, traditionally thought to serve a structural function providing support and strength to cells within tissues, is increasingly being recognized as having pleiotropic effects in development and growth. Elucidation of the role that the ECM plays in developmental processes has been significantly advanced by studying the phenotypic and developmental consequences of specific genetic alterations of ECM components in the mouse. These studies have revealed the enormous contribution of the ECM to the regulation of key processes in morphogenesis and organogenesis, such as cell adhesion, proliferation, specification, migration, survival, and differentiation. The ECM interacts with signaling molecules and morphogens thereby modulating their activities. This review considers these advances in our understanding of the function of ECM proteins during development, extending beyond their structural capacity, to embrace their new roles in intercellula signaling.     

Role of FoxO3a as a negative regulator of the cardiac myofibroblast conversion induced by TGF-β1

Cardiac fibroblasts (CFs) are necessary to maintain extracellular matrix (ECM) homeostasis in the heart. Normally, CFs are quiescent and secrete small amounts of ECM components, whereas, in pathological conditions, they differentiate into more active cells called cardiac myofibroblasts (CMF). CMF conversion is characteristic of cardiac fibrotic diseases, such as heart failure and diabetic cardiomyopathy. TGF-β1 is a key protein involved in CMF conversion. SMADs are nuclear factor proteins activated by TGF-β1 that need other proteins, such as forkhead box type O (FoxO) family members, to promote CMF conversion. FoxO1, a member of this family protein, is necessary for TGF-β1-induced CMF conversion, whereas the role of FoxO3a, another FoxO family member, is unknown. FoxO3a plays an important role in many fibrotic processes in the kidney and lung. However, the participation of FoxO3a in the conversion of CFs into CMF is not clear. In this paper, we demonstrate that TGF-β1 decreases the activation and expression of FoxO3a in CFs. FoxO3a regulation by TGF-β1 requires activated SMAD3, ERK1/2 and Akt. Furthermore, we show that FoxO1 is crucial in the FoxO3a regulation induced by TGF-β1, as shown by overexpressed FoxO1 enhancing and silenced FoxO1 suppressing the effects of TGF-β1 on FoxO3a. Finally, the regulation of TGF-β1-induced CMF conversion was enhanced by FoxO3a silencing and suppressed by inhibited FoxO3a degradation. Considering these collective findings, we suggest that FoxO3a acts as a negative regulator of the CMF conversion that is induced by TGF-β1.     

Matrix metalloproteinase expression in cardiac myocytes following myocardial infarction in the rabbit

Myocardial infarction (MI), leads to cardiac remodeling, thinning of the ventricle wall, ventricular dilation, and heart failure, and is a leading cause of death. Interactions between the contractile elements of the cardiac myocytes and the extracellular matrix (ECM) help maintain myocyte alignment required for the structural and functional integrity of the heart. Following MI, reorganization of the ECM and the myocytes occurs, contributing to loss of heart function. In certain pathological circumstances, the ECM is modulated such that the structure of the tissue becomes damaged. The matrix metalloproteinases (MMPs) are a family of enzymes that degrade molecules of the ECM. The present experiments were performed to define the time-course, isozyme subtypes, and cellular source of increased MMP expression that occurs following MI in an experimental rabbit model. Heart tissue samples from infarcted and sham animals were analyzed over a time-course of 1-14 days. By zymography, it was demonstrated that, unlike the sham controls, MMP-9 expression was induced within 24 hours following MI. MMP-3 expression, also absent in sham controls, was induced 2 days after MI. MMP-2 expression was detected in both the sham and infarcted samples and was modestly up-regulated following MI. Tissue inhibitor of metalloproteinase-1 (TIMP-1) expression was evaluated and shown to be down-regulated following MI, inverse of MMP-9 and MMP-3 expression. Further, MMP-9 and MMP-3 expression was detected by immunohistochemistry in myocytes within the infarct. Additional studies were conducted in which cultured rat cardiac myocytes were exposed to a hypoxic environment (2% O2) for 24 hours and the media analyzed for MMP expression. MMP-9 and MMP-3 were induced following exposure to hypoxia. It is speculated that the net increase in proteolytic activity by myocytes is a contributing factor leading to myocyte misalignment and slippage. Additional studies with a MMP inhibitor would elucidate this hypothesis.     

Zebrafish Crip2 Plays a Critical Role in Atrioventricular Valve Development by Downregulating the Expression of ECM Genes in the Endocardial Cushion

The initial step of atrioventricular (AV) valve development involves the deposition of extracellular matrix (ECM) components of the endocardial cushion and the endocardialmesenchymal transition. While the appropriately regulated expression of the major ECM components, Versican and Hyaluronan, that form the endocardial cushion is important for heart valve development, the underlying mechanism that regulates ECM gene expression remains unclear. We found that zebrafish crip2 expression is restricted to a subset of cells in the AV canal (AVC) endocardium at 55 hours post-fertilization (hpf). Knockdown of crip2 induced a heart-looping defect in zebrafish embryos, although the development of cardiac chambers appeared to be normal. In the AVC of Crip2-deficient embryos, the expression of both versican a and hyaluronan synthase 2 (has2) was highly upregulated, but the expression of bone morphogenetic protein 4 (bmp4) and T-box 2b (tbx2b) in the myocardium and of notch1b in the endocardium in the AVC did not change. Taken together, these results indicate that crip2 plays an important role in AV valve development by downregulating the expression of ECM components in the endocardial cushion.     

Fibronectin–integrin mediated signaling in human cervical cancer cells (SiHa)

Interaction between cell surface integrin receptors and extracellular matrix (ECM) components plays an important role in cell survival, proliferation, and migration, including tumor development and invasion of tumor cells. Matrix metalloproteinases (MMPs) are a family of metalloproteinases capable of digesting ECM components and are important molecules for cell migration. Binding of ECM to integrins initiates cascades of cell signaling events modulating expression and activity of different MMPs. The aim of this study is to investigate fibronectin–integrin-mediated signaling and modulation of MMPs. Our findings indicated that culture of human cervical cancer cell (SiHa) on fibronectin-coated surface perhaps sends signals via fibronectin–integrin-mediated signaling pathways recruiting focal adhesion kinase (FAK) extracellular signal regulated kinase (ERK), phosphatidyl inositol 3 kinase (PI-3K), integrin-linked kinase (ILK), nuclear factor-kappa B (NF-κB), and modulates expression and activation of mainly pro-MMP-9, and moderately pro-MMP-2 in serum-free culture medium.     

Cardiac myofibroblasts differentiated in 3D culture exhibit distinct changes in collagen I production, processing, and matrix deposition

Myofibroblasts are a differentiated fibroblast cell type characterized by increased contractile capacity and elevated production of extracellular matrix (ECM) proteins. In the heart, myofibroblast expression is implicated in fibrosis associated with pressure-overload hypertrophy, among other pathologies. Although enhanced expression of ECM proteins by myofibroblasts is established, few studies have addressed the nature of the ECM deposited by myofibroblasts. To characterize ECM production and assembly by cardiac myofibroblasts, we developed a three-dimensional (3D) culture system using primary cardiac fibroblasts seeded into a nylon mesh that allows us to reversibly interconvert between myofibroblast and fibroblast phenotypes. We report that an increase in collagen I production by myofibroblasts was accompanied by a significant increase in collagen deposition into insoluble ECM. Furthermore, myofibroblasts exhibited increased levels of procollagen alpha1(I) with C-propeptide attached (and N-propeptide removed) relative to procollagen alpha1(I) compared with fibroblast cultures. An increase in production of the myofibroblast-associated splice variant of fibronectin (EDA-Fn) was seen in myofibroblast 3D cultures. Because the regulation of procollagen I processing is known to have profound effects on ECM assembly, differences in procollagen I secretion and maturation coupled with expression of EDA-Fn are shown to contribute to the production of a distinct ECM by the cardiac myofibroblast.     

Extracellular Matrix in Neural Plasticity and Regeneration

The extracellular matrix (ECM) is a fundamental component of biological tissues. The ECM in the central nervous system (CNS) is unique in both composition and function. Functions such as learning, memory, synaptogenesis, and plasticity are regulated by numerous ECM molecules. The neural ECM acts as a non-specific physical barrier that modulates neuronal plasticity and axon regeneration. There are two specialized types of ECM in the CNS, diffuse perisynaptic ECM and condensed ECM, which selectively surround the perikaryon and initial part of dendritic trees in subtypes of neurons, forming perineuronal nets. This review presents the current knowledge about the role of important neuronal ECM molecules in maintaining the basic functions of a neuron, including electrogenesis and the ability to form neural circuits. The review mainly focuses on the role of ECM components that participate in the control of key events such as cell survival, axonal growth, and synaptic remodeling. Particular attention is drawn to the numerous molecular partners of the main ECM components. These regulatory molecules are integrated into the cell membrane or disposed into the matrix itself in solid or soluble form. The interaction of the main matrix components with molecular partners seems essential in molecular mechanisms controlling neuronal functions. Special attention is paid to the chondroitin sulfate proteoglycan 4, type 1 transmembrane protein, neural-glial antigen 2 (NG2/CSPG4), whose cleaved extracellular domain is such a molecular partner that it not only acts directly on neural and vascular cells, but also exerts its influence indirectly by binding to resident ECM molecules.

     

Lymphocyte interactions with extracellular matrix

To mediate an immune response, lymphocytes must be able to interact with and respond to the surrounding extracellular environment. In addition to cell surface molecules that facilitate adhesion of lymphocytes to other cells, recent studies have demonstrated that lymphocytes interact with glycoproteins and glycosaminoglycans that are major components of the extracellular matrix (ECM). Although many receptors mediating the effects of ECM components on lymphocyte function remain poorly defined, a number of lymphocyte ECM receptors have recently been identified; these include members of the integrin family of adhesion molecules as well as structurally unrelated molecules such as CD44 and CD26. Furthermore, as lymphocytes must be able to move between various microenvironments in vivo, they have proved to be an excellent cell type in which to identify and analyze various modes of regulation of cell-ECM interactions. As with other cell types, the ECM has been shown to have multiple effects on lymphocytes; functional analysis reveals effects of the ECM on lymphocyte migration, recognition/activation, and differentiation. These studies emphasize: 1) the importance of lymphocytes as a model system for identifying and analyzing ECM receptor expression and function, and 2) the multiple roles that the ECM plays in the function of the immune system in vivo.