Matrix Biology

Extracellular matrix-derived products modulate endothelial and progenitor cell migration and proliferation in vitro and stimulate regenerative healing in vivo.

Publication Date: 2010 Aug 23 PMID: 20797438
Authors: Vorotnikova, E. - McIntosh, D. - Dewilde, A. - Zhang, J. - Reing, J. E. - Zhang, L. - Cordero, K. - Bedelbaeva, K. - Gourevitch, D. - Heber-Katz, E. - Badylak, S. F. - Braunhut, S. J.
Journal: Matrix Biol

Most adult mammals heal without restorative replacement of lost tissue and instead form scar tissue at an injury site. One exception is the adult MRL/MpJ mouse that can regenerate ear and cardiac tissue after wounding with little evidence of scar tissue formation. Following production of a MRL mouse ear hole, 2mm in diameter, a structure rapidly forms at the injury site that resembles the amphibian blastema at a limb amputation site during limb regeneration. We have isolated MRL blastemal cells (MRL-B) from this structure and adapted them to culture. We demonstrate by RT-PCR that even after continuous culturing of these cells they maintain expression of several progenitor cell markers, including DLK (Pref-1), and Msx-1. We have isolated the underlying extracellular matrix (ECM) produced by these MRL-B cells using a new non-proteolytic method and studied the biological activities of this cell-free ECM. Multiplex microELISA analysis of MRL-B cell-free ECM vs. cells revealed selective enrichment of growth factors such as bFGF, HGF and KGF in the matrix compartment. The cell-free ECM, degraded by mild enzyme treatment, was active in promoting migration and proliferation of progenitor cells in vitro and accelerating wound closure in a mouse full thickness cutaneous wound assay in vivo. In vivo, a single application of MRL-B cell matrix-derived products to full thickness cutaneous wounds in non-regenerative mice, B6, induced re-growth of pigmented hair, dermis and epidermis at the wound site whereas scar tissue replaced these tissues at wound sites in mice treated with vehicle alone. These studies suggest that matrix-derived products can stimulate regenerative healing and avert scar tissue formation in adult mammals.

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Synthesis of embryonic tendon-like tissue by human marrow stromal/mesenchymal stem cells requires a three-dimensional environment and transforming growth factor ss3.

Publication Date: 2010 Aug 21 PMID: 20736064
Authors: Kapacee, Z. - Yeung, C. Y. - Lu, Y. - Crabtree, D. - Holmes, D. F. - Kadler, K. E.
Journal: Matrix Biol

Tendon-like tissue generated from stem cells in vitro has the potential to replace tendons and ligaments lost through injury and disease. However, thus far, no information has been available on the mechanism of tendon formation in vitro and how to accelerate the process. We show here that human mesenchymal stem cells (MSCs) and bone marrow-derived mononuclear cells (BM-MNCs) can generate tendon-like tissue in 7days mediated by transforming growth factor (TGF) beta3. MSCs cultured in fixed-length fibrin gels spontaneously synthesized narrow-diameter collagen fibrils and exhibited fibripositors (actin-rich, collagen fibril-containing plasma membrane protrusions) identical to those that occur in embryonic tendon. In contrast, BM-MNCs did not synthesize tendon-like tissue under these conditions. We performed real-time PCR analysis of MSCs and BM-MNCs. MSCs upregulated genes encoding type I collagen, TGFbeta3, and Smad2 at the time of maximum contraction of the tendon-like tissue (7days). Western blot analysis showed phosphorylation of Smad2 at maximum contraction. The TGFbeta inhibitor SB-431542, blocked the phosphorylation of Smad2 and stopped the formation of tendon-like tissue. Quantitative PCR showed that BM-MNCs expressed very low levels of TGFbeta3 compared to MSCs. Therefore we added exogenous TGFbeta3 protein to BM-MNCs in fibrin gels, which resulted in phosphorylation of Smad2, synthesis of collagen fibrils, the appearance of fibripositors at the plasma membrane, and the formation of tendon-like tissue. In conclusion, MSCs that self-generate TGFbeta signaling or the addition of TGFbeta3 protein to BM-MNCs in fixed-length fibrin gels spontaneously make embryonic tendon-like tissue in vitro within 7days.

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An experimental model for studying the biomechanics of embryonic tendon: Evidence that the development of mechanical properties depends on the actinomyosin machinery.

Publication Date: 2010 Aug 21 PMID: 20736063
Authors: Kalson, N. S. - Holmes, D. F. - Kapacee, Z. - Otermin, I. - Liu, Y. - Ennos, R. A. - Kadler, K. E.
Journal: Matrix Biol

Tendons attach muscles to bone and thereby transmit tensile forces during joint movement. However, a detailed understanding of the mechanisms that establish the mechanical properties of tendon has remained elusive because of the practical difficulties of studying tissue mechanics in vivo. Here we have performed a study of tendon-like constructs made by culturing embryonic tendon cells in fixed-length fibrin gels. The constructs display mechanical properties (toe-linear-fail stress-strain curve, stiffness, ultimate tensile strength, and failure strain) as well as collagen fibril volume fraction and extracellular matrix (ECM)/cell ratio that are statistically similar to those of embryonic chick metatarsal tendons. The development of mechanical properties during time in culture was abolished when the constructs were treated separately with Triton X-100 (to solubilise membranes), cytochalasin (to disassemble the actin cytoskeleton) and blebbistatin (a small molecule inhibitor of non-muscle myosin II). Importantly, these treatments had no effect on the mechanical properties of the constructs that existed prior to treatment. Live cell imaging and 14C-proline metabolic labeling showed that blebbistatin inhibited the contraction of the constructs without affecting cell viability, procollagen synthesis, or conversion of procollagen to collagen. In conclusion, the mechanical properties per se of the tendon constructs are attributable to the ECM generated by the cells but the improvement of mechanical properties during time in culture was dependent on non-muscle myosin II-derived forces.

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Nanofibers in a hyaluronan-based pericellular matrix.

Publication Date: 2010 Aug 20 PMID: 20732419
Authors: Gerlach, D. - Kaminski, T. - Perez-Willard, F. - Kirfel, G. - Gieselmann, V. - Kappler, J.
Journal: Matrix Biol

The extracellular matrix of the brain is a highly organized hyaluronan-based supramolecular assembly that is involved in neuronal pathfinding, cell migration, synaptogenesis and neuronal plasticity. Here, we analyze the structure of the hyaluronan-rich pericellular matrix of an oligodendroglial precursor cell line using helium ion beam scanning microscopy at a subnanometer resolution. We find that thin nanofibers are the ultimate building elements of this oligodendroglial pericellular matrix. These structures may participate in the regulation of oligodendroglial maturation and motility.

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Interaction between the extracellular matrix and lymphatics: Consequences for lymphangiogenesis and lymphatic function.

Publication Date: 2010 Aug 18 PMID: 20727409
Authors: Wiig, H. - Keskin, D. - Kalluri, R.
Journal: Matrix Biol

The lymphatic system is important for body fluid balance as well as immunological surveillance. Due to the identification of new molecular markers during the last decade, there has been a recent dramatic increase in our knowledge on the molecular mechanisms involved in lymphatic vessel growth (lymphangiogenesis) and lymphatic function. Here we review data showing that although it is often overlooked, the extracellular matrix plays an important role in the generation of new lymphatic vessels as a response to physiological and pathological stimuli. Extracellular matrix-lymphatic interactions as well as biophysical characteristics of the stroma have consequences for tumor formation, growth and metastasis. During the recent years, anti-lymphangiogenesis has emerged as an additional therapeutic modality to the clinically applied anti-angiogenesis strategy. Oppositely, enhancement of lymphangiogenesis in situations of lymph accumulation is seen as a promising strategy to a set of conditions where few therapeutic avenues are available. Knowledge on the interaction between the extracellular matrix and the lymphatics may enhance our understanding of the underlying mechanisms and may ultimately lead to better therapies for conditions where reduced or increased lymphatic function is the therapeutic target.

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Expression in SPARC-null mice of collagen type I lacking the globular domain of the alpha1(I) N-propeptide results in abdominal hernias and loss of dermal collagen.

Publication Date: 2010 Aug 11 PMID: 20708079
Authors: Card, L. - Henderson, N. - Zhang, Y. - Bornstein, P. - Bradshaw, A. D.
Journal: Matrix Biol

The sequence encoding the N-propeptide of collagen I is characterized by significant conservation of amino acids across species; however, the function of the N-propeptide remains poorly defined. Studies in vitro have suggested that one activity of this propeptide might be to act as a feedback inhibitor of collagen I synthesis. To determine whether the N-propeptide contributed to decreased collagen content in SPARC-null mice, mice carrying a deletion of exon 2, which encodes the globular domain of the N-propeptide of collagen I, were crossed to SPARC-null animals. Mice lacking SPARC and expressing collagen I without the globular domain of the N-propeptide were viable and fertile. However, a significant number of animals developed abdominal hernias within the first 2months of life with an approximate 20% penetrance (~35% of males). The dermis of SPARC-null/exon 2-deleted mice was thinner and contained fewer large collagen fibers in comparison with wild-type or in either single transgenic animal. The average collagen fibril diameter of exon 2-deleted mice did not significantly differ from wild-type mice (WT: 87.9nm versus exon 2-deleted: 88.2nm), whereas SPARC-null/exon 2-deleted fibrils were smaller than that of SPARC-null dermis (SPARC-null: 60.2nm, SPARC-null/exon 2-deleted: 40.8nm). As measured by hydroxyproline analysis, double transgenic skin biopsies contained significantly less collagen than those of wild-type, those of exon 2-deleted, and those of SPARC-null biopsies. Acetic acid extraction of collagen from skin biopsies revealed an increase in the proportion of soluble collagen in the SPARC-null/exon 2-deleted mice. These results support a function of the N-propeptide of collagen I in facilitating incorporation and stabilization of collagen I into the insoluble ECM and argue against a primary function of the N-propeptide as a negative regulator of collagen synthesis.

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Cryptic domains of tenascin-C differentially control fibronectin fibrillogenesis.

Publication Date: 2010 Aug 10 PMID: 20708078
Authors: To, W. S. - Midwood, K. S.
Journal: Matrix Biol

The three-dimensional organization of the ubiquitous extracellular matrix glycoprotein fibronectin regulates cell fate and morphogenesis during development; in particular tubule formation that constitutes the vasculature, lung and kidney. Tenascin-C is a matrix protein with a restricted expression pattern; it is specifically up-regulated at sites of fibronectin fibril assembly during development and in remodeling adult tissues. Here we demonstrate that specific domains of tenascin-C inhibit fibronectin matrix assembly whereas full-length tenascin-C does not. These domains act via distinct mechanisms: TNfn1-8 blocks fibrillogenesis by binding to fibronectin fibrils and preventing intermolecular fibronectin interactions whilst FBG acts independently of binding to fibronectin and instead is internalized and causes cytoskeletal re-organization. We also show that TNfn1-8 disrupts epithelial cell tubulogenesis. Our data demonstrate that tenascin-C contains cryptic sites which can control tissue levels of fibrillar fibronectin either by preventing de novo fibril assembly or reducing levels of deposited fibronectin. Exposure of these domains during tissue remodeling may provide a novel means of controlling fibronectin assembly.

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Nano-structure of the laminin gamma-1 short arm reveals an extended and curved multidomain assembly.

Publication Date: 2010 Aug 3 PMID: 20688161
Authors: Patel, T. R. - Morris, G. A. - Zwolanek, D. - Keene, D. R. - Li, J. - Harding, S. E. - Koch, M. - Stetefeld, J.
Journal: Matrix Biol

Laminins are multidomain glycoproteins that play important roles in development and maintenance of the extracellular matrix via their numerous interactions with other proteins. Several receptors for the laminin short arms revealed their importance in network formation and intercellular signaling. However, both the detailed structure of the laminin gamma-1 short arm and its organization within the complexes is poorly understood due to the complexity of the molecule and the lack of a high-resolution structure. The presented data provide the first subatomic resolution structure for the laminin gamma-1 short arm in solution. This was achieved using an integrated approach that combined a number of complementary biophysical techniques such as small angle X-ray scattering (SAXS), analytical ultracentrifugation, dynamic light scattering and electron microscopy. As a result of this study, we have obtained a significantly improved model for the laminin gamma-1 short arm that represents a major step forward in molecular understanding of laminin-mediated complex formations.

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Zebrafish type XVII collagen: Gene structures, expression profiles, and morpholino "knock-down" phenotypes.

Publication Date: 2010 Aug 4 PMID: 20670682
Authors: Kim, S. H. - Choi, H. Y. - So, J. H. - Kim, C. H. - Ho, S. Y. - Frank, M. - Li, Q. - Uitto, J.
Journal: Matrix Biol

The human COL17A1 gene encodes type XVII collagen (also known as the 180-kDa bullous pemphigoid antigen), an integral component of hemidesmosomes, attachment complexes providing integrity to the dermal-epidermal junction. Zebrafish, a useful model system to study skin development, displays fully developed hemidesmosomes at approximately 5days post-fertilization (dpf). We have identified two COL17A1 orthologues in the zebrafish genome, col17a1a and col17a1b, which are expressed in the skin and the neural system, respectively. The proteins coded by these genes have structural module organizations homologous to the human type XVII collagen. "Knock-down" of the expression of col17a1a with a specific morpholino targeting the 5' UTR of the gene resulted in a blistering phenotype and in perturbations in the basement membrane zone. "Knock-down" of col17a1b expression resulted in ablation or in marked reduction of neuromasts in the lateral line. Thus, zebrafish has two COL17A1 orthologues which may have evolved tissue-specific functions during vertebrate development. Collectively, zebrafish provides a model system to study the molecular aspects of skin development and offers insights into the corresponding human diseases.

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