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Matrix Biology
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1. Microvesicles shed by oligodendroglioma cells and rheumatoid synovial fibroblasts contain aggrecanase activity
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 4

Alessandra Lo Cicero, Iwona Majkowska, Hideaki Nagase, Italia Di Liegro, Linda Troeberg

Membrane microvesicle shedding is an active process and occurs in viable cells with no signs of apoptosis or necrosis. We report here that microvesicles shed by oligodendroglioma cells contain an ‘aggrecanase’ activity, cleaving aggrecan at sites previously identified as targets for adamalysin metalloproteinases with disintegrin and thrombospondin domains (ADAMTSs). Degradation was inhibited by EDTA, the metalloproteinase inhibitor GM6001 and by tissue inhibitor of metalloproteinases (TIMP)-3, but not by TIMP-1 or TIMP-2. This inhibitor profile indicates that the shed microvesicles contain aggrecanolytic ADAMTS(s) or related TIMP-3-sensitive metalloproteinase(s). The oligodendroglioma cells were shown to express the three most active aggrecanases, namely Adamts1, Adamts4 and Adamts5, suggesting that one or more of these enzymes may be responsible for the microvesicle activity. Microvesicles shed by rheumatoid synovial fibroblasts similarly degraded aggrecan in a TIMP-3-sensitive manner. Our findings raise the novel possibility that microvesicles may assist oligodendroglioma and rheumatoid synovial fibroblasts to invade through aggrecan-rich extracellular matrices.




2. Perlecan modulates VEGF signaling and is essential for vascularization in endochondral bone formation
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 4

Muneaki Ishijima, Nobuharu Suzuki, Kentaro Hozumi, Tomoya Matsunobu, Keisuke Kosaki, Haruka Kaneko, John R. Hassell, Eri Arikawa-Hirasawa, Yoshihiko Yamada

Perlecan (Hspg2) is a heparan sulfate proteoglycan expressed in basement membranes and cartilage. Perlecan deficiency (Hspg2-/-) in mice and humans causes lethal chondrodysplasia, which indicates that perlecan is essential for cartilage development. However, the function of perlecan in endochondral ossification is not clear. Here, we report the critical role of perlecan in VEGF signaling and angiogenesis in growth plate formation. The Hspg2-/- growth plate was significantly wider but shorter due to severely impaired endochondral bone formation. Hypertrophic chondrocytes were differentiated in Hspg2-/- growth plates; however, removal of the hypertrophic matrix and calcified cartilage was inhibited. Although the expression of MMP-13, CTGF, and VEGFA was significantly upregulated in Hspg2-/- growth plates, vascular invasion into the hypertrophic zone was impaired, which resulted in an almost complete lack of bone marrow and trabecular bone. We demonstrated that cartilage perlecan promoted activation of VEGF/VEGFR by binding to the VEGFR of endothelial cells. Expression of the perlecan transgene specific to the cartilage of Hspg2-/- mice rescued their perinatal lethality and growth plate abnormalities, and vascularization into the growth plate was restored, indicating that perlecan in the growth plate, not in endothelial cells, is critical in this process. These results suggest that perlecan in cartilage is required for activating VEGFR signaling of endothelial cells for vascular invasion and for osteoblast migration into the growth plate. Thus, perlecan in cartilage plays a critical role in endochondral bone formation by promoting angiogenesis essential for cartilage matrix remodeling and subsequent endochondral bone formation.




3. Connective tissue mineralization in Abcc6-/- mice, a model for pseudoxanthoma elasticum
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 4

N. Beril Kavukcuoglu, Qiaoli Li, Nancy Pleshko, Jouni Uitto

Pseudoxanthoma elasticum (PXE) is a heritable multisystem disorder characterized by ectopic mineralization. However, the structure of the mineral deposits, their interactions with the connective tissue matrix, and the details of the progressive maturation of the mineral crystals are currently unknown. In this study, we examined the mineralization processes in Abcc6 -/- mice, a model system for PXE, by energy dispersive X-ray and Fourier transform infrared imaging spectroscopy (FT-IRIS). The results indicated that the principal components of the mineral deposits were calcium and phosphate which co-localized within the histologically demonstrable lesions determined by topographic mapping. The Ca/P ratio increased in samples with progressive mineralization reaching the value comparable to that in endochondral bone. A progressive increase in mineralization was also reflected by increased mineral-to-matrix ratio determined by FT-IRIS. Determination of the mineral phases by FT-IRIS suggested progressive maturation of the mineral deposits from amorphous calcium phosphate to hydroxyapatite. These results provide critical information of the mechanisms of mineralization in PXE, with potential pharmacologic implications.




4. Sulfatases are determinants of alveolar formation
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 4

Emilio Arteaga-Solis, Carmine Settembre, Andrea Ballabio, Gerard Karsenty

Alveolar formation or alveolarization is orchestrated by a finely regulated and complex interaction between growth factors and extracellular matrix proteins. The lung parenchyma contains various extracellular matrix proteins including proteoglycans, which are composed of glycosaminoglycans (GAGs) linked to a protein core. Although GAGs are known to regulate growth factor distribution and activity according to their degree of sulfation the role of sulfated GAG in the respiratory system is not well understood. The degree of sulfation of GAGs is regulated in part, by sulfatases that remove sulfate groups. In vertebrates, the enzyme Sulfatase-Modifying Factor 1 (Sumf1) activates all sulfatases. Here we utilized mice lacking Sumf1 -/- to study the importance of proteoglycan desulfation in lung development. The Sumf1 -/- mice have normal lungs up until the onset of alveolarization at post-natal day 5 (P5). We detected increased deposition of sulfated GAG throughout the lung parenchyma and a decrease in alveolar septa formation. Moreover, stereological analysis showed that the alveolar volume is 20% larger in Sumf1 -/- as compared to wild type (WT) mice at P10 and P30. Additionally, pulmonary function test was consistent with increased alveolar volume. Genetic experiments demonstrate that in Sumf1 -/- mice arrest of alveolarization is independent of fibroblast growth factor signaling. In turn, the Sumf1 -/- mice have increased transforming growth factor ß (TGFß) signaling and in vivo injection of TGFß neutralizing antibody leads to normalization of alveolarization. Thus, absence of sulfatase activity increases sulfated GAG deposition in the lungs causing deregulation of TGFß signaling and arrest of alveolarization.




5. Time course involvement of matrix metalloproteinases in the vascular alterations of renovascular hypertension
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 4

Carla S. Ceron, Elen Rizzi, Danielle A. Guimaraes, Alisson Martins-Oliveira, Stefany B. Cau, Junia Ramos, Raquel F. Gerlach, Jose E. Tanus-Santos

Increased vascular matrix metalloproteinases (MMPs) levels play a role in late phases of hypertensive vascular remodeling. However, no previous study has examined the time course of MMPs in the various phases of two-kidney, one-clip hypertension (2K1C). We examined structural vascular changes, collagen and elastin content, vascular oxidative stress, and MMPs levels/activities during the development of 2K1C hypertension. Plasma angiotensin converting enzyme (ACE) activity was measured to assess renin-angiotensin system activation. Sham or 2K1C hypertensive rats were studied after 2, 4, 6, and 10weeks of hypertension. Systolic blood pressure (SBP) was monitored weekly. Morphometry of structural changes in the aortic wall was studied in hematoxylin/eosin, orcein and picrosirius red sections. Aortic NADPH activity and superoxide production was evaluated. Aortic gelatinolytic activity was determined by in situ zymography, and MMP-2, MMP-14, and tissue inhibitor of MMPs (TIMP)-2 levels were determined by gelatin zymography, immunofluorescence and immunohistochemistry. 2K1C hypertension was associated with increased ACE activity, which decreased to normal after 10weeks. We found increased aortic collagen and elastin content in the early phase of hypertension, which were associated with vascular hypertrophy, increased vascular MMP-2 and MMP-14 (but not TIMP-2) levels, and increased gelatinolytic activity, possibly as a result of increased vascular NADPH oxidase activity and oxidative stress. These results indicate that vascular remodeling of renovascular hypertension is an early process associated with early increases in MMPs activities, enhanced matrix deposition and oxidative stress. Using antioxidants or MMPs inhibitors in the early phase of hypertension may prevent the vascular alterations of hypertension.




6. CyrA, a matricellular protein that modulates cell motility in Dictyostelium discoideum
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 4

Robert J. Huber, Andres Suarez, Danton H. O'Day

CyrA, an extracellular matrix (slime sheath), calmodulin (CaM)-binding protein in Dictyostelium discoideum, possesses four tandem EGF-like repeats in its C-terminus and is proteolytically cleaved during asexual development. A previous study reported the expression and localization of CyrA cleavage products CyrA-C45 and CyrA-C40. In this study, an N-terminal antibody was produced that detected the full-length 63kDa protein (CyrA-C63). Western blot analyses showed that the intracellular expression of CyrA-C63 peaked between 12 and 16h of development, consistent with the time that cells are developing into a motile, multicellular slug. CyrA immunolocalization and CyrA-GFP showed that the protein localized to the endoplasmic reticulum, particularly its perinuclear component. CyrA-C63 secretion began shortly after the onset of starvation peaking between 8 and 16h of development. A pharmacological analysis showed that CyrA-C63 secretion was dependent on intracellular Ca2+ release and active CaM, PI3K, and PLA2. CyrA-C63 bound to CaM both intra- and extracellularly and both proteins were detected in the slime sheath deposited by migrating slugs. In keeping with its purported function, CyrA-GFP over-expression enhanced cAMP-mediated chemotaxis and CyrA-C45 was detected in vinculin B (VinB)-GFP immunoprecipitates, thus providing a link between the increase in chemotaxis and a specific cytoskeletal component. Finally, DdEGFL1-FITC was detected on the membranes of cells capped with concanavalin A suggesting that a receptor exists for this peptide sequence. Together with previous studies, the data presented here suggests that CyrA is a bona fide matricellular protein in D. discoideum.




7. ASMB Announcement
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 4








8. ISMB Announcement
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 4








9. Tumor suppression by collagen XV is independent of the restin domain
Publication year: 2012
Source:Matrix Biology

Michael J. Mutolo, Kirsten J. Morris, Shih-Hsing Leir, Thomas C. Caffrey, Marzena A. Lewandowska, Michael A. Hollingsworth, Ann Harris

Non-fibrillar collagen XV is a chondroitin sulfate modified glycoprotein that is associated with the basement membrane zone in many tissues. Its precise functions remain to be fully elucidated though it clearly plays a critical role in the structural integrity of the extracellular matrix. Loss of collagen XV from the basement membrane zone precedes invasion of a number of tumor types and we previously showed that collagen XV functions as a dose-dependent suppressor of tumorigenicity in cervical carcinoma cells. The carboxyl terminus of another non-fibrillar collagen (XVIII) is cleaved to produce endostatin, which has anti-angiogenic effects and thus may act as a tumor suppressor in vivo. Since collagen XV has structural similarity with collagen XVIII, its C-terminal restin domain could confer tumor suppressive functions on the molecule, though our previous data did not support this. We now show that expression of collagen XV enhances the adhesion of cervical carcinoma cells to collagen I in vitro as does the N-terminus and collagenous regions of collagen XV, but not the restin domain. Destruction of a cysteine residue in the collagenous region that is critical for intermolecular interactions of collagen XV abolished the enhanced adhesion to collagen I. Finally, we demonstrate that unlike full length collagen XV, expression of the restin domain alone does not suppress tumorigenicity of cervical carcinoma cells in vivo; hence, this process is dependent on functions and interactions of other parts of the protein.




10. Bone matrix osteonectin limits prostate cancer cell growth and survival
Publication year: 2012
Source:Matrix Biology

Kristina Kapinas, Katie M. Lowther, Catherine B. Kessler, Karissa Tilbury, Jay R. Lieberman, Jennifer S. Tirnauer, Paul Campagnola, Anne M. Delany

There is considerable interest in understanding prostate cancer metastasis to bone and the interaction of these cells with the bone microenvironment. Osteonectin/SPARC/BM-40 is a collagen binding matricellular protein that is enriched in bone. Its expression is increased in prostate cancer metastases, and it stimulates the migration of prostate carcinoma cells. However, the presence of osteonectin in cancer cells and the stroma may limit prostate tumor development and progression. To determine how bone matrix osteonectin affects the behavior of prostate cancer cells, we modeled prostate cancer cell-bone interactions using the human prostate cancer cell line PC-3, and mineralized matrices synthesized by wild type and osteonectin-null osteoblasts in vitro. We developed this in vitro system because the structural complexity of collagen matrices in vivo is not mimicked by reconstituted collagen scaffolds or by more complex substrates, like basement membrane extracts. Second harmonic generation imaging demonstrated that the wild type matrices had thick collagen fibers organized into longitudinal bundles, whereas osteonectin-null matrices had thinner fibers in random networks. Importantly, a mouse model of prostate cancer metastases to bone showed a collagen fiber phenotype similar to the wild type matrix synthesized in vitro. When PC-3 cells were grown on the wild type matrices, they displayed decreased cell proliferation, increased cell spreading, and decreased resistance to radiation-induced cell death, compared to cells grown on osteonectin-null matrix. Our data support the idea that osteonectin can suppress prostate cancer pathogenesis, expanding this concept to the microenvironment of skeletal metastases.




11. Matrilin-3 switches from anti- to pro-anabolic upon integration to the extracellular matrix
Publication year: 2012
Source:Matrix Biology

Jean-Baptiste Vincourt, Stéphanie Etienne, Laurent Grossin, Justine Cottet, Claudie Bantsimba-Malanda, Patrick Netter, Didier Mainard, Virginie Libante, Pierre Gillet, Jacques Magdalou

The extracellular matrix (ECM) has long been viewed primarily as an organized network of solid-phase ligands for integrin receptors. During degenerative processes, such as osteoarthritis, the ECM undergoes deterioration, resulting in its remodeling and in the release of some of its components. Matrilin-3 (MATN3) is an almost cartilage specific, pericellular protein acting in the assembly of the ECM of chondrocytes. In the past, MATN3 was found required for cartilage homeostasis, but also involved in osteoarthritis-related pro-catabolic functions. Here, to better understand the pathological and physiological functions of MATN3, its concentration as a circulating protein in articular fluids of human osteoarthritic patients was determined and its functions as a recombinant protein produced in human cells were investigated with particular emphasis on the physical state under which it is presented to chondrocytes. MATN3 down-regulated cartilage extracellular matrix (ECM) synthesis and up-regulated catabolism when administered as a soluble protein. When artificially immobilized, however, MATN3 induced chondrocyte adhesion via a a5ß1 integrin-dependent mechanism, AKT activation and favored survival and ECM synthesis. Furthermore, MATN3 bound directly to isolated a5ß1 integrin in vitro. TGFß1 stimulation of chondrocytes allowed integration of exogenous MATN3 into their ECM and ECM-integrated MATN3 induced AKT phosphorylation and improved ECM synthesis and accumulation. In conclusion, the integration of MATN3 to the pericellular matrix of chondrocytes critically determines the direction toward which MATN3 regulates cartilage metabolism. These data explain how MATN3 plays either beneficial or detrimental functions in cartilage and highlight the important role played by the physical state of ECM molecules.




12. Glycosaminoglycan backbone is not required for the modulation of hemostasis: Effect of different heparin derivatives and non-glycosaminoglycan analogs
Publication year: 2012
Source:Matrix Biology

Rodrigo I. Bouças, Thais R. Jarrouge-Bouças, Marcelo A. Lima, Edvaldo S. Trindade, Fabio A. Moraes, Renan P. Cavalheiro, Ivarne L.S. Tersariol, Debra Hoppenstead, Jawed Fareed, Helena B. Nader

Heparin and its derivatives are known to regulate a variety of pathophysiological events related to vascular biology. In the present manuscript we examine a variety of heparinomimetics biochemically (electrophoretic behavior and enzymatic degradation) and pharmacologically (in vitro anticoagulant activity and in vivo hemorrhagic and antithrombotic tests) as well as their interactions with cells from the vessel wall using a time resolved fluorometric method and confocal microscopy. Data were determined for unfractionated heparin (UFH), enoxaparin, synthetic heparin pentasaccharide, C3 heparin derived oligosaccharides and phosphosulfomannan (PI-88). While being structurally distinct from UFH, all compounds exhibited anticoagulant, antithrombotic and hemorrhagic activities. In addition, besides the pentasaccharide, they all stimulated the synthesis of an antithrombotic heparan sulfate present at the cell surface and secreted by endothelial cells. Also, like UFH, they interacted with both endothelial and smooth muscle cells and dislodged UFH from its binding sites in a dose dependent manner but, with distinct saturable curves showing that the binding of polymeric structures to extracellular matrix (ECM) proteins does not depend on a glycosaminoglycan backbone. The data also suggest a common pathway, which does not depend on the presence of the conventionally accepted antithrombin binding pentasaccharide, for ECM dependent activity of the heparinomimetic stimulated synthesis of antithrombotic heparan sulfate. Notably, although of similar molecular weight as well as polymeric backbone, the synthetic heparin pentasaccharide showed significant hemorrhagic action and negligible antithrombotic activity in a venous thrombosis model, contrasting with C3, that displayed negligible hemorrhagic effect and potent antithrombotic action. These results provide evidence that structurally unrelated polymers can elicit similar hemostatic activities and show that polymeric sequence is not always crucial for certain activities. The results also suggest that non-GAG structures may provide an alternative route for the pharmaceutical control of hemostasis.




13. From the editor's desk
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 3








14. Thrombospondins in physiology and disease: New tricks for old dogs
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 3

Joanne E. Murphy-Ullrich, Renato V. Iozzo






15. Adhesion-modulating/matricellular ECM protein families: A structural, functional and evolutionary appraisal
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 3

Deane F. Mosher, Josephine C. Adams

The thrombospondins are a family of secreted, oligomeric glycoproteins that interact with cell surfaces, multiple components of the extracellular matrix, growth factors and proteases. These interactions underlie complex roles in cell interactions and tissue homeostasis in animals. Thrombospondins have been grouped functionally with SPARCs, tenascins and CCN proteins as adhesion-modulating or matricellular components of the extracellular milieu. Although all these multi-domain proteins share various commonalities of domains, the grouping is not based on structural homologies. Instead, the terms emphasise the general observations that these proteins do not form large-scale ECM structures, yet act at cell surfaces and function in coordination with the structural ECM and associated extracellular proteins. The designation of adhesion-modulation thus depends on observed tissue and cell culture ECM distributions and on experimentally identified functional properties. To date, the evolutionary relationships of these proteins have not been critically compared: yet, knowledge of their evolutionary histories is clearly relevant to any consideration of functional similarities. In this article, we survey briefly the structural and functional knowledge of these protein families, consider the evolution of each family, and outline a perspective on their functional roles.




16. The matricellular protein thrombospondin-1 globally regulates cardiovascular function and responses to stress via CD47
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 3

David D. Roberts, Thomas W. Miller, Natasha M. Rogers, Mingyi Yao, Jeffrey S. Isenberg

Matricellular proteins play diverse roles in modulating cell behavior by engaging specific cell surface receptors and interacting with extracellular matrix proteins, secreted enzymes, and growth factors. Studies of such interactions involving thrombospondin-1 have revealed several physiological functions and roles in the pathogenesis of injury responses and cancer, but the relatively mild phenotypes of mice lacking thrombospondin-1 suggested that thrombospondin-1 would not be a central player that could be exploited therapeutically. Recent research focusing on signaling through its receptor CD47, however, has uncovered more critical roles for thrombospondin-1 in acute regulation of cardiovascular dynamics, hemostasis, immunity, and mitochondrial homeostasis. Several of these functions are mediated by potent and redundant inhibition of the canonical nitric oxide pathway. Conversely, elevated tissue thrombospondin-1 levels in major chronic diseases of aging may account for the deficient nitric oxide signaling that characterizes these diseases, and experimental therapeutics targeting CD47 show promise for treating such chronic diseases as well as acute stress conditions that are associated with elevated thrombospondin-1 expression.




17. Thrombospondins as key regulators of synaptogenesis in the central nervous system
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 3

W. Christopher Risher, Cagla Eroglu

Thrombospondins (TSPs) are a family of large, oligomeric multidomain glycoproteins that participate in a variety of biological functions as part of the extracellular matrix (ECM). Through their associations with a number of binding partners, TSPs mediate complex cell–cell and cell–matrix interactions in such diverse processes as angiogenesis, inflammation, osteogenesis, cell proliferation, and apoptosis. It was recently shown in the developing central nervous system (CNS) that TSPs promote the formation of new synapses, which are the unique cell–cell adhesions between neurons in the brain. This increase in synaptogenesis is mediated by the interaction between astrocyte-secreted TSPs and their neuronal receptor, calcium channel subunit a2d-1. The cellular and molecular mechanisms that underlie induction of synaptogenesis via this interaction are yet to be fully elucidated. This review will focus on what is known about TSP and synapse formation during development, possible roles for TSP following brain injury, and what the previously established actions of TSP in other biological tissues may tell us about the mechanisms underlying TSP's functions in CNS synaptogenesis.




18. Thrombospondin1 in tissue repair and fibrosis: TGF-ß-dependent and independent mechanisms
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 3

Mariya T. Sweetwyne, Joanne E. Murphy-Ullrich

Thrombospondin 1 (TSP1) plays major roles in both physiologic and pathologic tissue repair. TSP1 through its type 1 repeats is a known regulator of latent TGF-ß activation and plays a role in wound healing and fibrosis. Binding of the TSP N-terminal domain to cell surface calreticulin in complex with LDL-receptor related protein 1 stimulates intermediate cell adhesion, cell migration, anoikis resistance, collagen expression and matrix deposition in an in vivo model of the foreign body response. There is also emerging evidence that TSP EGF-like repeats alter endothelial cell–cell interactions and stimulate epithelial migration through transactivation of EGF receptors. The mechanisms underlying these functions of TSP1 and the implications for physiologic and pathologic wound repair and fibrosis will be discussed.




19. Expression of collagen VI a5 and a6 chains in human muscle and in Duchenne muscular dystrophy-related muscle fibrosis
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 3

Patrizia Sabatelli, Francesca Gualandi, Sudheer Kumar Gara, Paolo Grumati, Alessandra Zamparelli, Elena Martoni, Camilla Pellegrini, Luciano Merlini, Alessandra Ferlini, Paolo Bonaldo, Nadir Mario Maraldi, Mats Paulsson, Stefano Squarzoni, Raimund Wagener

Collagen VI is a major extracellular matrix (ECM) protein with a critical role in maintaining skeletal muscle functional integrity. Mutations in COL6A1, COL6A2 and COL6A3 genes cause Ullrich Congenital Muscular Dystrophy (UCMD), Bethlem Myopathy, and Myosclerosis. Moreover, Col6a1 -/- mice and collagen VI deficient zebrafish display a myopathic phenotype. Recently, two additional collagen VI chains were identified in humans, the a5 and a6 chains, however their distribution patterns and functions in human skeletal muscle have not been thoroughly investigated yet. By means of immunofluorescence analysis, the a6 chain was detected in the endomysium and perimysium, while the a5 chain labeling was restricted to the myotendinous junctions. In normal muscle cultures, the a6 chain was present in traces in the ECM, while the a5 chain was not detected. In the absence of ascorbic acid, the a6 chain was mainly accumulated into the cytoplasm of a sub-set of desmin negative cells, likely of interstitial origin, which can be considered myofibroblasts as they expressed a-smooth muscle actin. TGF-ß1 treatment, a pro-fibrotic factor which induces trans-differentiation of fibroblasts into myofibroblasts, increased the a6 chain deposition in the extracellular matrix after addition of ascorbic acid. In order to define the involvement of the a6 chain in muscle fibrosis we studied biopsies of patients affected by Duchenne Muscular Dystrophy (DMD). We found that the a6 chain was dramatically up-regulated in fibrotic areas where, in contrast, the a5 chain was undetectable. Our results show a restricted and differential distribution of the novel a6 and a5 chains in skeletal muscle when compared to the widely distributed, homologous a3 chain, suggesting that these new chains may play specific roles in specialized ECM structures. While the a5 chain may have a specialized function in tissue areas subjected to tensile stress, the a6 chain appears implicated in ECM remodeling during muscle fibrosis.




20. Maladaptive matrix remodeling and regional biomechanical dysfunction in a mouse model of aortic valve disease
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 3

Varun K. Krishnamurthy, Amy M. Opoka, Christine B. Kern, Farshid Guilak, Daria A. Narmoneva, Robert B. Hinton

Aortic valve disease (AVD) occurs in 2.5% of the general population and often requires surgical intervention. Aortic valve malformation (AVM) underlies the majority of cases, suggesting a developmental etiology. Elastin haploinsufficiency results in complex cardiovascular problems, and 20–45% of patients have AVM and/or AVD. Elastin insufficient (Eln+/-) mice demonstrate AVM and latent AVD due to abnormalities in the valve annulus region. The objective of this study was to examine extracellular matrix (ECM) remodeling and biomechanical properties in regional aortic valve tissue and determine the impact of early AVM on late AVD in the Eln+/- mouse model. Aortic valve ECM composition and remodeling from juvenile, adult, and aged stages were evaluated in Eln+/- mice using histology, ELISA, immunohistochemistry and gelatin zymography. Aortic valve tissue biomechanical properties were determined using micropipette aspiration. Cartilage-like nodules were demonstrated within the valve annulus region at all stages identifying a developmental abnormality preceding AVD. Interestingly, maladaptive ECM remodeling was observed in early AVM without AVD and worsened with late AVD, as evidenced by increased MMP-2 and MMP-9 expression and activity, as well as abnormalities in ADAMTS-mediated versican processing. Cleaved versican was increased in the valve annulus region of aged Eln+/- mice, and this abnormality correlated temporally with adverse alterations in valve tissue biomechanical properties and the manifestation of AVD. These findings identify maladaptive ECM remodeling in functional AVM as an early disease process with a progressive natural history, similar to that seen in human AVD, emphasizing the importance of the annulus region in pathogenesis. Combining molecular and engineering approaches provides complementary mechanistic insights that may be informative in the search for new therapeutic targets and durable valve bioprostheses.




21. New insights into cartilage repair — The role of migratory progenitor cells in osteoarthritis
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 3

Regina Gerter, Jenny Kruegel, Nicolai Miosge

Osteoarthritis is one of the most common musculo-skeletal diseases with a complex patholoy and a strong impact on cell biology, differentiation and migration behavior of mesenchymal stem cell-derived progenitor cells. In this review, we elucidate the influence of the pathologically altered extracellular matrix on progenitor cell behavior. Moreover, we discuss the modulation of progenitor cells especially of previously characterized chondrogenic progenitor cells (Koelling et al., 2009) in situ to enhance their regeneration potential. These options comprise the application of growth factors like fibroblast growth factor-2, a Runx-2 knock down and a contemporary anti-inflammatory therapy. This supports endogenous regeneration on behalf of the diseased osteoarthritic cartilage, which otherwise results mainly in an insufficient fibro-cartilaginous repair tissue. Furthermore, new results indicate a role of pericytes in osteoarthritis for these repair attempts. We discuss the biological mechanisms potentially leading to new therapeutic options in osteoarthritis to enhance regeneration in situ.




22. Disruption of the developmentally-regulated Col2a1 pre-mRNA alternative splicing switch in a transgenic knock-in mouse model
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 3

Renate Lewis, Soumya Ravindran, Louisa Wirthlin, Geoffrey Traeger, Russell J. Fernandes, Audrey McAlinden

The present study describes the generation of a knock-in mouse model to address the role of type II procollagen (Col2a1) alternative splicing in skeletal development and maintenance. Alternative splicing of Col2a1 precursor mRNA is a developmentally-regulated event that only occurs in chondrogenic tissue. Normally, chondroprogenitor cells synthesize predominantly exon 2-containing mRNA isoforms (type IIA and IID) while Col2a1 mRNA devoid of exon 2 (type IIB) is the major isoform produced by differentiated chondrocytes. Another isoform, IIC, has also been identified that contains a truncated exon 2 and is not translated into protein. The biological significance of this IIA/IID to IIB splicing switch is not known. Utilizing a splice site targeting knock-in approach, a 4 nucleotide mutation was created to convert the 5' splice site of Col2a1 exon 2 from a weak, non-consensus sequence to a strong, consensus splice site. This resulted in apparent expression of only the IIA mRNA isoform, as confirmed in vitro by splicing of a type II procollagen mini-gene containing the 5' splice site mutation. To test the splice site targeting approach in vivo, homozygote mice engineered to retain IIA exon 2 (Col2a1 +ex2 ) were generated. Chondrocytes from hindlimb epiphyseal cartilage of homozygote mice were shown to express only IIA mRNA and protein at all pre- and post-natal developmental stages analyzed (E12.5, E16.5, P0, P3, P7, P14, P28 and P70). As expected, type IIB procollagen was the major isoform produced in wild type cartilage at all post-natal time points. Col2a1 +ex2 homozygote mice are viable, appear healthy and display no overt phenotype to date. However, research is currently underway to investigate the biological consequence of persistent expression of the exon 2-encoded conserved cysteine-rich domain in post-natal skeletal tissues.




23. ASMB Announcement
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 3








24. From the editor's desk
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 2








25. A comprehensive model of hyaluronan turnover in the mouse
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 2

Laurence Jadin, Louis H. Bookbinder, Gregory I. Frost

The metabolism of hyaluronan (HA), especially its catabolism, is still far from being elucidated. Although several studies suggest that HA is degraded locally in tissues and through the lymphatic or circulatory systems, much needs to be learned about the enzymes, receptors and cell types that support this dynamic process. In the current work, the clearance of exogenously administered HA was examined in a C57BL/6 mouse model. Hyaluronidase-sensitive fluorescein-labeled 1.2MDa hyaluronan (flHA) was administered either intravenously (i.v.) or subcutaneously (s.c.) into wild type C57BL/6 mice. Plasma was sampled for pharmacokinetic analysis and tissues were harvested for histological examination of the cell types responsible for uptake using immunofluorescent localization and for size exclusion chromatography analysis. We observed that flHA could be degraded locally in the skin or be taken up by sinusoidal cells in lymph nodes, liver and spleen. I.v. administration of flHA revealed non-linear Michaelis–Menten pharmacokinetics compatible with a saturable, receptor-mediated clearance system (K m =11.6µg/ml±46.0%, V max =1.69µg/ml/min±59.7%). Through a combination of immunofluorescence microscopy, pharmacokinetic, and chromatographic analyses of labeled substrate in vivo, our results shed additional light on the mechanisms by which HA is catabolized in mammals, and serve as a basis for future studies.




26. Hyaluronan and versican in the control of human T-lymphocyte adhesion and migration
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 2

Stephen P. Evanko, Susan Potter-Perigo, Paul L. Bollyky, Gerald T. Nepom, Thomas N. Wight

The ability of lymphocytes to migrate freely through connective tissues is vital to efficient immune function. How the extracellular matrix (ECM) may affect T-cell adhesion and migration is not well understood. We have examined the adhesion and migration of activated human T-lymphocytes on ECM made by fibroblast-like synoviocytes and lung fibroblasts. These cells were minimally interactive until treated with a viral mimetic, Poly I:C. This treatment promoted myofibroblast formation and engendered a higher-order structured ECM, rich in versican and hyaluronan, to which T-cells avidly adhered in a hyaluronidase-sensitive manner. This Poly I:C-induced matrix impeded T-cell spreading and migration on and through synoviocyte monolayers, while hyaluronidase treatment or adding versican antibody during matrix formation reversed the effect on T-cell migration. Hyaluronidase also reversed the spread myofibroblast morphology. These data suggest that the viscous hyaluronan- and versican-rich matrix binds and constrains T-lymphocytes. Using purified matrix components and solid state matrices of defined composition, we uncovered a role for versican in modulating hyaluronan-T-cell interactions. Versican prevented T-cell binding to soluble hyaluronan, as well as the amoeboid shape change on hyaluronan-coated dishes and T-cell penetration of collagen gels. Together, these data suggest that hyaluronan and versican play a role in T-cell trafficking and function in inflamed tissues.




27. Effects of decorin and biglycan on human airway smooth muscle cell adhesion
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 2

Michelle L. D'Antoni, Paul-André Risse, Pasquale Ferraro, James G. Martin, Mara S. Ludwig

Growth on a decorin matrix results in decreased human airway smooth muscle cell (HASMC) number, by decreasing proliferation and increasing apoptosis. We questioned whether these effects were related to abnormal extracellular matrix (ECM)-cell adhesion. HASMCs were seeded on decorin, biglycan, collagen type I or plastic. Actin organization and focal adhesion formation were assessed by staining for filamentous (F) and globular (G) actin, and vinculin, respectively. Gene expression for focal adhesion proteins, ECM molecules and HASMC receptors was measured. Protein levels for fibronectin, a2, a5, av and ß3 integrin subunits and, focal adhesion kinase (FAK) were assessed. F-actin filaments were prominent in cells seeded on collagen I and plastic, less apparent in cells cultured on biglycan and faint in cells on decorin. Vinculin clustering was decreased in cells seeded on decorin and biglycan, as was vinculin gene expression. Compared to cells on plastic, cells on decorin had an increase in fibronectin gene expression. Seeding on decorin caused an increase in a2 integrin subunit and platelet-derived growth factor receptor A gene expression. There was also an increase in a2 and av integrin subunit protein. Finally, FAK protein levels in cells seeded on decorin or biglycan were decreased compared to cells seeded on plastic or collagen I. Cells grown on proteoglycan matrices demonstrate evidence of abnormalities during many of the key processes involved in normal cell adhesion. Upregulation of cell surface receptor proteins, such as a2 integrin subunit, may represent a compensatory mechanism to overcome poor adhesion induced by growth on these matrices.




28. Molecular responses of choroidal endothelial cells to elastin derived peptides through the elastin-binding protein (GLB1)
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 2

Jessica M. Skeie, Jasmine Hernandez, Aleksander Hinek, Robert F. Mullins

Purpose Neovascular AMD involves the activation of choroidal endothelial cells to increase their inflammatory and angiogenic behaviors. Elastin derived peptides (EDPs) can elicit some of these phenotypic changes in endothelial cells. This investigation was performed to follow up on those findings by determining a receptor for these peptides in the human eye as well as evaluating the effects of elevated EDPs on choroidal cells in vitro and in vivo. Methods The expression of elastin receptor genes including GLB1 was analyzed using reverse transcription PCR. Migration of choroidal endothelial cells was quantified in the presence of inhibitors to different EDP binding proteins. C57BL6 mice were injected with EDPs and studied by electroretinography, transmission electron microscopy, and microarray analysis. Results An alternatively spliced form of beta-galactosidase (GLB1) is present on human choroidal endothelial cells and acts as a receptor for EDPs. Elevated levels of circulating EDPs do not affect retinal function in the mouse, but do increase the expression and deposition of collagen IV in the RPE/choroid complex. Conclusions EDPs may play a role in neovascular AMD by binding to and inducing neovascular phenotypes in choroidal endothelial cells through their receptor, GLB1. These peptides also cause an increased mRNA expression and deposition of collagen IV in the RPE/choroid, which may alter diffusion properties between the retina and choriocapillaris.




29. The ?3 chain of laminin is widely but differentially expressed in murine basement membranes: Expression and functional studies
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 2

Yong N. Li, Stephanie Radner, Margaret M. French, Germán Pinzón-Duarte, Gerard H. Daly, Robert E. Burgeson, Manuel Koch, William J. Brunken

Laminins are heterotrimeric extracellular glycoproteins found in, but not confined to, basement membranes (BMs). They are important components in formation of the molecular networks of BMs as well as in cell polarity, cell differentiation and tissue morphogenesis. Each laminin is composed by an a, a ß and a ? chain. Previous studies have shown that the ?3 chain is partnered with either the ß1 chain (in placenta) or ß2 chain (in the CNS) (Libby et al., 2000). Several studies, including our own, suggested that the ?3 chain is expressed in both apical and basal compartments (Koch et al., 1999; Gersdorff et al., 2005; Yan and Cheng, 2006). This study investigates the expression pattern of the ?3 chain in mouse. We developed three new ?3-reactive antibodies, and we show that the ?3 chain is present in BMs. The distribution pattern is considerably more restricted than that of the ?1 chain and within any tissue there is differential deposition into BM compartments. This is particularly true in the retina and brain, where ?3 is uniquely expressed in a subset of the vascular basement membranes and the pial surface. We used conventional genetic ablation techniques to remove the ?3 chain in mice; unlike other laminin null mice (a5, ß2, ?1 nulls), these mice live a normal lifespan and have only minor abnormalities, the most striking of which are ectopic granule cells in the cerebellum and an apparent increase in capillary branching in the outer retina. These data support the suggestion that the ?3 chain is deposited in BMs and contributes some unique properties to their function, particularly in the nervous system.




30. Determination of a molecular shape for netrin-4 from hydrodynamic and small angle X-ray scattering measurements
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 2

Trushar R. Patel, Raphael Reuten, Shawn Xiong, Markus Meier, Donald J. Winzor, Manuel Koch, Jörg Stetefeld

As part of a continuing investigation of netrins, an emerging class of extracellular matrix proteins that are involved in axon guidance activity, we have used dynamic light scattering (DLS) and small angle X-ray scattering to investigate the solution conformation of a truncated version of netrin-4 (?netrin-4) that lacks the C-terminal portion. The protein is characterized by a hydrodynamic (Stokes) radius (r H ) of 4.60 (±0.20) nm, a radius of gyration (r G ) of 4.42 (±0.20) nm and a maximum particle dimension (D max ) of 16nm. More detailed ab initio modeling of the SAXS data indicates an extended rod like conformation for ?netrin-4 in solution—a concept supported by the excellent agreement observed between experimental parameter estimates and those calculated for the ab initio models for ?netrin-4 by the HYDROPRO program.




31. Viscoelastic properties of model segments of collagen molecules
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 2

Alfonso Gautieri, Simone Vesentini, Alberto Redaelli, Markus J. Buehler

Collagen is the prime construction material in vertebrate biology, determining the mechanical behavior of connective tissues such as tendon, bone and skin. Despite extensive efforts in the investigation of the origin of collagen unique mechanical properties, a deep understanding of the relationship between molecular structure and mechanical properties remains elusive, hindered by the complex hierarchical structure of collagen-based tissues. In particular, although extensive studies of viscoelastic properties have been pursued at the macroscopic (fiber/tissue) level, fewer investigations have been performed at the smaller scales, including in particular collagen molecules and fibrils. These scales are, however, important for a complete understanding of the role of collagen as an important constituent in the extracellular matrix. Here, using an atomistic modeling approach, we perform in silico creep tests of a collagen-like peptide, monitoring the strain-time response for different values of applied external load. The results show that individual collagen molecules exhibit a nonlinear viscoelastic behavior, with a Young's modulus increasing from 6 to 16GPa (for strains up to 20%), a viscosity of 3.84.±0.38Pa·s, and a relaxation time in the range of 0.24–0.64ns. The single molecule viscosity, for the first time reported here, is several orders of magnitude lower than the viscosity found for larger-scale single collagen fibrils, suggesting that the viscous behavior of collagen fibrils and fibers involves additional mechanisms, such as molecular sliding between collagen molecules within the fibril or the effect of relaxation of larger volumes of solvent. Based on our molecular modeling results we propose a simple structural model that describes collagen tissue as a hierarchical structure, providing a bottom-up description of elastic and viscous properties form the properties of the tissue basic building blocks.




32. ASMB Announcement
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 2








33. From the Editor's Desk Thank you!
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 1








34. Molecular functions of syndecan-1 in disease
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 1

Yvonne Hui-Fang Teng, Rafael S. Aquino, Pyong Woo Park

Syndecan-1 is a cell surface heparan sulfate proteoglycan that binds to many mediators of disease pathogenesis. Through these molecular interactions, syndecan-1 can modulate leukocyte recruitment, cancer cell proliferation and invasion, angiogenesis, microbial attachment and entry, host defense mechanisms, and matrix remodeling. The significance of syndecan-1 interactions in disease is underscored by the striking pathological phenotypes seen in the syndecan-1 null mice when they are challenged with disease-instigating agents or conditions. This review discusses the key molecular functions of syndecan-1 in modulating the onset, progression, and resolution of inflammatory diseases, cancer, and infection.




35. Laminin a1 is essential for mouse cerebellar development
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 1

Naoki Ichikawa-Tomikawa, Junko Ogawa, Vanessa Douet, Zhuo Xu, Yuji Kamikubo, Takashi Sakurai, Shinichi Kohsaka, Hideki Chiba, Nobutaka Hattori, Yoshihiko Yamada, Eri Arikawa-Hirasawa

Laminin a1 (Lama1), which is a subunit of laminin-1 (laminin-111), a heterotrimeric ECM protein, is essential for embryonic development and promotes neurite outgrowth in culture. Because the deletion of Lama1 causes lethality at early embryonic stages in mice, the in vivo role of Lama1 in neural development and functions has not yet been possible to determine. In this study, we generated conditional Lama1 knockout (Lama1 CKO ) mice in the epiblast lineage using Sox2-Cre mice. These Lama1 CKO mice survived, but displayed behavioral disorders and impaired formation of the cerebellum. Deficiency of Lama1 in the pial basement membrane of the meninges resulted in defects in the conformation of the meninges. During cerebellar development, Lama1 deficiency also caused a decrease in the proliferation and migration of granule cell precursors, disorganization of Bergmann glial fibers and endfeet, and a transient reduction in the activity of Akt. A marked reduction in numbers of dendritic processes in Purkinje cells was observed in Lama1 CKO mice. Together, these results indicate that Lama1 is required for cerebellar development and functions.




36. Drosophila basement membrane collagen col4a1 mutations cause severe myopathy
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 1

Ildikó Kelemen-Valkony, Márton Kiss, Judit Csiha, András Kiss, Urs Bircher, János Szidonya, Péter Maróy, Gábor Juhász, Orbán Komonyi, Katalin Csiszár, Mátyás Mink

Recent data from clinical and mammalian genetic studies indicate that COL4A1 mutations manifest with basement membrane defects that result in muscle weakness, cramps, contractures, dystrophy and atrophy. In-depth studies of mutant COL4A1-associated muscle phenotype, however, are lacking and significant details of the muscle-specific pathomechanisms remain unknown. In this study, we have used a comprehensive set of Drosophila col4a1 and col4a2 mutants and a series of genetic and mutational analyses, gene, protein expression, and immunohistochemistry experiments in order to establish a Drosophila model and address some of these questions. The Drosophila genome contains two type IV collagen genes, col4a1 and col4a2. Mutant heterozygotes of either gene are viable and fertile, whereas homozygotes are lethal. In complementation analysis of all known mutants of the locus and a complementation matrix derived from these data we have identified the dominant lesions within the col4a1, but not within the col4a2 gene. Expression of a col4a1 transgene partially rescued the dominant and recessive mutant col4a1 alleles but not the col4a2 mutations that were all recessive. Partial complementation suggested that col4a1 gene mutations have strong antimorph effect likely due to the incorporation of the mutant protein into the triple helix. In col4a1 mutants, morphological changes of the oviduct muscle included severe myopathy with centronuclear myofibers leading to gradual development of female sterility. In larval body wall muscles ultrastructural changes included disturbance of A and I bands between persisting Z bands. In the most severely affected DTS-L3 mutant, we have identified four missense mutations within the coding region of the col4a1 gene two of which affected the Y within the Gly-X-Y unit and a 3' UTR point mutation. In conclusion, our Drosophila mutant series may serve as an effective model to uncover the mechanisms by which COL4A1 mutations result in compromised myofiber–basement membrane interactions and aberrant muscle function.




37. Neutrophil elastase cleaves the murine hemidesmosomal protein BP180/type XVII collagen and generates degradation products that modulate experimental bullous pemphigoid
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 1

Lan Lin, Tomoko Betsuyaku, Lisa Heimbach, Ning Li, David Rubenstein, Steven D. Shapiro, Lijia An, George J. Giudice, Luis A. Diaz, Robert M. Senior, Zhi Liu

Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease associated with autoantibodies against the hemidesmosomal proteins BP180 and BP230. In the IgG passive transfer model of BP, blister formation is triggered by anti-BP180 IgG and depends on complement activation, mast cell degranulation, and neutrophil recruitment. Mice lacking neutrophil elastase (NE) do not develop experimental BP. Here, we demonstrated that NE degrades recombinant mouse BP180 within the immunodominant extracellular domain at amino acid positions 506 and 561, generating peptide p561 and peptide p506. Peptide p561 is chemotactic for neutrophils both in vitro and in vivo. Local injection of NE into B6 mice recruits neutrophils to the skin, and neutrophil infiltration is completely blocked by co-injection with the NE inhibitor a1-proteinase inhibitor. More importantly, NE directly cleaves BP180 in mouse and human skin, as well as the native human BP180 trimer molecule. These results demonstrate that (i) NE directly damages the extracellular matrix and (ii) NE degradation of mouse BP180 generates neutrophil chemotactic peptides that amplify disease severity at the early stage of the disease.




38. Glycomic analyses of ovarian follicles during development and atresia
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 1

Nicholas Hatzirodos, Julie Nigro, Helen F. Irving-Rodgers, Aditya V. Vashi, Katja Hummitzsch, Bruce Caterson, Thomas R. Sullivan, Raymond J. Rodgers

To examine the detailed composition of glycosaminoglycans during bovine ovarian follicular development and atresia, the specialized stromal theca layers were separated from the stratified epithelial granulosa cells of healthy (n=6) and atretic (n=6) follicles in each of three size ranges: small (3–5mm), medium (6-9mm) and large (10mm or more) (n=29 animals). Fluorophore-assisted carbohydrate electrophoresis analyses (on a per cell basis) and immunohistochemistry (n=14) were undertaken. We identified the major disaccharides in thecal layers and the membrana granulosa as chondroitin sulfate-derived ?uronic acid with 4-sulfated N-acetylgalactosamine and ?uronic acid with 6-sulfated N-acetylgalactosamine and the heparan sulfate-derived ?uronic acid with N-acetlyglucosamine, with elevated levels in the thecal layers. Increasing follicle size and atresia was associated with increased levels of some disaccharides. We concluded that versican contains 4-sulfated N-acetylgalactosamine and it is the predominant 4-sulfated N-acetylgalactosamine proteoglycan in antral follicles. At least one other non- or 6-sulfated N-acetylgalactosamine proteoglycan(s), which is not decorin or an inter-a-trypsin inhibitor family member, is present in bovine antral follicles and associated with hitherto unknown groups of cells around some larger blood vessels. These areas stained positively for chondroitin/dermatan sulfate epitopes [antibodies 7D4, 3C5, and 4C3], similar to stem cell niches observed in other tissues. The sulfation pattern of heparan sulfate glycosaminoglycans appears uniform across follicles of different sizes and in healthy and atretic follicles. The heparan sulfate products detected in the follicles are likely to be associated with perlecan, collagen XVIII or betaglycan.




39. Characterization of tissue biomechanics and mechanical signaling in uterine leiomyoma
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 1

John M. Norian, Carter M. Owen, Juan Taboas, Casey Korecki, Rocky Tuan, Minnie Malik, William H. Catherino, James H. Segars

Leiomyoma are common tumors arising within the uterus that feature excessive deposition of a stiff, disordered extracellular matrix (ECM). Mechanical stress is a critical determinant of excessive ECM deposition and increased mechanical stress has been shown to be involved in tumorigenesis. Here we tested the viscoelastic properties of leiomyoma and characterized dynamic and static mechanical signaling in leiomyoma cells using three approaches, including measurement of active RhoA. We found that the peak strain and pseudo-dynamic modulus of leiomyoma tissue was significantly increased relative to matched myometrium. In addition, leiomyoma cells demonstrated an attenuated response to applied cyclic uniaxial strain and to variation in substrate stiffness, relative to myometrial cells. However, on a flexible pronectin-coated silicone substrate, basal levels and lysophosphatidic acid-stimulated levels of activated RhoA were similar between leiomyoma and myometrial cells. In contrast, leiomyoma cells plated on a rigid polystyrene substrate had elevated levels of active RhoA, compared to myometrial cells. The results indicate that viscoelastic properties of the ECM of leiomyoma contribute significantly to the tumor's inherent stiffness and that leiomyoma cells have an attenuated sensitivity to mechanical cues. The findings suggest there may be a fundamental alteration in the communication between the external mechanical environment (extracellular forces) and reorganization of the actin cytoskeleton mediated by RhoA in leiomyoma cells. Additional research will be needed to elucidate the mechanism(s) responsible for the attenuated mechanical signaling in leiomyoma cells.




40. Random phage-epitope library based identification of a peptide antagonist of Mac-1 ß2 integrin ligand binding
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 1

Mehdi Houimel, Luca Mazzucchelli

The leukocyte ß2 integrin Mac-1 (CD11b/CD18) plays a pivotal role in inflammation and host defense. To develop peptide antagonists selectively inhibiting the function of Mac-1, we used a random constrained 6-mer (cys-6aa-cys) peptide library to map the structural features of CD11b, by determining the epitope of neutralizing monoclonal antibody mAb 44a (anti-CD11b). We have used a stringent phage display strategy, which resulted in the identification of one disulfide C-RLKEKH-C constrained peptide by direct biopanning of library on decreasing amounts of purified mAb 44a. The selected peptide mimics a discontinuous epitope, a peculiar shape on the CD11b-I-domain surface. Competitive ELISA experiments with different Mac-1 ligands showed that C-RLKEKH-C is able to bind to fibrinogen, iC3b, and C1q. Furthermore, the monomeric circular peptide C-RLKEKH-C, was effective in blocking the interaction between 125I-fibrinogen and Mac-1 (IC50 =3.35±0.1×10-6 M), and inhibited the adhesion of human neutrophils to fibrinogen and iC3b. These data provide information about the relative location of amino acids on the I-domain surface using mAb 44a imprint of the CD11b protein. The derived mimotope may help in the design of future anti-inflammatory therapeutic agents that can act as specific therapeutic agents targeting PMNs mediated inflammation.




41. ASMB Announcement
Publication year: 2012
Source:Matrix Biology, Volume 31, Issue 1








42. Cell-matrix adhesions in 3D
Publication year: 2011
Source:Matrix Biology, Volume 30, Issues 7–8

Jill S. Harunaga, Kenneth M. Yamada

Cells in a three-dimensional (3D) extracellular matrix environment often display different properties and behavior compared to cells cultured on a two-dimensional (2D) substrate. Recent studies characterizing the cell-matrix adhesions formed by cells within a 3D matrix have arrived at contradictory conclusions regarding the presence and composition of adhesions. Here we review this literature, and provide a comparative compilation of information found in published studies from the 3D cell-matrix adhesion field in order to identify shared and divergent conclusions and conceptually important areas that require further research. Although there is a general consensus that discrete cell-matrix adhesions exist in various 3D matrix environments, there are specific exceptions, particularly in cells undergoing amoeboid migration. There are also technical issues to consider when imaging adhesions in 3D matrix; for example, over-expression of a cytoskeletal cell adhesion component can potentially cloud the visualization of adhesions and even alter the mode of cell migration. Properties such as stiffness and local matrix topography may also affect the composition of cell-matrix adhesions. For example, even though cells contain integrin-based 3D adhesions, there can be substantial variability within these adhesions in the presence of force-dependent cytoskeletal components such as vinculin. These new findings and ideas provide promising new leads for understanding the regulation and function of cell-matrix adhesions in 3D matrix.




43. Ucmaa (Grp-2) is required for zebrafish skeletal development. Evidence for a functional role of its glutamate ?-carboxylation
Publication year: 2011
Source:Matrix Biology, Volume 30, Issues 7–8

Cristian Dan Neacsu, Melanie Grosch, Mercedes Tejada, Andreas Winterpacht, Mats Paulsson, Raimund Wagener, Andreas Tagariello

UCMA (alternatively named GRP) is a novel member of the family of ?-carboxyglutamate (Gla) containing proteins that is mainly expressed in cartilage. We have used the zebrafish as a model organism to study UCMA function. Due to the whole genome duplication two Ucma genes are present in zebrafish, ucmaa and ucmab, located on chromosomes 25 and 4, respectively. UCMA gene structure, alternative splicing and protein sequence are highly conserved between mammals and zebrafish and Ucmaa and Ucmab are expressed in zebrafish skeletal tissues. Ucmaa is first detected in the notochord at 18 hpf and expression continues during notochord development. In addition, it is widely present in the developing craniofacial cartilage. In contrast, the weakly expressed Ucmab can be first detected at specific sites in the craniofacial cartilage at 96 hpf, but not in notochord. Knockdown of ucmaa leads to severe growth retardation and perturbance of skeletal development. The cartilage of the morphants has a decreased aggrecan and collagen II content. Similar malformations were observed when glutamate ?-carboxylation was inhibited by warfarin treatment, indicating that glutamate ?-carboxylation is crucial for Ucma function and pointing to a role of UCMA in the pathogenesis of “warfarin embryopathies” and other human skeletal diseases.




44. Morphogenetic roles of perlecan in the tooth enamel organ: An analysis of overexpression using transgenic mice
Publication year: 2011
Source:Matrix Biology, Volume 30, Issues 7–8

Hiroko Ida-Yonemochi, Ichiro Satokata, Hayato Ohshima, Toshiya Sato, Minesuke Yokoyama, Yoshihiko Yamada, Takashi Saku

Perlecan, a heparan sulfate proteoglycan, is enriched in the intercellular space of the enamel organ. To understand the role of perlecan in tooth morphogenesis, we used a keratin 5 promoter to generate transgenic (Tg) mice that over-express perlecan in epithelial cells, and examined their tooth germs at tissue and cellular levels. Immunohistochemistry showed that perlecan was more strongly expressed in the enamel organ cells of Tg mice than in wild-type mice. Histopathology showed wider intercellular spaces in the stellate reticulum of the Tg molars and loss of cellular polarity in the enamel organ, especially in its cervical region. Hertwig's epithelial root sheath (HERS) cells in Tg mice were irregularly aligned due to excessive deposits of perlecan along the inner, as well as on the outer sides of the HERS. Tg molars had dull-ended crowns and outward-curved tooth roots and their enamel was poorly crystallized, resulting in pronounced attrition of molar cusp areas. In Tg mice, expression of integrin ß1 mRNA was remarkably higher at E18, while expression of bFGF, TGF-ß1, DSPP and Shh was more elevated at P1. The overexpression of perlecan in the enamel organ resulted in irregular morphology of teeth, suggesting that the expression of perlecan regulates growth factor signaling in a stage-dependent manner during each step of the interaction between ameloblast-lineage cells and mesenchymal cells.




45. The effect of hyaluronan on airway mucus transport and airway epithelial barrier integrity: Potential application to the cytoprotection of airway tissue
Publication year: 2011
Source:Matrix Biology, Volume 30, Issues 7–8

Jean-Marie Zahm, Magali Milliot, Anthony Bresin, Christelle Coraux, Philippe Birembaut

The lubricating abilities and the protective functions of hyaluronan, a structural component of interstitial and connective tissues, were assessed in in vitro models of airway mucus transport and epithelial barrier. We found that hyaluronan enhanced the transport of airway mucus by cilia and by cough: the lower the hyaluronan molecular weight, the higher the increase. By immunofluorescence and western blot, we observed a significant dose-dependent (0.1, 1, 5 and 10mg/ml) increase by low molecular weight hyaluronan (40kDa) in the expression of tight junction proteins such as ZO-1, as well as an increase in the trans-epithelial resistance. Incubation of airway epithelial cells with hyaluronan 40kDa also significantly increased the gap junction functionality. Finally, we demonstrated that hyaluronan 40kDa protects the airway epithelium against injury induced by bacterial products during infection. These results demonstrate that the expression and functionality of intercellular adhesion molecules are increased by hyaluronan which can also act as a lubricant at the airway epithelium surface and suggest that hyaluronan may play a therapeutic role in a variety of respiratory diseases.




46. Interstitial fibrosis is associated with increased COL1A2 transcription in AA-injured renal tubular epithelial cells in vivo
Publication year: 2011
Source:Matrix Biology, Volume 30, Issues 7–8

Maria Fragiadaki, Abigail S. Witherden, Tomoyo Kaneko, Sonali Sonnylal, Charles D. Pusey, George Bou-Gharios, Roger M. Mason

Accumulation of type I collagen is a key event in renal interstitial fibrosis. As there is no effective treatment, understanding the site where collagen is transcribed and the factors driving it in response to disease in vivo is critical for designing future therapies. The present research investigated the transcriptional activity of the COL1A2 gene in a mouse model of progressive fibrosis induced by aristolochic acid (aristolochic acid nephropathy, AAN). To achieve this we genetically modified mice to express a reporter gene (LacZ) and CCN2 (connective tissue growth factor) under the transcriptional control of the COL1A2 promoter /enhancer sequences. Using these mice we asked where is collagen actively transcribed and secondly, what is the role of CCN2 in AAN. Here, we report that de-novo transcription of the COL1A2 gene occurred predominantly in damaged tubular epithelial cells during progressive interstitial fibrosis in vivo. The activation of COL1A2 was studied by detection of the reporter gene LacZ and COL1A2 mRNA in interstitial, glomerular, vascular, and tubular epithelial tissue from laser capture microscopy. We also demonstrated that LacZ-positive cells co-express E-Cadherin a marker of epithelial origin which is consistent with an epithelial phenotype which is capable of collagen expression during injury. There was no evidence of detachment of these cells from tubules to become myofibroblasts. Moreover, we showed that the transgenic mice show a modest enhancement of CCN2 expression; however fibrosis induced by AA is the same in transgenics and controls suggesting that CCN2, at this level of expression, is not sufficient to enhance fibrogenesis. Overall our study provides a better understanding into the expression patterns and roles of two major extracellular matrix proteins: type I collagen and CCN2.




47. Peptide from the C-terminal domain of tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) inhibits membrane activation of matrix metalloproteinase-2 (MMP-2)
Publication year: 2011
Source:Matrix Biology, Volume 30, Issues 7–8

Xiaoping Xu, Margarita Mikhailova, Zhihua Chen, Sanjay Pal, Trista K. Robichaud, Eileen M. Lafer, Sam Baber, Bjorn Steffensen

Cellular activation of latent matrix metalloproteinase-2 (proMMP-2) requires formation of a cell membrane-associated activation complex that involves specific binding between the hemopexin domain of proMMP-2 (PEX) and the C-terminal domain of tissue inhibitor of matrix metalloproteinases-2 (C-TIMP-2). In this study, we tested the feasibility of inhibiting activation of proMMP-2 by exogenous inhibitors, which block the binding between PEX and TIMP-2. The recombinant C-TIMP-2 and synthetic peptides from C-TIMP-2 were used as inhibitors for proMMP-2 activation. Recombinant C-TIMP-2 bound specifically to both the catalytically inactive MMP-2E404A and the C-terminal domain of MMP-2 (PEX) in a concentration dependent manner with apparent K d of 3.9×10-7 M and 1.7×10-7 M, respectively. Moreover, C-TIMP-2 competed the binding between MMP-2E404A and full-length TIMP-2. Finally, activity assays showed that addition of C-TIMP-2 to HT-1080 fibrosarcoma cells inhibited proMMP-2 activation in a concentration-dependent manner. We then designed a synthetic peptide, P175L, consisting of 20 residues from the PEX-binding tail region of C-TIMP-2. P175L bound PEX and inhibited cell membrane-mediated activation of proMMP-2 in a concentration dependent manner. Deletion of the last 9 tail residues of C-TIMP-2 in P175L abrogated the inhibitory activities of the peptide showing that these residues were essential for function. Overall, these experiments have demonstrated that proMMP-2 activation can be inhibited by exogenous inhibitors which points to a potential strategy for MMP-2 specific inhibition.




48. Emerging roles for ADAMTS5 during development and disease
Publication year: 2011
Source:Matrix Biology, Volume 30, Issues 5–6

Christopher Kintakas, Daniel R. McCulloch






49. Caveolin-1 modulates TGF-ß1 signaling in cardiac remodeling
Publication year: 2011
Source:Matrix Biology, Volume 30, Issues 5–6

Shelley K. Miyasato, Jorik Loeffler, Ralph Shohet, Jianhua Zhang, Merry Lindsey, Claude Jourdan Le Saux

The cardiac response to myocardial injury includes fibrotic and hypertrophic processes and a key mediator in this response is transforming growth factor-ß1 (TGF-ß1). Caveolin-1 (cav1), the main structural protein of caveolae, is an inhibitor of the TGF-ß1 signaling pathway. To examine the role of cav1 in cardiac repair, cav1 deficient (Cav1 -/- ) and wild type (WT) mice were subjected to cryoinjury of the left ventricle (LV). At baseline the two groups exhibited no inflammation, similar collagen content, and similar cardiac function. After injury, Cav1 -/- animals displayed enhanced TGF-ß1 signaling, as reflected by a 3-fold increase in the activation of the Smad2-dependent pathway and more widespread collagen deposition in the heart. Qualitative and quantitative analyses indicated that collagen deposition peaked in the WT LV 14days after injury, accompanied by increased mRNA abundance for procol1a2 (2-fold) and procol3a1 (3-fold). Collagen deposition was further enhanced in Cav1 -/- mice, which was accompanied by reduced expression of matrix metalloproteinases MMP-8 (3-fold) and -13 mRNA (2-fold). The levels of expression of inflammatory markers of acute phase were similar between the strains However, macrophage clearance in the damaged region was delayed in Cav1 -/- mice. We observed a 4-fold decrease in collagen deposition in Cav1 -/- mice injected with a cav1 scaffolding domain peptide (CSD) and a 2-fold decrease in WT mice treated with the CSD. We conclude that cav1 has a direct role in reducing TGF-ß1 signaling and as such might be an appropriate target for therapies to influence cardiac remodeling.




50. Implication of the oligomeric state of the N-terminal PTX3 domain in cumulus matrix assembly
Publication year: 2011
Source:Matrix Biology, Volume 30, Issues 5–6

Elena Ievoli, Ragnar Lindstedt, Antonio Inforzato, Antonella Camaioni, Francesca Palone, Anthony J. Day, Alberto Mantovani, Giovanni Salvatori, Antonietta Salustri

Pentraxin 3 (PTX3) plays a key role in the formation of the hyaluronan-rich matrix of the cumulus oophorus surrounding ovulated eggs that is required for successful fertilization and female fertility. PTX3 is a multimeric protein consisting of eight identical protomers held together by a combination of non-covalent interactions and disulfide bonds. Recent findings suggest that the oligomeric status of PTX3 is important for stabilizing the cumulus matrix. Because the role of PTX3 in the cumulus resides in the unique N-terminal sequence of the protomer, we investigated further this issue by testing the ability of distinct Cys/Ser mutants of recombinant N-terminal region of PTX3 (N_PTX3) with different oligomeric arrangement to promote in vitro normal expansion in cumuli from Ptx3-null mice. Here we report that the dimer of the N_PTX3 is unable to rescue cumulus matrix organization, and that the tetrameric assembly of the protein is the minimal oligomeric state required for accomplishing this function. We have previously demonstrated that PTX3 binds to HCs of IaI and TSG-6, which are essential for cumulus matrix formation and able to interact with hyaluronan. Interestingly, here we show by solid-phase binding experiments that the dimer of the N_PTX3 retains the ability to bind to both IaI and TSG-6, suggesting that the octameric structure of PTX3 provides multiple binding sites for each of these ligands. These findings support the hypothesis that PTX3 contributes to cumulus matrix organization by cross-linking HA polymers through interactions with multiple HCs of IaI and/or TSG-6. The N-terminal PTX3 tetrameric oligomerization was recently reported to be also required for recognition and inhibition of FGF2. Given that this growth factor has been detected in the mammalian preovulatory follicle, we wondered whether FGF2 negatively influences cumulus expansion and PTX3 may also serve in vivo to antagonize its activity. We found that a molar excess of FGF2, above PTX3 binding capacity, does not affect in vitro cumulus matrix formation thus ruling out this possibility. In conclusion, the data strength the view that PTX3 acts as a nodal molecule in cross-linking HA in the matrix.




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