The Experts below are selected from a list of 798 Experts worldwide ranked by ideXlab platform

Dieter P. Reinhardt - One of the best experts on this subject based on the ideXlab platform.

  • adamts10 inactivation in mice leads to persistence of ocular microfibrils subsequent to reduced Fibrillin 2 cleavage
    Matrix Biology, 2019
    Co-Authors: Lauren W Wang, Dieter P. Reinhardt, Wendy E Kutz, Timothy J Mead, Lauren C Beene, Shweta Singh, Michael W Jenkins, Suneel S Apte
    Abstract:

    Abstract Mutations in the secreted metalloproteinase ADAMTS10 cause recessive Weill-Marchesani syndrome (WMS), comprising ectopia lentis, short stature, brachydactyly, thick skin and cardiac valve anomalies. Dominant WMS caused by FBN1 mutations is clinically similar and affects Fibrillin-1 microfibrils, which are a major component of the ocular zonule. ADAMTS10 was previously shown to enhance Fibrillin-1 assembly in vitro. Here, Adamts10 null mice were analyzed to determine the impact of ADAMTS10 deficiency on Fibrillin microfibrils in vivo. An intragenic lacZ reporter identified widespread Adamts10 expression in the eye, musculoskeletal tissues, vasculature, skin and lung. Adamts10−/− mice had reduced viability on the C57BL/6 background, and although surviving mice were slightly smaller and had stiff skin, they lacked brachydactyly and cardiovascular defects. Ectopia lentis was not observed in Adamts10−/− mice, similar to Fbn1−/− mice, most likely because the mouse zonule contains Fibrillin-2 in addition to Fibrillin-1. Unexpectedly, in contrast to wild-type eyes, Adamts10−/− zonule fibers were thicker and immunostained strongly with Fibrillin-2 antibodies into adulthood, whereas Fibrillin-1 staining was reduced. Furthermore, Fibrillin-2 staining of hyaloid vasculature remnants persisted post-natally in Adamts10−/− eyes. ADAMTS10 was found to cleave Fibrillin-2, providing an explanation for persistence of Fibrillin-2 at these sites. Thus, analysis of Adamts10−/− mice led to identification of Fibrillin-2 as a novel ADAMTS10 substrate and defined a proteolytic mechanism for clearance of ocular Fibrillin-2 at the end of the juvenile period.

  • Lessons from tracheal tube development for understanding congenital tracheal malformations.
    European Respiratory Journal, 2019
    Co-Authors: Heena Kumra, Neha E.h. Dinesh, Dieter P. Reinhardt
    Abstract:

    Novel functions of the extracellular protein Fibrillin-2 were discovered in tracheal tube development: Fibrillin-2 has important roles in tracheal cartilage development and smooth muscle cell orientation and polarity, all required for tracheal contractionhttp://ow.ly/rn3Z30nId3N

  • Fibrillin 2 and tenascin c bridge the age gap in lung epithelial regeneration
    Biomaterials, 2017
    Co-Authors: Sarah E Gilpin, Dieter P. Reinhardt, Qiyao Li, Daniele Evangelistaleite, Brian L Frey
    Abstract:

    Abstract Organ engineering based on native matrix scaffolds involves combining regenerative cell populations with corresponding biological matrices to form functional grafts on-demand. The extracellular matrix (ECM) that is retained following lung decellularization provides essential structure and biophysical cues for whole organ regeneration after recellularization. The unique ECM composition in the early post-natal lung, during active alveologenesis, may possess distinct signals that aid in driving cell adhesion, survival, and proliferation. We evaluated the behavior of basal epithelial stem cells (BESCs) isolated from adult human lung tissue, when cultured on acellular ECM derived from neonatal (aged

  • Unusual life cycle and impact on microfibril assembly of ADAMTS17, a secreted metalloprotease mutated in genetic eye disease.
    Scientific Reports, 2017
    Co-Authors: Dirk Hubmacher, Dieter P. Reinhardt, Michael Schneider, Steven J. Berardinelli, Hideyuki Takeuchi, Belinda Willard, Robert S. Haltiwanger, Suneel S Apte
    Abstract:

    Secreted metalloproteases have diverse roles in the formation, remodeling, and the destruction of extracellular matrix. Recessive mutations in the secreted metalloprotease ADAMTS17 cause ectopia lentis and short stature in humans with Weill-Marchesani-like syndrome and primary open angle glaucoma and ectopia lentis in dogs. Little is known about this protease or its connection to Fibrillin microfibrils, whose major component, Fibrillin-1, is genetically associated with ectopia lentis and alterations in height. Fibrillin microfibrils form the ocular zonule and are present in the drainage apparatus of the eye. We show that recombinant ADAMTS17 has unique characteristics and an unusual life cycle. It undergoes rapid autocatalytic processing in trans after its secretion from cells. Secretion of ADAMTS17 requires O-fucosylation and its autocatalytic activity does not depend on propeptide processing by furin. ADAMTS17 binds recombinant Fibrillin-2 but not Fibrillin-1 and does not cleave either. It colocalizes to Fibrillin-1 containing microfibrils in cultured fibroblasts and suppresses Fibrillin-2 (FBN2) incorporation in microfibrils, in part by transcriptional downregulation of Fbn2 mRNA expression. RNA in situ hybridization detected Adamts17 expression in specific structures in the eye, skeleton and other organs, where it may regulate the Fibrillin isoform composition of microfibrils.

  • Human eye development is characterized by coordinated expression of Fibrillin isoforms.
    Investigative Ophthalmology & Visual Science, 2014
    Co-Authors: Dirk Hubmacher, Dieter P. Reinhardt, Thomas Plesec, Katja Schenke-layland, Suneel S Apte
    Abstract:

    PURPOSE: Mutations in human Fibrillin-1 and -2, which are major constituents of tissue microfibrils, can affect multiple ocular components, including the ciliary zonule, lens, drainage apparatus, cornea, and retina. However, the expression pattern of the three human Fibrillins and an integral microfibrillar component, MAGP1, during human eye development is not known. METHODS: We analyzed sections from human eyes at gestational weeks (GWs) 6, 8, and 11 and at 1 and 3 years of age with antibodies specific for each human Fibrillin isoform or MAGP1, using immunofluorescence microscopy. RESULTS: During embryonic development, each Fibrillin isoform was detected in vascular structures bridging the ciliary body and the developing lens, hyaloid vasculature, and retina. In addition, they were present in the developing corneal basement membranes and lens capsule. MAGP1 codistributed with the Fibrillin isoforms. In contrast, the juvenile zonule was composed of Fibrillin-1 microfibrils containing MAGP1, but Fibrillin-2 was absent and Fibrillin-3 was only sparsely detected. CONCLUSIONS: Fibrillin-1, -2, and, unique to humans, Fibrillin-3 are found in various ocular structures during human embryonic eye development, whereas Fibrillin-1 dominates the postnatal zonule. We speculate that vasculature spanning the ciliary body and lens, which elaborates Fibrillin-2 and -3, may provide an initial scaffold for Fibrillin assembly and zonule formation.

Francesco Ramirez - One of the best experts on this subject based on the ideXlab platform.

  • Extracellular Microfibrils Control Osteoblast-supported Osteoclastogenesis by Restricting TGF Stimulation of
    2020
    Co-Authors: Harikiran Nistala, Sui Lee-arteaga, Silvia Smaldone, Gabriella Siciliano, Francesco Ramirez
    Abstract:

    Mutations in Fibrillin-1 or Fibrillin-2, the major structural components of extracellular microfibrils, cause pleiotropic manifestations in Marfan syndrome and congenital contractural arachnodactyly, respectively. We recently found that Fibrillin-1 and Fibrillin-2 control bone formation by regulating osteoblast differentiation through the differential modulation of endogenous TGF and bone morphogenetic protein signals. Here, we describe in vivo and ex vivo experiments that implicate the Fibrillins as negative regulators of bone resorption. Adult Fbn2 / mice display a greater than normal osteolytic response

  • Abnormal Activation of BMP Signaling Causes Myopathy in Fbn2 Null Mice
    PLOS Genetics, 2015
    Co-Authors: Gerhard Sengle, Noe L. Charbonneau, Francesco Ramirez, Eric J Carlson, Sara F Tufa, Valerie M Carlberg, Douglas R. Keene, Silvia Smaldone, Lynn Y. Sakai
    Abstract:

    Fibrillins are large extracellular macromolecules that polymerize to form the backbone structure of connective tissue microfibrils. Mutations in the gene for Fibrillin-1 cause the Marfan syndrome, while mutations in the gene for Fibrillin-2 cause Congenital Contractural Arachnodactyly. Both are autosomal dominant disorders, and both disorders affect musculoskeletal tissues. Here we show that Fbn2 null mice (on a 129/Sv background) are born with reduced muscle mass, abnormal muscle histology, and signs of activated BMP signaling in skeletal muscle. A delay in Myosin Heavy Chain 8, a perinatal myosin, was found in Fbn2 null forelimb muscle tissue, consistent with the notion that muscle defects underlie forelimb contractures in these mice. In addition, white fat accumulated in the forelimbs during the early postnatal period. Adult Fbn2 null mice are already known to demonstrate persistent muscle weakness. Here we measured elevated creatine kinase levels in adult Fbn2 null mice, indicating ongoing cycles of muscle injury. On a C57Bl/6 background, Fbn2 null mice showed severe defects in musculature, leading to neonatal death from respiratory failure. These new findings demonstrate that loss of Fibrillin-2 results in phenotypes similar to those found in congenital muscular dystrophies and that FBN2 should be considered as a candidate gene for recessive congenital muscular dystrophy. Both in vivo and in vitro evidence associated muscle abnormalities and accumulation of white fat in Fbn2 null mice with abnormally activated BMP signaling. Genetic rescue of reduced muscle mass and accumulation of white fat in Fbn2 null mice was accomplished by deleting a single allele of Bmp7. In contrast to other reports that activated BMP signaling leads to muscle hypertrophy, our findings demonstrate the exquisite sensitivity of BMP signaling to the Fibrillin-2 extracellular environment during early postnatal muscle development. New evidence presented here suggests that Fibrillin-2 can sequester BMP complexes in a latent state.

  • Establishment of Fibrillin-deficient osteoprogenitor cell lines identifies molecular abnormalities associated with extracellular matrix perturbation of osteogenic differentiation
    Cell and Tissue Research, 2011
    Co-Authors: Silvia Smaldone, Luca Carta, Francesco Ramirez
    Abstract:

    Fibrillin-1 and Fibrillin-2 are structural components of the extracellular matrix which are also involved in modulating local TGFβ and BMP bioavailability. Loss of Fibrillin-1 or Fibrillin-2 is associated with perturbed osteoblast maturation principally as the result of unbalanced TGFβ and BMP signaling. Here, we demonstrated that stable expression of small hairpin RNAs against Fibrillin-1(Fbn1) or Fibrillin-2 (Fbn2) transcripts in the clonal osteoprogenitor cell line Kusa-A1 led to the same phenotypic and molecular manifestations as germline Fbn1- or Fbn2-null mutations in primary calvarial osteoblast cultures. Proof-of-concept experiments are also presented showing that Fbn1- or Fbn2-silenced Kusa-A1 cell lines are suitable models to identify candidate determinants of osteogenesis which are under the control of extracellular microfibrils. Specific findings included: the inference of a potential role for Fibrillin-1-mediated cell–matrix interactions in regulating Kusa-A1 proliferation; the possibility of Fibrillin-2 involvement in modulating the activity of transcription factor Runx2 by restricting microRNA expression and/or processing; and the suggestion that Fibrillin-1 and Fibrillin-2 influence Notch signaling indirectly by differentially regulating BMP signaling. Collectively, the data reiterated the notion that Fibrillin-1 and Fibrillin-2 exert opposite effects on osteoblast differentiation through the discrete modulation of a broad network of interacting signaling molecules.

  • Material and mechanical properties of bones deficient for Fibrillin-1 or Fibrillin-2 microfibrils.
    Matrix Biology, 2011
    Co-Authors: Emilio Arteaga-solis, Nancy Pleshko, Lee Sui-arteaga, Mitchell B. Schaffler, Karl J. Jepsen, Francesco Ramirez
    Abstract:

    The contribution of non-collagenous components of the extracellular matrix to bone strength is largely undefined. Here we report that deficiency of Fibrillin-1 or Fibrillin-2 microfibrils causes distinct changes in bone material and mechanical properties. Morphometric examination of mice with hypomorphic or null mutations in Fibrillin-1 or Fibrillin-2, respectively, revealed appreciable differences in the postnatal shaping and growth of long bones. Fourier transform infrared imaging spectroscopy indicated that Fibrillin-1 plays a predominantly greater role than Fibrillin-2 in determining the material properties of bones. Biomechanical tests demonstrated that Fibrillin-2 exerts a greater positive influence on the mechanical properties of bone than Fibrillin-1 assemblies. Published evidence indirectly supports the notion that the above findings are mostly, if not exclusively, related to the differential control of TGFβ family signaling by Fibrillin proteins. Our study therefore advance our understanding of the role that extracellular microfibrils play in bone physiology and implicitly, in the pathogenesis of bone loss in human diseases caused by mutations in Fibrillin-1 or -2.

  • extracellular microfibrils control osteoblast supported osteoclastogenesis by restricting tgfβ stimulation of rankl production
    Journal of Biological Chemistry, 2010
    Co-Authors: Harikiran Nistala, Silvia Smaldone, Sui Leearteaga, Gabriella Siciliano, Francesco Ramirez
    Abstract:

    Mutations in Fibrillin-1 or Fibrillin-2, the major structural components of extracellular microfibrils, cause pleiotropic manifestations in Marfan syndrome and congenital contractural arachnodactyly, respectively. We recently found that Fibrillin-1 and Fibrillin-2 control bone formation by regulating osteoblast differentiation through the differential modulation of endogenous TGFβ and bone morphogenetic protein signals. Here, we describe in vivo and ex vivo experiments that implicate the Fibrillins as negative regulators of bone resorption. Adult Fbn2−/− mice display a greater than normal osteolytic response to locally implanted lipopolysaccharide-coated titanium particles. Although isolated cultures of Fbn2−/− preosteoclasts exhibited normal differentiation and activity, these features were substantially augmented when mutant or wild-type preosteoclasts were co-cultured with Fbn2−/− but not wild-type osteoblasts. Greater osteoclastogenic potential of Fbn2−/− osteoblasts was largely accounted for by up-regulation of the Rankl gene secondary to heightened TGFβ activity. This conclusion was based on the findings that blockade of TGFβ signaling blunts Rankl up-regulation in Fbn2−/− osteoblasts and bones and that systemic TGFβ antagonism improves locally induced osteolysis in Fbn2−/− mice. Abnormally high Rankl expression secondary to elevated TGFβ activity was also noted in cultured osteoblasts from Fbn1−/− mice. Collectively our data demonstrated that extracellular microfibrils balance local catabolic and anabolic signals during bone remodeling in addition to implying distinct mechanisms of bone loss in Marfan syndrome and congenital contractural arachnodactyly.

Robert P Mecham - One of the best experts on this subject based on the ideXlab platform.

  • Ocular Phenotype of Fbn2-Null MiceOcular Phenotype of Fbn2-Null Mice
    Investigative Ophthalmology & Visual Science, 2013
    Co-Authors: Yidong Tu, Robert P Mecham, Steven Bassnett
    Abstract:

    PURPOSE: Fibrillin-2 (Fbn2) is the dominant Fibrillin isoform expressed during development of the mouse eye. To test its role in morphogenesis, we examined the ocular phenotype of Fbn2(-/-) mice. METHODS: Ocular morphology was assessed by confocal microscopy using antibodies against microfibril components. RESULTS: Fbn2(-/-) mice had a high incidence of anterior segment dysgenesis. The iris was the most commonly affected tissue. Complete iridal coloboma was present in 37% of eyes. Dyscoria, corectopia and pseudopolycoria were also common (43% combined incidence). In wild-type (WT) mice, Fibrillin-2-rich microfibrils are prominent in the pupillary membrane (PM) during development. In Fbn2-null mice, the absence of Fbn2 was partially compensated for by increased expression of Fibrillin-1, although the resulting PM microfibrils were disorganized, compared with WTs. In colobomatous adult Fbn2(-/-) eyes, the PM failed to regress normally, especially beneath the notched region of the iris. Segments of the ciliary body were hypoplastic, and zonular fibers, although relatively plentiful, were unevenly distributed around the lens equator. In regions where the zonular fibers were particularly disturbed, the synchronous differentiation of the underlying lens fiber cells was affected. CONCLUSIONS: Fbn2 has an indispensable role in ocular morphogenesis in mice. The high incidence of iris coloboma in Fbn2-null animals implies a previously unsuspected role in optic fissure closure. The observation that fiber cell differentiation was disturbed in Fbn2(-/-) mice raises the possibility that the attachment of zonular fibers to the lens surface may help specify the equatorial margin of the lens epithelium.

  • essential role for Fibrillin 2 in zebrafish notochord and vascular morphogenesis
    Developmental Dynamics, 2008
    Co-Authors: John M Gansner, Erik C Madsen, Robert P Mecham, Jonathan D Gitlin
    Abstract:

    Recent studies demonstrate that lysyl oxidase cuproenzymes are critical for zebrafish notochord formation, but the molecular mechanisms of copper-dependent notochord morphogenesis are incompletely understood. We, therefore, conducted a forward genetic screen for zebrafish mutants that exhibit notochord sensitivity to lysyl oxidase inhibition, yielding a mutant with defects in notochord and vascular morphogenesis, puff daddygw1 (pfdgw1). Meiotic mapping and cloning reveal that the pfdgw1 phenotype results from disruption of the gene encoding the extracellular matrix protein Fibrillin-2, and the spatiotemporal expression of Fibrillin-2 is consistent with the pfdgw1 phenotype. Furthermore, each aspect of the pfdgw1 phenotype is recapitulated by morpholino knockdown of Fibrillin-2. Taken together, the data reveal a genetic interaction between Fibrillin-2 and the lysyl oxidases in notochord formation and demonstrate the importance of Fibrillin-2 in specific early developmental processes in zebrafish. Developmental Dynamics 237:2844–2861, 2008. © 2008 Wiley-Liss, Inc.

  • identification of a major microfibril associated glycoprotein 1 binding domain in Fibrillin 2
    Journal of Biological Chemistry, 2004
    Co-Authors: Claudio C Werneck, Timothy M Ritty, Barbara Crippes Trask, Thomas J Broekelmann, Timothy M Trask, Fernando Segade, Robert P Mecham
    Abstract:

    Using yeast two-hybrid, ligand blotting, and solid phase binding assays, we have shown that microfibrilassociated glycoprotein-1 (MAGP-1) interacts with the 8-cysteine motif of Fibrillin-2 encoded by exon 24. Binding to this sequence was demonstrated for full-length MAGP-1 as well as for the MAGP-1 matrix-binding domain encoded by exons 7 and 8. The matrix-binding domain, but not the full-length protein, also bound to regions of Fibrillin-2 defined by exons 16 and 17, exon 20, and exons 23 and 24. Interestingly, no binding was detected to sequences near the N or C terminus where MAGP-1 and MAGP-2, respectively, were shown to interact with Fibrillin-1. The localization of MAGP-1 binding to the 8-Cys domain encoded by exon 24 suggests that the bead structure of microfibrils consists of exon 24 and portions of the central region of Fibrillin-2. Exon 24 in Fibrillin lies in the region of the molecule where mutations produce the most severe phenotypes associated with Marfan syndrome (Fibrillin-1) and congenital contractural arachnodactyly (Fibrillin-2). It is possible that these mutations alter the ability of Fibrillin to bind MAGP-1, which may contribute to the severity of the disease.

  • Fibrillin 2 defects impair elastic fiber assembly in a homocysteinemic chick model
    Journal of Nutrition, 2002
    Co-Authors: C H Hill, Robert P Mecham, Barry Starcher
    Abstract:

    Homocysteinemia in humans is associated with vascular complications that increase the risk for atherosclerosis and stroke. Animal studies have shown that the disease is multifactorial and includes lesions associated with the elastin component of the extracellular matrix. In the following experiments we have used the aortas from rapidly growing chicks to assess the cause of the elastin defects resulting from homocysteinemia. Day-old chicks were fed diets containing varying amounts of DL-methionine, DL-homocysteine, homocysteine thiolactone or DL-cysteine for periods up to 9 wk. Three weeks after feeding 2% DL-methionine the plasma methionine was elevated 20-fold, whereas plasma homocysteine was more than 3-fold normal plasma values. The aortas showed severe histopathology, evidenced by the pronounced separation of elastic lamellae with marked smooth muscle proliferation and, in some instances, aneurysms. There was no evidence of decreased desmosine content or a significant reduction in lysyl oxidase in the aortas from the treated groups compared to those from controls. Increasing other dietary factors such as the vitamins required for methionine metabolism had no effect on the development of the vascular lesions. Twenty to 30% of the chicks fed the high methionine diets exhibited severe neurological problems, expressed as tonic contractions or seizures. Electron microscopy revealed disordered aortic elastic fibrils, associated with either an absence of or disrupted assembly of microfibrils. Immunohistochemical studies demonstrated a loss of Fibrillin-2 immunoreactivity in the aortas of chicks fed 2% methionine. The studies suggest that elevated plasma methionine or its metabolites disrupt normal microfibril configuration, leading to the assembly of aberrant elastic fibers. J. Nutr. 132: 2143-2150, 2002.

  • interaction of tropoelastin with the amino terminal domains of Fibrillin 1 and Fibrillin 2 suggests a role for the Fibrillins in elastic fiber assembly
    Journal of Biological Chemistry, 2000
    Co-Authors: Timothy M Trask, Timothy M Ritty, Barbara Crippes Trask, Joel Rosenbloom, William R Abrams, Robert P Mecham
    Abstract:

    Alignment of tropoelastin molecules during the process of elastogenesis is thought to require Fibrillin-containing microfibrils. In this study, we have demonstrated that amino-terminal domains of two microfibrillar proteins, Fibrillin-1 and Fibrillin-2, interact with tropoelastin in solid phase binding assays. The tropoelastin-binding site was localized to a region beginning at the glycine-rich and proline-rich regions of Fibrillin-2 and Fibrillin-1, respectively, and continuing through the second 8-cysteine domain. Characterization of the binding requirements using the Fibrillin-2 construct found that a folded, secondary structure was necessary for binding. Furthermore, binding between tropoelastin and Fibrillin was mediated by ionic interactions involving the lysine side chains of tropoelastin. The importance of the lysine side chains was corroborated by the finding that the Fibrillin-2 construct did not bind to mature elastin, whose lysine side chains have been modified to form cross-links. Interestingly, there was no interaction between the Fibrillin constructs and tropoelastin in solution phase, suggesting that binding of tropoelastin to a solid substrate exposes a cryptic binding site. These results suggest that Fibrillin plays an important role in elastic fiber assembly by binding tropoelastin and perhaps facilitating side chain alignment for efficient cross-linking.

Kazuharu Irie - One of the best experts on this subject based on the ideXlab platform.

  • Expression of Fibrillins and tropoelastin by human gingival and periodontal ligament fibroblasts in vitro.
    Journal of Periodontal Research, 2020
    Co-Authors: Eichi Tsuruga, Kazuharu Irie, Yasunori Sakakura, Toshihiko Yajima
    Abstract:

    : The elastic system fibers consist of three different types, oxytalan, elaunin and elastic fibers, which differ in the relative content of microfibrils and elastin. In periodontal tissues, oxytalan fibers are known to be distributed in the periodontal ligament and gingiva, while elaunin and elastic fibers are present only in the gingiva. We examined the in vitro synthesis of microfibrils and elastin by human gingival fibroblasts (HGF) and periodontal ligament fibroblasts (HPLF). The two kinds of HGF and HPLF were cultured in MEM containing 10% newborn calf serum for 30 days. Since Fibrillin-1 and Fibrillin-2 are the major components of microfibrils involved in elastogenesis, we investigated the synthesis of Fibrillins and tropoelastin in the conditioned medium of HGF and HPLF. Western blot analysis revealed Fibrillin-1 and Fibrillin-2 to occur in the HGF and HPLF culture medium, HGF exhibiting a higher level of synthesis than HPLF. Tropoelastin, on the other hand, was detected only in the medium of HGF after day 24. In addition, analysis of RNA extracted from HGF and HPLF on day 30 showed that only HGF expressed mRNA encoding tropoelastin. Immunohistochemically, accumulation of tropoelastin in the perinuclear area was found only in HGF. These results show that HGF expressed microfibrils and elastin, while HPLF expressed only microfibrils for the experimental period, and suggest a biochemical basis for the different distribution of elastic system fibers of the gingiva and periodontal ligament in vivo.

  • Fibrillin 2 degradation by matrix metalloproteinase 2 in periodontium
    Journal of Dental Research, 2007
    Co-Authors: Eichi Tsuruga, Kazuharu Irie, Toshihiko Yajima
    Abstract:

    Elastic system fibers, comprised of microfibrils and tropoelastin, are extracellular components of periodontal tissue. During development, the microfibrils act as a template on which tropoelastin is deposited. However, the process of elastic system fiber remodeling is not fully understood. Therefore, we examined whether matrix metalloproteinases (MMPs) are involved in the remodeling of Fibrillins (major components of microfibrils) by human gingival fibroblasts and periodontal ligament (PDL) fibroblasts. Gingival and PDL fibroblasts were cultured for 6 weeks. In some cultures, MMP inhibitor or tissue inhibitor of matrix metalloproteinsase-2 (TIMP-2) was added to the medium for an additional 2 weeks. Active MMP-2 (62 kDa) appeared as cell-membrane-associated or in extracellular matrix only in PDL fibroblast cell layers. The addition of MMP inhibitor or TIMP-2 significantly increased Fibrillin-2 accumulation in PDL fibroblast cell layers, and decreased the amount of Fibrillin-2 fragments, suggesting that act...

  • microfibril associated glycoprotein 1 and Fibrillin 2 are associated with tropoelastin deposition in vitro
    The International Journal of Biochemistry & Cell Biology, 2005
    Co-Authors: Eichi Tsuruga, Toshihiko Yajima, Kazuharu Irie
    Abstract:

    Elastic system fibers consist of microfibrils and tropoelastin. During development, microfibrils act as a template on which tropoelastin is deposited. Microfibril-associated glycoprotein-1 (MAGP-1) and Fibrillin-2, the major components of microfibrils, provide the likely template for tropoelastin deposition. In this study, we used the RNA interference (RNAi) technique to establish MAGP-1 and Fibrillin-2 gene-specific knock-downs individually in elastin-producing cells (human gingival fibroblasts). We then examined the extracellular deposition of tropoelastin by western blotting. These two genes were specifically suppressed to <30% of the control level, and this was responsible for the diminution of tropoelastin deposition. An immunofluorescence study also confirmed that RNAi-mediated down-regulation of MAGP-1 or Fibrillin-2 led to the loss of tropoelastin immunoreactivity. These results suggest that MAGP-1 and Fibrillin-2 are, directly or indirectly, associated with the extracellular deposition of tropoelastin during elastic fiber formation in human gingival fibroblasts in vitro.

  • Microfibril-associated glycoprotein-1 and Fibrillin-2 are associated with tropoelastin deposition in vitro
    The International Journal of Biochemistry & Cell Biology, 2004
    Co-Authors: Eichi Tsuruga, Toshihiko Yajima, Kazuharu Irie
    Abstract:

    Elastic system fibers consist of microfibrils and tropoelastin. During development, microfibrils act as a template on which tropoelastin is deposited. Microfibril-associated glycoprotein-1 (MAGP-1) and Fibrillin-2, the major components of microfibrils, provide the likely template for tropoelastin deposition. In this study, we used the RNA interference (RNAi) technique to establish MAGP-1 and Fibrillin-2 gene-specific knock-downs individually in elastin-producing cells (human gingival fibroblasts). We then examined the extracellular deposition of tropoelastin by western blotting. These two genes were specifically suppressed to

  • gene expression and accumulation of Fibrillin 1 Fibrillin 2 and tropoelastin in cultured periodontal fibroblasts
    Journal of Dental Research, 2002
    Co-Authors: Eichi Tsuruga, Kazuharu Irie, Toshihiko Yajima
    Abstract:

    The elastic system fibers consist of three types—oxytalan, elaunin, and elastic fibers—differing in their relative microfibril and elastin contents. All three types are found in human gingiva, but human periodontal ligaments contain only elastin-free fibers. We examined cultured human gingival fibroblasts (HGF) and cultured human periodontal ligament fibroblasts (HPLF) to determine the gene expression of Fibrillin-1 and Fibrillin-2 (the major components of microfibrils) and of tropoelastin. In addition, we assessed the degree of accumulation of these proteins in the extracellular matrix. Northern blot analysis revealed that the level of expression of Fibrillin-1 and Fibrillin-2 was higher in HGF than in HPLF. However, examination of matrix samples from HGF and HPLF cell layers showed that there was no difference in Fibrillin-1 accumulation, although Fibrillin-2 accumulated to a much greater extent in the HGF-derived matrix. Tropoelastin was expressed only in and around HGF. These results show a correlatio...

Timothy M Ritty - One of the best experts on this subject based on the ideXlab platform.

  • bone and soft connective tissue alterations result from loss of Fibrillin 2 expression
    Matrix Biology, 2008
    Co-Authors: Rajeev K Boregowda, Emmanuel M Paul, Jason D White, Timothy M Ritty
    Abstract:

    Fibrillins 1, 2 and 3 make up a family of genes that encode large, cysteine-rich extracellular matrix glycoproteins found in connective tissues, lung, blood vessels and other extensible tissues. Fibrillins 1 and 2 have both overlapping as well as separate distributions in human embryonic and adult tissues. Fibrillin-containing microfibrils are known to modulate morphogenetic events by proper targeting of growth factors to the extracellular matrix. Mutation of the Fibrillin-2 gene causes a genetic disorder, congenital contractural arachnodactyly (CCA), that results in flexion contractures. Previously, we have shown a distinct Fibrillin-2 distribution in the pericellular matrix of interior tenocytes and later demonstrated a unique Fibrillin-2 containing structure that runs along the tendon cell arrays in the canine flexor tendon. We hypothesized that loss of these Fibrillin-2 containing structures might affect normal tendon development. To test our hypothesis, connective tissues from mice null for Fibrillin-2 gene expression were studied. Murine flexor digitorum longus tendons were evaluated for total collagen content, and the intermolecular collagen cross-links hydroxylysyl and lysyl pyridinoline. The results show decreased collagen cross-links in Fibrillin-2 null mice, however total collagen content remained the same when compared to wild type. Bone morphology was studied using micro computed tomography (CT). Fibrillin-2 null mice display a focal area of decreased bone length in the extremities as compared to wild type mice. Together, these results demonstrate a role for Fibrillin-2 in bone and soft connective tissue morphological and biochemical processes.

  • identification of a major microfibril associated glycoprotein 1 binding domain in Fibrillin 2
    Journal of Biological Chemistry, 2004
    Co-Authors: Claudio C Werneck, Timothy M Ritty, Barbara Crippes Trask, Thomas J Broekelmann, Timothy M Trask, Fernando Segade, Robert P Mecham
    Abstract:

    Using yeast two-hybrid, ligand blotting, and solid phase binding assays, we have shown that microfibrilassociated glycoprotein-1 (MAGP-1) interacts with the 8-cysteine motif of Fibrillin-2 encoded by exon 24. Binding to this sequence was demonstrated for full-length MAGP-1 as well as for the MAGP-1 matrix-binding domain encoded by exons 7 and 8. The matrix-binding domain, but not the full-length protein, also bound to regions of Fibrillin-2 defined by exons 16 and 17, exon 20, and exons 23 and 24. Interestingly, no binding was detected to sequences near the N or C terminus where MAGP-1 and MAGP-2, respectively, were shown to interact with Fibrillin-1. The localization of MAGP-1 binding to the 8-Cys domain encoded by exon 24 suggests that the bead structure of microfibrils consists of exon 24 and portions of the central region of Fibrillin-2. Exon 24 in Fibrillin lies in the region of the molecule where mutations produce the most severe phenotypes associated with Marfan syndrome (Fibrillin-1) and congenital contractural arachnodactyly (Fibrillin-2). It is possible that these mutations alter the ability of Fibrillin to bind MAGP-1, which may contribute to the severity of the disease.

  • tendon cell array isolation reveals a previously unknown Fibrillin 2 containing macromolecular assembly
    Structure, 2003
    Co-Authors: Timothy M Ritty, Robyn Roth, John E Heuser
    Abstract:

    Within tendon, between collagen fascicles, cells are organized in linear arrays surrounded by a specialized environment of extracellular matrix (ECM) proteins that are largely unidentified. Our goal was to identify interfascicular, pericellular ECM components and provide additional resolution to the organization of the pericellular matrix. To this end, we employed a combination of enzymatic digestion, mechanical disruption, and differential sedimentation to demonstrate for the first time that it possible to liberate living linear tendon cell arrays from whole tendon. Here, we identify type VI collagen, versican, and Fibrillin-2 as components of the immediate pericellular ECM of linearly arrayed tendon cells. Additionally, a unique Fibrillin-2-containing macromolecular assembly is described in detail for the first time. This new structure is unlike any previously described Fibrillin-containing macromolecular assembly. Having a largely constant diameter, it runs axially along tendon cell arrays and can exceed 1000 microm in length.

  • interaction of tropoelastin with the amino terminal domains of Fibrillin 1 and Fibrillin 2 suggests a role for the Fibrillins in elastic fiber assembly
    Journal of Biological Chemistry, 2000
    Co-Authors: Timothy M Trask, Timothy M Ritty, Barbara Crippes Trask, Joel Rosenbloom, William R Abrams, Robert P Mecham
    Abstract:

    Alignment of tropoelastin molecules during the process of elastogenesis is thought to require Fibrillin-containing microfibrils. In this study, we have demonstrated that amino-terminal domains of two microfibrillar proteins, Fibrillin-1 and Fibrillin-2, interact with tropoelastin in solid phase binding assays. The tropoelastin-binding site was localized to a region beginning at the glycine-rich and proline-rich regions of Fibrillin-2 and Fibrillin-1, respectively, and continuing through the second 8-cysteine domain. Characterization of the binding requirements using the Fibrillin-2 construct found that a folded, secondary structure was necessary for binding. Furthermore, binding between tropoelastin and Fibrillin was mediated by ionic interactions involving the lysine side chains of tropoelastin. The importance of the lysine side chains was corroborated by the finding that the Fibrillin-2 construct did not bind to mature elastin, whose lysine side chains have been modified to form cross-links. Interestingly, there was no interaction between the Fibrillin constructs and tropoelastin in solution phase, suggesting that binding of tropoelastin to a solid substrate exposes a cryptic binding site. These results suggest that Fibrillin plays an important role in elastic fiber assembly by binding tropoelastin and perhaps facilitating side chain alignment for efficient cross-linking.

  • n terminal domains of Fibrillin 1 and Fibrillin 2 direct the formation of homodimers a possible first step in microfibril assembly
    Biochemical Journal, 1999
    Co-Authors: Timothy M Trask, Thomas J Broekelmann, Timothy M Ritty, Clarina Tisdale, Robert P Mecham
    Abstract:

    Aggregation of Fibrillin molecules via disulphide bonds is postulated to be an early step in microfibril assembly. By expressing fragments of Fibrillin 1 and Fibrillin 2 in a mammalian expression system, we found that the N-terminal region of each protein directs the formation of homodimers and that disulphide bonds stabilize this interaction. A large fragment of Fibrillin 1 containing much of the region downstream from the N-terminus remained as a monomer when expressed in the same cell system, indicating that this region of the protein lacks dimerization domains. This finding also confirms that the overexpression of Fibrillin fragments does not in itself lead to spurious dimer formation. Pulse-chase analysis demonstrated that dimer formation occurred intracellularly, suggesting that the process of Fibrillin aggregation is initiated early after biosynthesis of the molecules. These findings also implicate the N-terminal region of Fibrillin 1 and Fibrillin 2 in directing the formation of a dimer intermediate that aggregates to form the functional microfibril.