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    ABSTRACT: Realizing the full therapeutic potential of mesenchymal stromal/stem cells (MSCs) awaits improved understanding of mechanisms controlling their fate. Using MSCs cultured as spheroids to recapitulate a three-dimensional cellular environment, we show that perturbing the mesenchymal regulators, platelet-derived growth factor (PDGF) receptors or fibronectin, reverts MSCs towards mesodermal progenitors with endothelial potential that can potently induce neovascularization in vivo. MSCs within untreated spheroids retain their mesenchymal spindle-shape with abundant smooth muscle alpha-actin filaments and fibronectin-rich matrix. Inhibiting PDGF receptors or depleting fibronectin induces rounding and depletes smooth muscle alpha-actin expression; these cells have characteristics of mesenchymoangioblasts, with enhanced expression of mesendoderm and endoderm transcription factors, prominent up-regulation of E-cadherin, and Janus kinase signaling-dependent expression of Oct4A and Nanog. PDGF receptor-inhibited spheroids also upregulate endothelial markers PECAM-1 and VE-cadherin and secrete many angiogenic factors, and in vivo they potently stimulate neovascularization, and their MSCs integrate within functional blood vessels that are perfused by the circulation. Thus MSC potency and vascular induction are regulated by perturbing mesenchymal fate. Stem Cells 2013.
    Stem Cells 03/2014; 32(3). DOI:10.1002/stem.1538
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    ABSTRACT: Collagen fibrils can exceed thousands of microns in length and are therefore the longest, largest, and most size-pleomorphic protein polymers in vertebrates; thus, knowing how cells transport collagen fibrils is essential for a more complete understanding of protein transport and its role in tissue morphogenesis. Here, we identified newly formed collagen fibrils being transported at the surface of embryonic tendon cells in vivo by using serial block face-scanning electron microscopy of the cell-matrix interface. Newly formed fibrils ranged in length from ∼1 to ∼30 µm. The shortest (1-10 µm) occurred in intracellular fibricarriers; the longest (∼30 µm) occurred in plasma membrane fibripositors. Fibrils and fibripositors were reduced in numbers when collagen secretion was blocked. ImmunoEM showed the absence of lysosomal-associated membrane protein 2 on fibricarriers and fibripositors and there was no effect of leupeptin on fibricarrier or fibripositor number and size, suggesting that fibricarriers and fibripositors are not part of a fibril degradation pathway. Blebbistatin decreased fibricarrier number and increased fibripositor length; thus, nonmuscle myosin II (NMII) powers the transport of these compartments. Inhibition of dynamin-dependent endocytosis with dynasore blocked fibricarrier formation and caused accumulation of fibrils in fibripositors. Data from fluid-phase HRP electron tomography showed that fibricarriers could originate at the plasma membrane. We propose that NMII-powered transport of newly formed collagen fibrils at the plasma membrane is fundamental to the development of collagen fibril-rich tissues. A NMII-dependent cell-force model is presented as the basis for the creation and dynamics of fibripositor structures.
    Proceedings of the National Academy of Sciences 11/2013; 110(49). DOI:10.1073/pnas.1314348110
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    ABSTRACT: Integrins are adhesion receptors that allow cells to sense and respond to microenvironmental signals encoded by the extracellular matrix. They are crucial for the adhesion, survival, proliferation, differentiation and migration of most cell types. In cell cycle regulation, integrin-mediated signals from the local niche constitute a spatial checkpoint to allow cells to progress from G1 to S phase, and are as important as temporal growth factors signals. Proliferation is altered in diseases such as cancer and fibrosis, so understanding how integrins contribute to this process will provide novel strategies for therapy. Here we consider recent studies to elucidate mechanisms of integrin-dependent cell cycle progression and discuss perspectives for future study.
    Matrix biology: journal of the International Society for Matrix Biology 10/2013; 34. DOI:10.1016/j.matbio.2013.10.011
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    ABSTRACT: Disease mechanisms leading to different forms of chondrodysplasia include extracellular matrix (ECM) alterations and intracellular stress leading to abnormal changes to chondrocyte proliferation and survival. Delineating the relative contribution of these two disease mechanisms is a major challenge in understanding disease pathophysiology in genetic skeletal diseases and a prerequisite for developing effective therapies. To determine the influence of intracellular stress and changes in chondrocyte phenotype to the development of chondrodysplasia we targeted the expression of the G2320R mutant form of thyroglobulin to the endoplasmic reticulum (ER) of resting and proliferating chondrocytes. Previous studies on this mutant protein have shown that it induces intracellular aggregates and causes cell stress and death in the thyroid gland. The expression and retention of this exogenous mutant protein in resting and proliferating chondrocytes resulted in a chronic cell stress response, growth plate dysplasia and reduced bone growth without inducing any alterations to the architecture and organization of the cartilage ECM. More significantly, the decreased bone growth appeared to be the direct result of reduced chondrocyte proliferation in the proliferative zone of transgenic mice growth plates without transcriptional activation of a classical unfolded protein response (UPR) or apoptosis. Overall, these data show that mutant protein retention in the ER of resting and proliferative zone chondrocytes is sufficient to cause disrupted bone growth. The specific disease pathways triggered by mutant protein retention do not necessarily involve a prototypic UPR, but all pathways impact upon chondrocyte proliferation in the cartilage growth plate.
    Disease Models and Mechanisms 09/2013; 6(6). DOI:10.1242/dmm.013342
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    ABSTRACT: Inhibition of αvβ3 or expression of mutant p53 promotes invasion into fibronectin (FN)-containing extracellular matrix (ECM) by enhancing Rab-coupling protein (RCP)-dependent recycling of α5β1 integrin. RCP and α5β1 cooperatively recruit receptor tyrosine kinases, including EGFR1, to regulate their trafficking and downstream signaling via protein kinase B (PKB)/Akt, which, in turn, promotes invasive migration. In this paper, we identify a novel PKB/Akt substrate, RacGAP1, which is phosphorylated as a consequence of RCP-dependent α5β1 trafficking. Phosphorylation of RacGAP1 promotes its recruitment to IQGAP1 at the tips of invasive pseudopods, and RacGAP1 then locally suppresses the activity of the cytoskeletal regulator Rac and promotes the activity of RhoA in this subcellular region. This Rac to RhoA switch promotes the extension of pseudopodial processes and invasive migration into FN-containing matrices, in a RhoA-dependent manner. Thus, the localized endocytic trafficking of α5β1 within the tips of invasive pseudopods elicits signals that promote the reorganization of the actin cytoskeleton, protrusion, and invasion into FN-rich ECM.
    The Journal of Cell Biology 09/2013; 202(6). DOI:10.1083/jcb.201302041
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    ABSTRACT: Pseudoachondroplasia and multiple epiphyseal dysplasia are genetic skeletal diseases resulting from mutations in cartilage structural proteins. Electron microscopy and immunohistochemistry previously showed that the appearance of the cartilage extracellular matrix (ECM) in targeted mouse models of these diseases is disrupted; however, the precise changes in ECM organization and the pathological consequences remain unknown. Our aim was to determine the effects of matrilin-3 and COMP mutations on the composition and extractability of ECM components to inform how these detrimental changes might influence cartilage organization and degeneration. Cartilage was sequentially extracted using increasing denaturants and the extraction profiles of specific proteins determined using SDS-PAGE/Western blotting. Furthermore, the relative composition of protein pools was determined using mass spectrometry for a non-biased semi-quantitative analysis. Western blotting revealed changes in the extraction of matrilins, COMP and collagen IX in mutant cartilage. Mass spectrometry confirmed quantitative changes in the extraction of structural and non-structural ECM proteins, including proteins with roles in cellular processes such as protein folding and trafficking. In particular, genotype-specific differences in the extraction of collagens XII and XIV and tenascins C and X were identified; interestingly, increased expression of several of these genes has recently been implicated in susceptibility and/or progression of murine osteoarthritis. We demonstrated that mutation of matrilin-3 and COMP caused changes in the extractability of other cartilage proteins and that proteomic analyses of Matn3 V194D, Comp T585M and Comp DelD469 mouse models revealed both common and discrete disease signatures that provide novel insight into skeletal disease mechanisms and cartilage degradation.
    08/2013; 2(8):802-11. DOI:10.1242/bio.20135280
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    ABSTRACT: Mutant matrilin-3 (V194D) forms non-native disulphide bonded aggregates in the rER of chondrocytes from cell and mouse models of multiple epiphyseal dysplasia (MED). Intracellular retention of mutant matrilin-3 causes ER stress and induces an unfolded protein response (UPR) including the upregulation of two genes recently implicated in ER stress: Armet and Creld2. Nothing is known about the role of Armet and Creld2 in human genetic diseases.In this study we used a variety of cell and mouse models of chondrodysplasia to determine the genotype-specific expression profiles of Armet and Creld2. We also studied their interactions with various mutant proteins and investigated their potential roles as protein disulphide isomerases (PDI).Armet and Creld2 were up-regulated in cell and/or mouse models of chondrodysplasias caused by mutations in Matn3 and Col10a1, but not Comp. Intriguingly, both Armet and Creld2 were also secreted into the ECM of these disease models following ER stress. Armet and Creld2 interacted with mutant matrilin-3, but not with COMP, thereby validating the genotype-specific expression. Substrate trapping experiments confirmed Creld2 processed PDI-like activity, thus identifying a putative functional role. Finally, alanine substitution of the two terminal cysteine residues from the A-domain of V194D matrilin-3 prevented aggregation, promoted mutant protein secretion and reduced the levels of Armet and Creld2 in a cell culture model.We demonstrate that Armet and Creld2 are genotype-specific ER stress response proteins with substrate specificities and that aggregation of mutant matrilin-3 is a key disease trigger in MED that could be exploited as a potential therapeutic target.
    Human Molecular Genetics 08/2013; 22(25). DOI:10.1093/hmg/ddt383
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    ABSTRACT: This article explores new ideas about how the ECM-integrin axis controls normal and malignant breast biology. We discuss the role of integrins in mammary stem cells, and how cell-matrix interactions regulate ductal and alveolar development and function. We also examine the contribution of integrins to tissue disorganisation and metastasis, and how an altered stromal and ECM tumour microenvironment affects the cancer cell niche both within primary tumours and at distant sites. Finally, we mention novel strategies for integrin-directed breast cancer treatment.
    Current opinion in cell biology 07/2013; DOI:10.1016/j.ceb.2013.06.010
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    ABSTRACT: Collagen fibrils are the major tensile element in vertebrate tissues, in which they occur as ordered bundles in the extracellular matrix. Abnormal fibril assembly and organization results in scarring, fibrosis, poor wound healing and connective tissue diseases. Transmission electron microscopy (TEM) is used to assess the formation of the fibrils, predominantly by measuring fibril diameter. Here we describe a protocol for measuring fibril diameter as well as fibril volume fraction, mean fibril length, fibril cross-sectional shape and fibril 3D organization, all of which are major determinants of tissue function. Serial-section TEM (ssTEM) has been used to visualize fibril 3D organization in vivo. However, serial block face-scanning electron microscopy (SBF-SEM) has emerged as a time-efficient alternative to ssTEM. The protocol described below is suitable for preparing tissues for TEM and SBF-SEM (by 3View). We describe how to use 3View for studying collagen fibril organization in vivo and show how to find and track individual fibrils. The overall time scale is ∼8 d from isolating the tissue to having a 3D image stack.
    Nature Protocol 06/2013; 8(7):1433-1448. DOI:10.1038/nprot.2013.086
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    ABSTRACT: This review discusses recent advances in our understanding of adhesion receptor trafficking in vitro, and extrapolates them as far as what is currently possible towards an understanding of migration in three dimensions in vivo. Our specific focus is the mechanisms for endocytosis and recycling of the two major classes of cell-matrix adhesion receptors, integrins and syndecans. We review the signalling networks that are employed to regulate trafficking and conversely the effects of trafficking on signalling itself. We then define the contribution that this element of the migration process makes to processes such as wound healing and tumour invasion.
    Current opinion in cell biology 06/2013; 25(5). DOI:10.1016/j.ceb.2013.05.008
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