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Calligaris M, Spanò DP, Puccio MC, Müller SA, Bonelli S, Lo Pinto M, Zito G, Blobel CP, Lichtenthaler SF, Troeberg L, Scilabra SD. Development of a Proteomic Workflow for the Identification of Heparan Sulphate Proteoglycan-Binding Substrates of ADAM17. Proteomics 2024; 24:e202400076. [PMID: 39318062 DOI: 10.1002/pmic.202400076] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2024] [Revised: 08/29/2024] [Accepted: 08/29/2024] [Indexed: 09/26/2024]
Abstract
Ectodomain shedding, which is the proteolytic release of transmembrane proteins from the cell surface, is crucial for cell-to-cell communication and other biological processes. The metalloproteinase ADAM17 mediates ectodomain shedding of over 50 transmembrane proteins ranging from cytokines and growth factors, such as TNF and EGFR ligands, to signalling receptors and adhesion molecules. Yet, the ADAM17 sheddome is only partly defined and biological functions of the protease have not been fully characterized. Some ADAM17 substrates (e.g., HB-EGF) are known to bind to heparan sulphate proteoglycans (HSPG), and we hypothesised that such substrates would be under-represented in traditional secretome analyses, due to their binding to cell surface or pericellular HSPGs. Thus, to identify novel HSPG-binding ADAM17 substrates, we developed a proteomic workflow that involves addition of heparin to solubilize HSPG-binding proteins from the cell layer, thereby allowing their mass spectrometry detection by heparin-treated secretome (HEP-SEC) analysis. Applying this methodology to murine embryonic fibroblasts stimulated with an ADAM17 activator enabled us to identify 47 transmembrane proteins that were shed in response to ADAM17 activation. This included known HSPG-binding ADAM17 substrates (i.e., HB-EGF, CX3CL1) and 14 novel HSPG-binding putative ADAM17 substrates. Two of these, MHC-I and IL1RL1, were validated as ADAM17 substrates by immunoblotting.
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Affiliation(s)
- Matteo Calligaris
- Department of Research IRCCS ISMETT (Istituto Mediterraneo per i Trapianti e Terapie ad Alta Specializzazione), Proteomics Group of Ri.MED Foundation, Palermo, Italy
- Department of Pharmacy, University of Pisa, Pisa, Italy
| | - Donatella Pia Spanò
- Department of Research IRCCS ISMETT (Istituto Mediterraneo per i Trapianti e Terapie ad Alta Specializzazione), Proteomics Group of Ri.MED Foundation, Palermo, Italy
- STEBICEF (Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche), Università degli Studi di Palermo, Palermo, Italy
| | - Maria Chiara Puccio
- Department of Research IRCCS ISMETT (Istituto Mediterraneo per i Trapianti e Terapie ad Alta Specializzazione), Proteomics Group of Ri.MED Foundation, Palermo, Italy
| | - Stephan A Müller
- Neuroproteomics Department, German Center for Neurodegenerative Diseases (DZNE), Munich, Germany
| | - Simone Bonelli
- Department of Research IRCCS ISMETT (Istituto Mediterraneo per i Trapianti e Terapie ad Alta Specializzazione), Proteomics Group of Ri.MED Foundation, Palermo, Italy
- STEBICEF (Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche), Università degli Studi di Palermo, Palermo, Italy
| | - Margot Lo Pinto
- Department of Research IRCCS ISMETT (Istituto Mediterraneo per i Trapianti e Terapie ad Alta Specializzazione), Proteomics Group of Ri.MED Foundation, Palermo, Italy
| | - Giovanni Zito
- Department of Research, IRCCS ISMETT (Istituto Mediterraneo per i Trapianti e Terapie ad Alta Specializzazione), Palermo, Italy
| | - Carl P Blobel
- Arthritis and Tissue Degeneration Program, Hospital for Special Surgery, New York, USA
- School of Medicine, Technical University Munich, Munich, Germany
- Department of Biochemistry, Cell and Molecular Biology, Weill Cornell Medicine, New York, USA
| | - Stefan F Lichtenthaler
- Neuroproteomics Department, German Center for Neurodegenerative Diseases (DZNE), Munich, Germany
- School of Medicine, Technical University Munich, Munich, Germany
- Neuroproteomics, School of Medicine, Klinikum rechts der Isar, Technische Universität München, Munich, Germany
- Munich Cluster for Systems Neurology (SyNergy), Munich, Germany
| | - Linda Troeberg
- Norwich Medical School, University of East Anglia, Norwich, UK
| | - Simone Dario Scilabra
- Department of Research IRCCS ISMETT (Istituto Mediterraneo per i Trapianti e Terapie ad Alta Specializzazione), Proteomics Group of Ri.MED Foundation, Palermo, Italy
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The extracellular matrix of hematopoietic stem cell niches. Adv Drug Deliv Rev 2022; 181:114069. [PMID: 34838648 PMCID: PMC8860232 DOI: 10.1016/j.addr.2021.114069] [Citation(s) in RCA: 36] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2021] [Revised: 11/18/2021] [Accepted: 11/21/2021] [Indexed: 12/21/2022]
Abstract
Comprehensive overview of different classes of ECM molecules in the HSC niche. Overview of current knowledge on role of biophysics of the HSC niche. Description of approaches to create artificial stem cell niches for several application. Importance of considering ECM in drug development and testing. Hematopoietic stem cells (HSCs) are the life-long source of all types of blood cells. Their function is controlled by their direct microenvironment, the HSC niche in the bone marrow. Although the importance of the extracellular matrix (ECM) in the niche by orchestrating niche architecture and cellular function is widely acknowledged, it is still underexplored. In this review, we provide a comprehensive overview of the ECM in HSC niches. For this purpose, we first briefly outline HSC niche biology and then review the role of the different classes of ECM molecules in the niche one by one and how they are perceived by cells. Matrix remodeling and the emerging importance of biophysics in HSC niche function are discussed. Finally, the application of the current knowledge of ECM in the niche in form of artificial HSC niches for HSC expansion or targeted differentiation as well as drug testing is reviewed.
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Cleavage of the Perlecan-Semaphorin 3A-Plexin A1-Neuropilin-1 (PSPN) Complex by Matrix Metalloproteinase 7/Matrilysin Triggers Prostate Cancer Cell Dyscohesion and Migration. Int J Mol Sci 2021; 22:ijms22063218. [PMID: 33809984 PMCID: PMC8004947 DOI: 10.3390/ijms22063218] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2021] [Revised: 03/15/2021] [Accepted: 03/15/2021] [Indexed: 12/11/2022] Open
Abstract
The Perlecan-Semaphorin 3A-Plexin A1-Neuropilin-1 (PSPN) Complex at the cell surface of prostate cancer (PCa) cells influences cell–cell cohesion and dyscohesion. We investigated matrix metalloproteinase-7/matrilysin (MMP-7)’s ability to digest components of the PSPN Complex in bone metastatic PCa cells using in silico analyses and in vitro experiments. Results demonstrated that in addition to the heparan sulfate proteoglycan, perlecan, all components of the PSPN Complex were degraded by MMP-7. To investigate the functional consequences of PSPN Complex cleavage, we developed a preformed microtumor model to examine initiation of cell dispersion after MMP-7 digestion. We found that while perlecan fully decorated with glycosaminoglycan limited dispersion of PCa microtumors, MMP-7 initiated rapid dyscohesion and migration even with perlecan present. Additionally, we found that a bioactive peptide (PLN4) found in perlecan domain IV in a region subject to digestion by MMP-7 further enhanced cell dispersion along with MMP-7. We found that digestion of the PSPN Complex with MMP-7 destabilized cell–cell junctions in microtumors evidenced by loss of co-registration of E-cadherin and F-actin. We conclude that MMP-7 plays a key functional role in PCa cell transition from a cohesive, indolent phenotype to a dyscohesive, migratory phenotype favoring production of circulating tumor cells and metastasis to bone.
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Elgundi Z, Papanicolaou M, Major G, Cox TR, Melrose J, Whitelock JM, Farrugia BL. Cancer Metastasis: The Role of the Extracellular Matrix and the Heparan Sulfate Proteoglycan Perlecan. Front Oncol 2020; 9:1482. [PMID: 32010611 PMCID: PMC6978720 DOI: 10.3389/fonc.2019.01482] [Citation(s) in RCA: 104] [Impact Index Per Article: 20.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2019] [Accepted: 12/10/2019] [Indexed: 12/12/2022] Open
Abstract
Cancer metastasis is the dissemination of tumor cells to new sites, resulting in the formation of secondary tumors. This process is complex and is spatially and temporally regulated by intrinsic and extrinsic factors. One important extrinsic factor is the extracellular matrix, the non-cellular component of tissues. Heparan sulfate proteoglycans (HSPGs) are constituents of the extracellular matrix, and through their heparan sulfate chains and protein core, modulate multiple events that occur during the metastatic cascade. This review will provide an overview of the role of the extracellular matrix in the events that occur during cancer metastasis, primarily focusing on perlecan. Perlecan, a basement membrane HSPG is a key component of the vascular extracellular matrix and is commonly associated with events that occur during the metastatic cascade. Its contradictory role in these events will be discussed and we will highlight the recent advances in cancer therapies that target HSPGs and their modifying enzymes.
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Affiliation(s)
- Zehra Elgundi
- Graduate School of Biomedical Engineering, UNSW Sydney, Sydney, NSW, Australia
| | - Michael Papanicolaou
- The Garvan Institute of Medical Research and The Kinghorn Cancer Centre, UNSW Sydney, Darlinghurst, NSW, Australia.,School of Life Sciences, University of Technology Sydney, Sydney, NSW, Australia
| | - Gretel Major
- The Garvan Institute of Medical Research and The Kinghorn Cancer Centre, UNSW Sydney, Darlinghurst, NSW, Australia
| | - Thomas R Cox
- The Garvan Institute of Medical Research and The Kinghorn Cancer Centre, UNSW Sydney, Darlinghurst, NSW, Australia.,St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW, Australia
| | - James Melrose
- Graduate School of Biomedical Engineering, UNSW Sydney, Sydney, NSW, Australia.,Raymond Purves Bone and Joint Research Laboratories, Kolling Institute of Medical Research, Royal North Shore Hospital, University of Sydney, St Leonards, NSW, Australia
| | - John M Whitelock
- Graduate School of Biomedical Engineering, UNSW Sydney, Sydney, NSW, Australia
| | - Brooke L Farrugia
- Graduate School of Biomedical Engineering, UNSW Sydney, Sydney, NSW, Australia.,Department of Biomedical Engineering, Melbourne School of Engineering, The University of Melbourne, Melbourne, VIC, Australia
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Vögtle T, Sharma S, Mori J, Nagy Z, Semeniak D, Scandola C, Geer MJ, Smith CW, Lane J, Pollack S, Lassila R, Jouppila A, Barr AJ, Ogg DJ, Howard TD, McMiken HJ, Warwicker J, Geh C, Rowlinson R, Abbott WM, Eckly A, Schulze H, Wright GJ, Mazharian A, Fütterer K, Rajesh S, Douglas MR, Senis YA. Heparan sulfates are critical regulators of the inhibitory megakaryocyte-platelet receptor G6b-B. eLife 2019; 8:e46840. [PMID: 31436532 PMCID: PMC6742478 DOI: 10.7554/elife.46840] [Citation(s) in RCA: 42] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2019] [Accepted: 08/21/2019] [Indexed: 02/06/2023] Open
Abstract
The immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor G6b-B is critical for platelet production and activation. Loss of G6b-B results in severe macrothrombocytopenia, myelofibrosis and aberrant platelet function in mice and humans. Using a combination of immunohistochemistry, affinity chromatography and proteomics, we identified the extracellular matrix heparan sulfate (HS) proteoglycan perlecan as a G6b-B binding partner. Subsequent in vitro biochemical studies and a cell-based genetic screen demonstrated that the interaction is specifically mediated by the HS chains of perlecan. Biophysical analysis revealed that heparin forms a high-affinity complex with G6b-B and mediates dimerization. Using platelets from humans and genetically modified mice, we demonstrate that binding of G6b-B to HS and multivalent heparin inhibits platelet and megakaryocyte function by inducing downstream signaling via the tyrosine phosphatases Shp1 and Shp2. Our findings provide novel insights into how G6b-B is regulated and contribute to our understanding of the interaction of megakaryocytes and platelets with glycans.
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Affiliation(s)
- Timo Vögtle
- Institute of Cardiovascular Sciences, College of Medical and Dental SciencesUniversity of BirminghamBirminghamUnited Kingdom
| | - Sumana Sharma
- Cell Surface Signalling LaboratoryWellcome Trust Sanger InstituteCambridgeUnited Kingdom
| | - Jun Mori
- Institute of Cardiovascular Sciences, College of Medical and Dental SciencesUniversity of BirminghamBirminghamUnited Kingdom
| | - Zoltan Nagy
- Institute of Cardiovascular Sciences, College of Medical and Dental SciencesUniversity of BirminghamBirminghamUnited Kingdom
| | - Daniela Semeniak
- Institute of Experimental BiomedicineUniversity Hospital WürzburgWürzburgGermany
| | - Cyril Scandola
- Université de Strasbourg, Institut National de la Santé et de la Recherche Médicale, Etablissement Français du Sang Grand Est, Unité Mixte de Recherche-S 1255, Fédération de Médecine Translationnelle de StrasbourgStrasbourgFrance
| | - Mitchell J Geer
- Institute of Cardiovascular Sciences, College of Medical and Dental SciencesUniversity of BirminghamBirminghamUnited Kingdom
| | - Christopher W Smith
- Institute of Cardiovascular Sciences, College of Medical and Dental SciencesUniversity of BirminghamBirminghamUnited Kingdom
| | - Jordan Lane
- Sygnature Discovery LimitedNottinghamUnited Kingdom
| | | | - Riitta Lassila
- Coagulation Disorders Unit, Department of Hematology, Comprehensive Cancer CenterUniversity of Helsinki, Helsinki University HospitalHelsinkiFinland
- Aplagon OyHelsinkiFinland
| | - Annukka Jouppila
- Coagulation Disorders UnitHelsinki University Hospital Research InstituteHelsinkiFinland
| | - Alastair J Barr
- Department of Biomedical Science, Faculty of Science & TechnologyUniversity of WestminsterLondonUnited Kingdom
| | - Derek J Ogg
- Peak Proteins LimitedAlderley ParkCheshireUnited Kingdom
| | - Tina D Howard
- Peak Proteins LimitedAlderley ParkCheshireUnited Kingdom
| | | | - Juli Warwicker
- Peak Proteins LimitedAlderley ParkCheshireUnited Kingdom
| | - Catherine Geh
- Peak Proteins LimitedAlderley ParkCheshireUnited Kingdom
| | | | - W Mark Abbott
- Peak Proteins LimitedAlderley ParkCheshireUnited Kingdom
| | - Anita Eckly
- Université de Strasbourg, Institut National de la Santé et de la Recherche Médicale, Etablissement Français du Sang Grand Est, Unité Mixte de Recherche-S 1255, Fédération de Médecine Translationnelle de StrasbourgStrasbourgFrance
| | - Harald Schulze
- Institute of Experimental BiomedicineUniversity Hospital WürzburgWürzburgGermany
| | - Gavin J Wright
- Cell Surface Signalling LaboratoryWellcome Trust Sanger InstituteCambridgeUnited Kingdom
| | - Alexandra Mazharian
- Institute of Cardiovascular Sciences, College of Medical and Dental SciencesUniversity of BirminghamBirminghamUnited Kingdom
| | - Klaus Fütterer
- School of Biosciences, College of Life and Environmental SciencesUniversity of BirminghamBirminghamUnited Kingdom
| | - Sundaresan Rajesh
- Institute of Cancer and Genomic Sciences, College of Medical and Dental SciencesUniversity of BirminghamBirminghamUnited Kingdom
| | - Michael R Douglas
- Institute of Inflammation and Ageing, College of Medical and Dental SciencesUniversity of BirminghamBirminghamUnited Kingdom
- Department of NeurologyDudley Group NHS Foundation TrustDudleyUnited Kingdom
- School of Life and Health SciencesAston UniversityBirminghamUnited Kingdom
| | - Yotis A Senis
- Institute of Cardiovascular Sciences, College of Medical and Dental SciencesUniversity of BirminghamBirminghamUnited Kingdom
- Université de Strasbourg, Institut National de la Santé et de la Recherche Médicale, Etablissement Français du Sang Grand Est, Unité Mixte de Recherche-S 1255, Fédération de Médecine Translationnelle de StrasbourgStrasbourgFrance
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6
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Grindel BJ, Martinez JR, Tellman TV, Harrington DA, Zafar H, Nakhleh L, Chung LW, Farach-Carson MC. Matrilysin/MMP-7 Cleavage of Perlecan/HSPG2 Complexed with Semaphorin 3A Supports FAK-Mediated Stromal Invasion by Prostate Cancer Cells. Sci Rep 2018; 8:7262. [PMID: 29740048 PMCID: PMC5940808 DOI: 10.1038/s41598-018-25435-3] [Citation(s) in RCA: 31] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2017] [Accepted: 04/19/2018] [Indexed: 12/13/2022] Open
Abstract
Interrupting the interplay between cancer cells and extracellular matrix (ECM) is a strategy to halt tumor progression and stromal invasion. Perlecan/heparan sulfate proteoglycan 2 (HSPG2) is an extracellular proteoglycan that orchestrates tumor angiogenesis, proliferation, differentiation and invasion. Metastatic prostate cancer (PCa) cells degrade perlecan-rich tissue borders to reach bone, including the basement membrane, vasculature, reactive stromal matrix and bone marrow. Domain IV-3, perlecan’s last 7 immunoglobulin repeats, mimics native proteoglycan by promoting tumoroid formation. This is reversed by matrilysin/matrix metalloproteinase-7 (MMP-7) cleavage to favor cell dispersion and tumoroid dyscohesion. Both perlecan and Domain IV-3 induced a strong focal adhesion kinase (FAK) dephosphorylation/deactivation. MMP-7 cleavage of perlecan reversed this, with FAK in dispersed tumoroids becoming phosphorylated/activated with metastatic phenotype. We demonstrated Domain IV-3 interacts with the axon guidance protein semaphorin 3A (Sema3A) on PCa cells to deactivate pro-metastatic FAK. Sema3A antibody mimicked the Domain IV-3 clustering activity. Direct binding experiments showed Domain IV-3 binds Sema3A. Knockdown of Sema3A prevented Domain IV-3-induced tumoroid formation and Sema3A was sensitive to MMP-7 proteolysis. The perlecan-Sema3A complex abrogates FAK activity and stabilizes PCa cell interactions. MMP-7 expressing cells destroy the complex to initiate metastasis, destroy perlecan-rich borders, and favor invasion and progression to lethal bone disease.
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Affiliation(s)
- Brian J Grindel
- Department of BioSciences, Rice University, Houston, TX, 77005, USA.,Department of Diagnostic and Biomedical Sciences, University of Texas Health Science Center at Houston, School of Dentistry, Houston, TX, 77054, USA.,Department of Cancer Systems Imaging, Division of Diagnostic Imaging, The University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA
| | - Jerahme R Martinez
- Department of BioSciences, Rice University, Houston, TX, 77005, USA.,Department of Mechanical Engineering, University of Delaware, Newark, DE, 19706, USA
| | - Tristen V Tellman
- Department of Diagnostic and Biomedical Sciences, University of Texas Health Science Center at Houston, School of Dentistry, Houston, TX, 77054, USA
| | - Daniel A Harrington
- Department of BioSciences, Rice University, Houston, TX, 77005, USA.,Department of Diagnostic and Biomedical Sciences, University of Texas Health Science Center at Houston, School of Dentistry, Houston, TX, 77054, USA
| | - Hamim Zafar
- Department of Computer Science, Rice University, Houston, TX, 77005, USA
| | - Luay Nakhleh
- Department of Computer Science, Rice University, Houston, TX, 77005, USA
| | - Leland W Chung
- Uro-Oncology Research Program, Samuel Oschin Comprehensive Cancer Institute at Cedars-Sinai Medical Center, Los Angeles, CA, 90048, USA
| | - Mary C Farach-Carson
- Department of BioSciences, Rice University, Houston, TX, 77005, USA. .,Department of Diagnostic and Biomedical Sciences, University of Texas Health Science Center at Houston, School of Dentistry, Houston, TX, 77054, USA.
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7
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Papy-Garcia D, Albanese P. Heparan sulfate proteoglycans as key regulators of the mesenchymal niche of hematopoietic stem cells. Glycoconj J 2017; 34:377-391. [PMID: 28577070 DOI: 10.1007/s10719-017-9773-8] [Citation(s) in RCA: 39] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2016] [Revised: 05/01/2017] [Accepted: 05/04/2017] [Indexed: 12/21/2022]
Abstract
The complex microenvironment that surrounds hematopoietic stem cells (HSCs) in the bone marrow niche involves different coordinated signaling pathways. The stem cells establish permanent interactions with distinct cell types such as mesenchymal stromal cells, osteoblasts, osteoclasts or endothelial cells and with secreted regulators such as growth factors, cytokines, chemokines and their receptors. These interactions are mediated through adhesion to extracellular matrix compounds also. All these signaling pathways are important for stem cell fates such as self-renewal, proliferation or differentiation, homing and mobilization, as well as for remodeling of the niche. Among these complex molecular cues, this review focuses on heparan sulfate (HS) structures and functions and on the role of enzymes involved in their biosynthesis and turnover. HS associated to core protein, constitute the superfamily of heparan sulfate proteoglycans (HSPGs) present on the cell surface and in the extracellular matrix of all tissues. The key regulatory effects of major medullar HSPGs are described, focusing on their roles in the interactions between hematopoietic stem cells and their endosteal niche, and on their ability to interact with Heparin Binding Proteins (HBPs). Finally, according to the relevance of HS moieties effects on this complex medullar niche, we describe recent data that identify HS mimetics or sulfated HS signatures as new glycanic tools and targets, respectively, for hematopoietic and mesenchymal stem cell based therapeutic applications.
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Affiliation(s)
- Dulce Papy-Garcia
- CRRET Laboratory, Université Paris Est, EA 4397 Université Paris Est Créteil, ERL CNRS 9215, F-94010, Créteil, France
| | - Patricia Albanese
- CRRET Laboratory, Université Paris Est, EA 4397 Université Paris Est Créteil, ERL CNRS 9215, F-94010, Créteil, France.
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8
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Grindel B, Li Q, Arnold R, Petros J, Zayzafoon M, Muldoon M, Stave J, Chung LWK, Farach-Carson MC. Perlecan/HSPG2 and matrilysin/MMP-7 as indices of tissue invasion: tissue localization and circulating perlecan fragments in a cohort of 288 radical prostatectomy patients. Oncotarget 2016; 7:10433-47. [PMID: 26862737 PMCID: PMC4891130 DOI: 10.18632/oncotarget.7197] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2015] [Accepted: 01/01/2016] [Indexed: 12/27/2022] Open
Abstract
Prostate cancer (PCa) cells use matrix metalloproteinases (MMPs) to degrade tissue during invasion. Perlecan/HSPG2 is degraded at basement membranes, in reactive stroma and in bone marrow during metastasis. We previously showed MMP-7 efficiently degrades perlecan. We now analyzed PCa tissue and serum from 288 prostatectomy patients of various Gleason grades to decipher the relationship between perlecan and MMP-7 in invasive PCa. In 157 prostatectomy specimens examined by tissue microarray, perlecan levels were 18% higher than their normal counterparts. In Gleason grade 4 tissues, MMP-7 and perlecan immunostaining levels were highly correlated with each other (average correlation coefficient of 0.52) in PCa tissue, regardless of grade. Serial sections showed intense, but non-overlapping, immunostaining for MMP-7 and perlecan at adjacent borders, reflecting the protease-substrate relationship. Using a capture assay, analysis of 288 PCa sera collected at prostatectomy showed elevated levels of perlecan fragments, with most derived from domain IV. Perlecan fragments in PCa sera were associated with overall MMP-7 staining levels in PCa tissues. Domain IV perlecan fragments were present in stage IV, but absent in normal, sera, suggesting perlecan degradation during metastasis. Together, perlecan fragments in sera and MMP-7 in tissues of PCa patients are measures of invasive PCa.
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Affiliation(s)
- Brian Grindel
- Department of BioSciences, Rice University, Houston, TX 77005, USA
| | - Quanlin Li
- Biostatistics and Bioinformatics Research Center, Samuel Oschin Comprehensive Cancer Institute, Cedars Sinai Medical Center, Los Angeles, CA 90048, USA
| | - Rebecca Arnold
- Emory University Departments of Urology, Pathology and Laboratory Medicine and Hematology and Medical Oncology, Atlanta, GA 30322, USA
| | - John Petros
- Emory University Departments of Urology, Pathology and Laboratory Medicine and Hematology and Medical Oncology, Atlanta, GA 30322, USA.,The Atlanta Veteran Affairs Medical Center, Decatur, GA 30033, USA
| | - Majd Zayzafoon
- Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35233, USA
| | - Mark Muldoon
- Strategic Diagnostics Inc., Newark, DE 19702, USA.,Romer Labs Technology, Inc., Newark, DE 19713, USA
| | - James Stave
- Strategic Diagnostics Inc., Newark, DE 19702, USA.,CD Diagnostics, Claymont, DE 19703, USA
| | - Leland W K Chung
- Uro-Oncology Research Program, Samuel Oschin Comprehensive Cancer Institute at Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA
| | - Mary C Farach-Carson
- Department of BioSciences, Rice University, Houston, TX 77005, USA.,Department of Bioengineering, Rice University, Houston, TX 77005, USA
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9
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DeCarlo AA, Whitelock JM. The Role of Heparan Sulfate and Perlecan in Bone-regenerative Procedures. J Dent Res 2016; 85:122-32. [PMID: 16434729 DOI: 10.1177/154405910608500203] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022] Open
Abstract
Tissue engineering, grafting procedures, regeneration, and tissue remodeling are developing therapeutic modalities with great potential medical value, but these regenerative modalities are not as effective or predictable as clinicians and patients would like. Greater understanding of growth factors, cytokines, extracellular matrix molecules, and their roles in cell-mediated healing processes have made these regenerative therapies more clinically viable and will continue advancing the fields of tissue engineering and grafting. However, millions of oral and non-oral bone-grafting procedures are performed annually, and only a small percentage yield the most desirable results. Here we review the heparan-sulfate-decorated extracellular biomolecule named perlecan and the research relating to its potential as an adjunct in bone-regenerative procedures. The review includes an overview of bone graft substitutes and biological adjuncts to bone-regenerative procedures in medicine as they apply to periodontal disease, alveolar ridge augmentation, and barrier membrane therapy. Perlecan is discussed as a potential biological adjunct in terms of growth factor sequestration and delivery, and promoting cell adhesion, proliferation, differentiation, and angiogenesis. Further, we propose delivery and application schemes for perlecan and/or its domains in bone-regenerative procedures, with particular emphasis on its heparan-sulfate-decorated domain I. The perlecan molecule, with its heparan sulfate glycosylation, may provide a multi-faceted approach for the delivery of a more comprehensive stimulus than other single potential adjuncts currently available for bone-regenerative procedures.
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Affiliation(s)
- A A DeCarlo
- Agenta Biotechnologies, Inc., OADI Technology Center, Birmingham, AL 35211, USA.
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10
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Poluzzi C, Iozzo RV, Schaefer L. Endostatin and endorepellin: A common route of action for similar angiostatic cancer avengers. Adv Drug Deliv Rev 2016; 97:156-73. [PMID: 26518982 DOI: 10.1016/j.addr.2015.10.012] [Citation(s) in RCA: 98] [Impact Index Per Article: 10.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2015] [Revised: 10/15/2015] [Accepted: 10/16/2015] [Indexed: 12/17/2022]
Abstract
Traditional cancer therapy typically targets the tumor proper. However, newly-formed vasculature exerts a major role in cancer development and progression. Autophagy, as a biological mechanism for clearing damaged proteins and oxidative stress products released in the tumor milieu, could help in tumor resolution by rescuing cells undergoing modifications or inducing autophagic-cell death of tumor blood vessels. Cleaved fragments of extracellular matrix proteoglycans are emerging as key players in the modulation of angiogenesis and endothelial cell autophagy. An essential characteristic of cancer progression is the remodeling of the basement membrane and the release of processed forms of its constituents. Endostatin, generated from collagen XVIII, and endorepellin, the C-terminal segment of the large proteoglycan perlecan, possess a dual activity as modifiers of both angiogenesis and endothelial cell autophagy. Manipulation of these endogenously-processed forms, located in the basement membrane within tumors, could represent new therapeutic approaches for cancer eradication.
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Affiliation(s)
- Chiara Poluzzi
- Pharmazentrum Frankfurt/ZAFES, Institut für Allgemeine Pharmakologie und Toxikologie, Klinikum der Goethe-Universität Frankfurt am Main, Frankfurt am Main, Germany
| | - Renato V Iozzo
- Department of Pathology, Anatomy and Cell Biology, and the Cancer Cell Biology and Signaling Program, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, USA
| | - Liliana Schaefer
- Pharmazentrum Frankfurt/ZAFES, Institut für Allgemeine Pharmakologie und Toxikologie, Klinikum der Goethe-Universität Frankfurt am Main, Frankfurt am Main, Germany.
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Melrose J, Shu C, Whitelock JM, Lord MS. The cartilage extracellular matrix as a transient developmental scaffold for growth plate maturation. Matrix Biol 2016; 52-54:363-383. [PMID: 26807757 DOI: 10.1016/j.matbio.2016.01.008] [Citation(s) in RCA: 56] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2015] [Revised: 01/19/2016] [Accepted: 01/19/2016] [Indexed: 10/22/2022]
Abstract
The cartilage growth plate is a specialized developmental tissue containing characteristic zonal arrangements of chondrocytes. The proliferative and differentiative states of chondrocytes are tightly regulated at all stages including the initial limb bud and rudiment cartilage stages of development, the establishment of the primary and secondary ossification centers, development of the growth plates and laying down of bone. A multitude of spatio-temporal signals, including transcription factors, growth factors, morphogens and hormones, control chondrocyte maturation and terminal chondrocyte differentiation/hypertrophy, cell death/differentiation, calcification and vascular invasion of the growth plate and bone formation during morphogenetic transition of the growth plate. This involves hierarchical, integrated signaling from growth and factors, transcription factors, mechanosensory cues and proteases in the extracellular matrix to regulate these developmental processes to facilitate progressive changes in the growth plate culminating in bone formation and endochondral ossification. This review provides an overview of selected components which have particularly important roles in growth plate biology including collagens, proteoglycans, glycosaminoglycans, growth factors, proteases and enzymes.
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Affiliation(s)
- James Melrose
- Raymond Purves Bone and Joint Research Laboratory, Kolling Institute, Northern Sydney Local Health District, St Leonards, NSW 2065, Australia; Sydney Medical School, Northern, The University of Sydney, Royal North Shore Hospital, St Leonards, NSW 2065, Australia; Graduate School of Biomedical Engineering, University of New South Wales, Sydney, NSW 2052, Australia
| | - Cindy Shu
- Raymond Purves Bone and Joint Research Laboratory, Kolling Institute, Northern Sydney Local Health District, St Leonards, NSW 2065, Australia
| | - John M Whitelock
- Sydney Medical School, Northern, The University of Sydney, Royal North Shore Hospital, St Leonards, NSW 2065, Australia
| | - Megan S Lord
- Sydney Medical School, Northern, The University of Sydney, Royal North Shore Hospital, St Leonards, NSW 2065, Australia.
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12
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Hematopoietic Stem and Progenitor Cell Expansion in Contact with Mesenchymal Stromal Cells in a Hanging Drop Model Uncovers Disadvantages of 3D Culture. Stem Cells Int 2015; 2016:4148093. [PMID: 26839560 PMCID: PMC4709770 DOI: 10.1155/2016/4148093] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2015] [Accepted: 10/12/2015] [Indexed: 01/19/2023] Open
Abstract
Efficient ex vivo expansion of hematopoietic stem cells with a concomitant preservation of stemness and self-renewal potential is still an unresolved ambition. Increased numbers of methods approaching this issue using three-dimensional (3D) cultures were reported. Here, we describe a simplified 3D hanging drop model for the coculture of cord blood-derived CD34+ hematopoietic stem and progenitor cells (HSPCs) with bone marrow-derived mesenchymal stromal cells (MSCs). When seeded as a mixed cell suspension, MSCs segregated into tight spheroids. Despite the high expression of niche-specific extracellular matrix components by spheroid-forming MSCs, HSPCs did not migrate into the spheroids in the initial phase of coculture, indicating strong homotypic interactions of MSCs. After one week, however, HSPC attachment increased considerably, leading to spheroid collapse as demonstrated by electron microscopy and immunofluorescence staining. In terms of HSPC proliferation, the conventional 2D coculture system was superior to the hanging drop model. Furthermore, expansion of primitive hematopoietic progenitors was more favored in 2D than in 3D, as analyzed in colony-forming assays. Conclusively, our data demonstrate that MSCs, when arranged with a spread (monolayer) shape, exhibit better HSPC supportive qualities than spheroid-forming MSCs. Therefore, 3D systems are not necessarily superior to traditional 2D culture in this regard.
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13
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Klein G, Schmal O, Aicher WK. Matrix metalloproteinases in stem cell mobilization. Matrix Biol 2015; 44-46:175-83. [PMID: 25617493 DOI: 10.1016/j.matbio.2015.01.011] [Citation(s) in RCA: 45] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2014] [Revised: 01/15/2015] [Accepted: 01/15/2015] [Indexed: 01/05/2023]
Abstract
Hematopoietic stem cells (HSCs) have the capability to migrate back and forth between their preferred microenvironment in bone marrow niches and the peripheral blood, but under steady-state conditions only a marginal number of stem cells can be found in the circulation. Different mobilizing agents, however, which create a highly proteolytic milieu in the bone marrow, can drastically increase the number of circulating HSCs. Among other proteases secreted and membrane-bound matrix metalloproteinases (MMPs) are known to be involved in the induced mobilization process and can digest niche-specific extracellular matrix components and cytokines responsible for stem cell retention to the niches. Iatrogenic stem cell mobilization and stem cell homing to their niches are clinically employed on a routine basis, although the exact mechanisms of both processes are still not fully understood. In this review we provide an overview on the various roles of MMPs in the induced release of HSCs from the bone marrow.
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Affiliation(s)
- Gerd Klein
- Center for Medical Research, Department of Internal Medicine, Section for Transplantation Immunology and Immunohematology, University of Tübingen, Germany.
| | - Olga Schmal
- Center for Medical Research, Department of Internal Medicine, Section for Transplantation Immunology and Immunohematology, University of Tübingen, Germany
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14
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Kawahara R, Granato DC, Carnielli CM, Cervigne NK, Oliveria CE, Martinez CAR, Yokoo S, Fonseca FP, Lopes M, Santos-Silva AR, Graner E, Coletta RD, Leme AFP. Agrin and perlecan mediate tumorigenic processes in oral squamous cell carcinoma. PLoS One 2014; 9:e115004. [PMID: 25506919 PMCID: PMC4266612 DOI: 10.1371/journal.pone.0115004] [Citation(s) in RCA: 46] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2014] [Accepted: 11/17/2014] [Indexed: 01/03/2023] Open
Abstract
Oral squamous cell carcinoma is the most common type of cancer in the oral cavity, representing more than 90% of all oral cancers. The characterization of altered molecules in oral cancer is essential to understand molecular mechanisms underlying tumor progression as well as to contribute to cancer biomarker and therapeutic target discovery. Proteoglycans are key molecular effectors of cell surface and pericellular microenvironments, performing multiple functions in cancer. Two of the major basement membrane proteoglycans, agrin and perlecan, were investigated in this study regarding their role in oral cancer. Using real time quantitative PCR (qRT-PCR), we showed that agrin and perlecan are highly expressed in oral squamous cell carcinoma. Interestingly, cell lines originated from distinct sites showed different expression of agrin and perlecan. Enzymatically targeting chondroitin sulfate modification by chondroitinase, oral squamous carcinoma cell line had a reduced ability to adhere to extracellular matrix proteins and increased sensibility to cisplatin. Additionally, knockdown of agrin and perlecan promoted a decrease on cell migration and adhesion, and on resistance of cells to cisplatin. Our study showed, for the first time, a negative regulation on oral cancer-associated events by either targeting chondroitin sulfate content or agrin and perlecan levels.
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Affiliation(s)
- Rebeca Kawahara
- Laboratório de Espectrometria de Massas, Laboratório Nacional de Biociências, LNBio, CNPEM, Campinas, Brazil
| | - Daniela C. Granato
- Laboratório de Espectrometria de Massas, Laboratório Nacional de Biociências, LNBio, CNPEM, Campinas, Brazil
| | - Carolina M. Carnielli
- Laboratório de Espectrometria de Massas, Laboratório Nacional de Biociências, LNBio, CNPEM, Campinas, Brazil
| | - Nilva K. Cervigne
- Faculdade de Odontologia de Piracicaba, Universidade Estadual de Campinas, UNICAMP, Piracicaba, Brazil
| | - Carine E. Oliveria
- Faculdade de Odontologia de Piracicaba, Universidade Estadual de Campinas, UNICAMP, Piracicaba, Brazil
| | - César A. R. Martinez
- Laboratório de Espectrometria de Massas, Laboratório Nacional de Biociências, LNBio, CNPEM, Campinas, Brazil
| | - Sami Yokoo
- Laboratório de Espectrometria de Massas, Laboratório Nacional de Biociências, LNBio, CNPEM, Campinas, Brazil
| | - Felipe P. Fonseca
- Faculdade de Odontologia de Piracicaba, Universidade Estadual de Campinas, UNICAMP, Piracicaba, Brazil
| | - Marcio Lopes
- Faculdade de Odontologia de Piracicaba, Universidade Estadual de Campinas, UNICAMP, Piracicaba, Brazil
| | - Alan R. Santos-Silva
- Faculdade de Odontologia de Piracicaba, Universidade Estadual de Campinas, UNICAMP, Piracicaba, Brazil
| | - Edgard Graner
- Faculdade de Odontologia de Piracicaba, Universidade Estadual de Campinas, UNICAMP, Piracicaba, Brazil
| | - Ricardo D. Coletta
- Faculdade de Odontologia de Piracicaba, Universidade Estadual de Campinas, UNICAMP, Piracicaba, Brazil
| | - Adriana Franco Paes Leme
- Laboratório de Espectrometria de Massas, Laboratório Nacional de Biociências, LNBio, CNPEM, Campinas, Brazil
- * E-mail:
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15
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Grindel BJ, Martinez JR, Pennington CL, Muldoon M, Stave J, Chung LW, Farach-Carson MC. Matrilysin/matrix metalloproteinase-7(MMP7) cleavage of perlecan/HSPG2 creates a molecular switch to alter prostate cancer cell behavior. Matrix Biol 2014; 36:64-76. [PMID: 24833109 DOI: 10.1016/j.matbio.2014.04.005] [Citation(s) in RCA: 58] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2013] [Revised: 04/16/2014] [Accepted: 04/17/2014] [Indexed: 01/17/2023]
Abstract
Perlecan/HSPG2, a large heparan sulfate (HS) proteoglycan, normally is expressed in the basement membrane (BM) underlying epithelial and endothelial cells. During prostate cancer (PCa) cell invasion, a variety of proteolytic enzymes are expressed that digest BM components including perlecan. An enzyme upregulated in invasive PCa cells, matrilysin/matrix metalloproteinase-7 (MMP-7), was examined as a candidate for perlecan proteolysis both in silico and in vitro. Purified perlecan showed high sensitivity to MMP-7 digestion even when fully decorated with HS or when presented in native context connected with other BM proteins. In both conditions, MMP-7 produced discrete perlecan fragments corresponding to an origin in immunoglobulin (Ig) repeat region domain IV. While not predicted by in silico analysis, MMP-7 cleaved every subpart of recombinantly generated perlecan domain IV. Other enzymes relevant to PCa that were tested had limited ability to cleave perlecan including prostate specific antigen, hepsin, or fibroblast activation protein α. A long C-terminal portion of perlecan domain IV, Dm IV-3, induced a strong clustering phenotype in the metastatic PCa cell lines, PC-3 and C4-2. MMP-7 digestion of Dm IV-3 reverses the clustering effect into one favoring cell dispersion. In a C4-2 Transwell® invasion assay, perlecan-rich human BM extract that was pre-digested with MMP-7 showed loss of barrier function and permitted a greater level of cell penetration than untreated BM extract. We conclude that enzymatic processing of perlecan in the BM or territorial matrix by MMP-7 as occurs in the invasive tumor microenvironment acts as a molecular switch to alter PCa cell behavior and favor cell dispersion and invasiveness.
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Affiliation(s)
- B J Grindel
- Department of Biochemistry and Cell Biology, Rice University, Houston, TX, 77005, USA
| | - J R Martinez
- Department of Biochemistry and Cell Biology, Rice University, Houston, TX, 77005, USA
| | - C L Pennington
- Shared Equipment Authority, Rice University, Houston, TX 77005
| | - M Muldoon
- Strategic Diagnostics Inc, Newark, DE, 19702, USA
| | - J Stave
- Strategic Diagnostics Inc, Newark, DE, 19702, USA
| | - L W Chung
- Uro-Oncology Research Program, Samuel Oschin Comprehensive Cancer Institute at Cedars-Sinai Medical Center, Los Angeles, CA, 90048, USA
| | - M C Farach-Carson
- Department of Biochemistry and Cell Biology, Rice University, Houston, TX, 77005, USA; Department of Bioengineering, Rice University, Houston, TX 77005, USA.
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16
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Grigorian M, Liu T, Banerjee U, Hartenstein V. The proteoglycan Trol controls the architecture of the extracellular matrix and balances proliferation and differentiation of blood progenitors in the Drosophila lymph gland. Dev Biol 2013; 384:301-12. [PMID: 23510717 PMCID: PMC4278754 DOI: 10.1016/j.ydbio.2013.03.007] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2012] [Revised: 03/07/2013] [Accepted: 03/09/2013] [Indexed: 10/27/2022]
Abstract
The heparin sulfate proteoglycan Terribly Reduced Optic Lobes (Trol) is the Drosophila melanogaster homolog of the vertebrate protein Perlecan. Trol is expressed as part of the extracellular matrix (ECM) found in the hematopoietic organ, called the lymph gland. In the normal lymph gland, the ECM forms thin basement membranes around individual or small groups of blood progenitors. The pattern of basement membranes, reported by Trol expression, is spatio-temporally correlated to hematopoiesis. The central, medullary zone which contain undifferentiated hematopoietic progenitors has many, closely spaced membranes. Fewer basement membranes are present in the outer, cortical zone, where differentiation of blood cells takes place. Loss of trol causes a dramatic change of the ECM into a three-dimensional, spongy mass that fills wide spaces scattered throughout the lymph gland. At the same time proliferation is reduced, leading to a significantly smaller lymph gland. Interestingly, differentiation of blood progenitors in trol mutants is precocious, resulting in the break-down of the usual zonation of the lymph gland. which normally consists of an immature center (medullary zone) where cells remain undifferentiated, and an outer cortical zone, where differentiation sets in. We present evidence that the effect of Trol on blood cell differentiation is mediated by Hedgehog (Hh) signaling, which is known to be required to maintain an immature medullary zone. Overexpression of hh in the background of a trol mutation is able to rescue the premature differentiation phenotype. Our data provide novel insight into the role of the ECM component Perlecan during Drosophila hematopoiesis.
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Affiliation(s)
- Melina Grigorian
- Department of Molecular, Cell and Developmental Biology, University of California Los Angeles, Los Angeles, CA 90095, USA.
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17
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Dang Z, Kuffová L, Liu L, Forrester JV. Soluble antigen traffics rapidly and selectively from the corneal surface to the eye draining lymph node and activates T cells when codelivered with CpG oligonucleotides. J Leukoc Biol 2013; 95:431-40. [PMID: 24295832 DOI: 10.1189/jlb.0612294] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
The transport of antigen to the secondary lymphoid tissue is a central component in the initiation of the adaptive immune response. The mechanism of antigen delivery to the DLN from the avascular cornea has not been fully explored. Previous studies in the mouse have shown that cell-associated corneal antigen is delivered within 6 h to the eye draining SM DLN via DCs and macrophages. In this study, we used a system in which antigen and the processed p-MHCII complexes derived from the antigen could be tracked in vivo. We report that soluble antigen applied to an abraded cornea in the mouse is transported rapidly (within 30 min) to the SM DLN, where a proportion is taken up by resident DCs and presented as p-MHCII complexes, while the larger part is cleared by 8 h. At a later time, a second wave of antigen transport in migratory DCs enters the DLN and participates in further continued antigen presentation. With the use of an antigen-specific TCR transgenic mouse system, we demonstrate that T cell activation does not occur during the early stages of soluble antigen delivery to LN, even though p-MHCII complexes are generated. Antigen-specific T cell activation occurs in the later, presumed cell-associated phase but requires codelivery of a "danger" signal, such as the TLR ligand CpG. We suggest that the early delivery of soluble antigen is more likely to induce T cell nonresponsiveness (anergy) unless presented in the context of an innate-immune cell activation (danger) signal.
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Affiliation(s)
- Zexu Dang
- 2.Division of Applied Medicine, School of Medicine and Dentistry, Institute of Medical Science, Foresterhill, University of Aberdeen, Aberdeen AB25 2ZD, Scotland, UK.
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18
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Ishijima M, Suzuki N, Hozumi K, Matsunobu T, Kosaki K, Kaneko H, Hassell JR, Arikawa-Hirasawa E, Yamada Y. Perlecan modulates VEGF signaling and is essential for vascularization in endochondral bone formation. Matrix Biol 2012; 31:234-45. [PMID: 22421594 DOI: 10.1016/j.matbio.2012.02.006] [Citation(s) in RCA: 61] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2011] [Revised: 02/27/2012] [Accepted: 02/28/2012] [Indexed: 10/28/2022]
Abstract
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.
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Affiliation(s)
- Muneaki Ishijima
- Laboratory of Cell and Developmental Biology, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892-4370, USA
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19
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Copland IB, Galipeau J. Death and inflammation following somatic cell transplantation. Semin Immunopathol 2011; 33:535-50. [DOI: 10.1007/s00281-011-0274-8] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2010] [Accepted: 04/14/2011] [Indexed: 12/13/2022]
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20
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Ellis AL, Pan W, Yang G, Jones K, Chuang C, Whitelock JM, DeCarlo AA. Similarity of recombinant human perlecan domain 1 by alternative expression systems bioactive heterogenous recombinant human perlecan D1. BMC Biotechnol 2010; 10:66. [PMID: 20828410 PMCID: PMC2944331 DOI: 10.1186/1472-6750-10-66] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2010] [Accepted: 09/09/2010] [Indexed: 11/16/2022] Open
Abstract
Background Heparan sulfate glycosaminoglycans are diverse components of certain proteoglycans and are known to interact with growth factors as a co-receptor necessary to induce signalling and growth factor activity. In this report we characterize heterogeneously glycosylated recombinant human perlecan domain 1 (HSPG2 abbreviated as rhPln.D1) synthesized in either HEK 293 cells or HUVECs by transient gene delivery using either adenoviral or expression plasmid technology. Results By SDS-PAGE analysis following anion exchange chromatography, the recombinant proteoglycans appeared to possess glycosaminoglycan chains ranging, in total, from 6 kDa to >90 kDa per recombinant. Immunoblot analysis of enzyme-digested high Mr rhPln.D1 demonstrated that the rhPln.D1 was synthesized as either a chondroitin sulfate or heparan sulfate proteoglycan, in an approximately 2:1 ratio, with negligible hybrids. Secondary structure analysis suggested helices and sheets in both recombinant species. rhPln.D1 demonstrated binding to rhFGF-2 with an apparent kD of 2 ± 0.2 nM with almost complete susceptibility to digestion by heparinase III in ligand blot analysis but not to chondroitinase digestion. Additionally, we demonstrate HS-mediated binding of both rhPln.D1 species to several other GFs. Finally, we corroborate the augmentation of FGF-mediated cell activation by rhPln.D1 and demonstrate mitogenic signalling through the FGFR1c receptor. Conclusions With importance especially to the emerging field of DNA-based therapeutics, we have shown here that proteoglycan synthesis, in different cell lines where GAG profiles typically differ, can be directed by recombinant technology to produce populations of bioactive recombinants with highly similar GAG profiles.
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Affiliation(s)
- April L Ellis
- Agenta Biotechnologies, Inc., Innovation Depot, Birmingham, AL 35203, USA
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21
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Nigro J, White JF, Ramshaw JA, Haylock DN, Nilsson SK, Werkmeister JA. The effect of bovine endosteum-derived particles on the proliferation of human mesenchymal stem cells. Biomaterials 2010; 31:5689-99. [DOI: 10.1016/j.biomaterials.2010.03.054] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2010] [Accepted: 03/21/2010] [Indexed: 01/22/2023]
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Iozzo RV, Zoeller JJ, Nyström A. Basement membrane proteoglycans: modulators Par Excellence of cancer growth and angiogenesis. Mol Cells 2009; 27:503-13. [PMID: 19466598 PMCID: PMC6712562 DOI: 10.1007/s10059-009-0069-0] [Citation(s) in RCA: 175] [Impact Index Per Article: 10.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2009] [Accepted: 04/25/2009] [Indexed: 01/13/2023] Open
Abstract
Proteoglycans located in basement membranes, the nanostructures underling epithelial and endothelial layers, are unique in several respects. They are usually large, elongated molecules with a collage of domains that share structural and functional homology with numerous extracellular matrix proteins, growth factors and surface receptors. They mainly carry heparan sulfate side chains and these contribute not only to storing and preserving the biological activity of various heparan sulfate-binding cytokines and growth factors, but also in presenting them in a more "active configuration" to their cognate receptors. Abnormal expression or deregulated function of these proteoglycans affect cancer and angiogenesis, and are critical for the evolution of the tumor microenvironment. This review will focus on the functional roles of the major heparan sulfate proteoglycans from basement membrane zones: perlecan, agrin and collagen XVIII, and on their roles in modulating cancer growth and angiogenesis.
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Affiliation(s)
- Renato V Iozzo
- Department of Pathology, Anatomy and Cell Biology, and the Cancer Cell Biology and Signaling Program, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
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Rodgers KD, San Antonio JD, Jacenko O. Heparan sulfate proteoglycans: a GAGgle of skeletal-hematopoietic regulators. Dev Dyn 2008; 237:2622-42. [PMID: 18629873 PMCID: PMC2651149 DOI: 10.1002/dvdy.21593] [Citation(s) in RCA: 55] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
This review summarizes our current understanding of the presence and function of heparan sulfate proteoglycans (HSPGs) in skeletal development and hematopoiesis. Although proteoglycans (PGs) comprise a large and diverse group of cell surface and matrix molecules, we chose to focus on HSPGs owing to their many proposed functions in skeletogenesis and hematopoiesis. Specifically, we discuss how HSPGs play predominant roles in establishing and regulating niches during skeleto-hematopoietic development by participating in distinct developmental processes such as patterning, compartmentalization, growth, differentiation, and maintenance of tissues. Special emphasis is placed on our novel hypothesis that mechanistically links endochondral skeletogenesis to the establishment of the hematopoietic stem cell (HSC) niche in the marrow. HSPGs may contribute to these developmental processes through their unique abilities to establish and mediate morphogen, growth factor, and cytokine gradients; facilitate signaling; provide structural stability to tissues; and act as molecular filters and barriers.
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Affiliation(s)
- Kathryn D Rodgers
- Department of Animal Biology, Division of Biochemistry, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania 19104-6046, USA.
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24
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Bix G, Iozzo RV. Novel interactions of perlecan: unraveling perlecan's role in angiogenesis. Microsc Res Tech 2008; 71:339-48. [PMID: 18300285 DOI: 10.1002/jemt.20562] [Citation(s) in RCA: 79] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
Abstract
Perlecan, a highly conserved and ubiquitous basement membrane heparan sulfate proteoglycan, is essential for life, inasmuch as its absence results in embryonic lethality in mice and C. elegans, and neonatal lethality in humans. Perlecan plays an essential role in vasculogenesis and chondrogenesis, as well as in pathological states where these processes are maladapted. Although a large body of evidence supports a pro-angiogenic role for perlecan, recent findings suggests that portions of the perlecan protein core can be antiangiogenic, requiring a further evaluation of the functioning of this complex molecule. This review is focused on the genetics of mammalian and nonmammalian perlecan, the elucidation of its novel interacting partners and its role in angiogenesis. By more fully understanding perlecan's functioning in angiogenesis, we may gain invaluable insight that could lead to therapeutic interventions in cancer and other pathologic states.
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Affiliation(s)
- Gregory Bix
- Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA
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25
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Rodgers KD, Sasaki T, Aszodi A, Jacenko O. Reduced perlecan in mice results in chondrodysplasia resembling Schwartz-Jampel syndrome. Hum Mol Genet 2007; 16:515-28. [PMID: 17213231 DOI: 10.1093/hmg/ddl484] [Citation(s) in RCA: 58] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/14/2022] Open
Abstract
Perlecan knock-in mice were developed to model Schwartz-Jampel syndrome (SJS), a skeletal disease resulting from decreased perlecan. Two mouse strains were generated: those carrying a C-to-Y mutation at residue 1532 and the neomycin cassette (C1532Yneo) and those harboring the mutation alone (C1532Y). Immunostaining, biochemistry, size measurements, skeletal studies and histology revealed Hspg2 transcriptional changes in C1532Yneo mice, leading to reduced perlecan secretion and a skeletal disease phenotype characteristic of SJS patients. Skeletal disease features include smaller size, impaired mineralization, misshapen bones, flat face and joint dysplasias reminiscent of osteoarthritis and osteonecrosis. Moreover, C1532Yneo mice displayed transient expansion of hypertrophic cartilage in the growth plate concomitant with radial trabecular bone orientation. In contrast, C1532Y mice, harboring only the mutation associated with SJS, displayed a mild phenotype, inconsistent with SJS. These studies question the C1532Y mutation as the sole causative factor of SJS in the human family harboring this alteration and imply that transcriptional changes leading to perlecan reduction may represent the disease mechanism for SJS.
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Affiliation(s)
- Kathryn D Rodgers
- Department of Animal Biology, University of Pennsylvania School of Veterinary Medicine, 3800 Spruce Street, Rosenthal Room 152, Pennsylvania, PA 19104-6046, USA.
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26
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Sher I, Zisman-Rozen S, Eliahu L, Whitelock JM, Maas-Szabowski N, Yamada Y, Breitkreutz D, Fusenig NE, Arikawa-Hirasawa E, Iozzo RV, Bergman R, Ron D. Targeting perlecan in human keratinocytes reveals novel roles for perlecan in epidermal formation. J Biol Chem 2005; 281:5178-87. [PMID: 16269412 DOI: 10.1074/jbc.m509500200] [Citation(s) in RCA: 78] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
Heparin-binding growth factors are crucial for the formation of human epidermis, but little is known about the role of heparan sulfate proteoglycans in this process. Here we investigated the role of the heparan sulfate proteoglycan, perlecan, in the formation of human epidermis, by utilizing in vitro engineered human skin. By disrupting perlecan expression either in the dermis or the epidermis, we found that epidermally derived perlecan is essential for epidermal formation. Perlecan-deficient keratinocytes formed a strikingly thin and poorly organized epidermis because of premature apoptosis and failure to complete their stratification program. Exogenous perlecan fully restored epidermal formation. Perlecan deposition in the basement membrane zone correlated with formation of multilayered epidermis. Perlecan deficiency, however, had no effect on the lining and deposition of major basement membrane components as was evident by a continuous linear staining of laminin and collagen IV. Similarly, perlecan deficiency did not affect the distribution of beta1 integrin. Addition of the perlecan ligand, fibroblast growth factor 7, protected perlecan-deficient keratinocytes from cell death and improved the thickness of the epidermis. Taken together, our results revealed novel roles for perlecan in epidermal formation. Perlecan regulates both the survival and terminal differentiation steps of keratinocytes. Our results suggested a model whereby perlecan regulates these processes via controlling the bioavailability of perlecan-binding soluble factors involved in epidermal morphogenesis.
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Affiliation(s)
- Ifat Sher
- Department of Biology, Technion, Israel Institute of Technology, 32000 Haifa, Israel
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Whitelock JM, Iozzo RV. Heparan Sulfate: A Complex Polymer Charged with Biological Activity. Chem Rev 2005; 105:2745-64. [PMID: 16011323 DOI: 10.1021/cr010213m] [Citation(s) in RCA: 321] [Impact Index Per Article: 16.1] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Affiliation(s)
- John M Whitelock
- Graduate School of Biomedical Engineering, University of New South Wales, Kensington, Sydney, New South Wales 2052, Australia.
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Han B, Liu J, Ma MJ, Zhao L. Clinicopathological significance of heparanase and basic fibroblast growth factor expression in human esophageal cancer. World J Gastroenterol 2005; 11:2188-92. [PMID: 15810091 PMCID: PMC4305794 DOI: 10.3748/wjg.v11.i14.2188] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: Human heparanase is an endo-D-glucuronidase that degrades heparan sulfate/heparin and has been implicated in a variety of biological processes. The objective was to investigate the expression of heparanase (Hps) and basic fibroblast growth factor (bFGF) and their relationship to neoangiogenesis and metastasis of human esophageal carcinoma.
METHODS: Seventy-nine patients who had undergone esophageal resection for esophageal carcinoma without preoperative treatment were included in the present study. Immunohistochemistry was used to study the expression of Hps, bFGF and microvessel density (MVD) in 79 cases of esoph-ageal carcinoma. bFGF and Hps were quantitatively detected with immunohistochemistry in 79 cases of human esopha-geal carcinoma and 19 cases of adjacent normal human esophageal carcinoma. Cd34 was used to explore the MVD as a marker of endothelial cells.
RESULTS: Hps and bFGF expression in tumor tissue, being remarkably higher than that in normal esophageal tissue, were significantly correlated with clinicopathological features (depth of invasion, lymph-node metastasis and TNM stage) and MVD.
CONCLUSION: The results of this study suggest that the coexpression of Hps and bFGF plays a key role in angiogenesis, invasion and metastasis of esophageal carcinoma. Hps and bFGF may serve as a predictor of progression in esophageal carcinoma. The expression of heparanase in esophageal carcinoma enhances growth, invasion, and angiogenesis of the tumor, and bFGF seems to be a potent antigenic factor for esophageal carcinoma.
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Affiliation(s)
- Biao Han
- First Affiliated Hospital, Lanzhou, Medical College, Lanzhou 730000, Gansu Province, China
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29
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Sixt M, Kanazawa N, Selg M, Samson T, Roos G, Reinhardt DP, Pabst R, Lutz MB, Sorokin L. The Conduit System Transports Soluble Antigens from the Afferent Lymph to Resident Dendritic Cells in the T Cell Area of the Lymph Node. Immunity 2005; 22:19-29. [PMID: 15664156 DOI: 10.1016/j.immuni.2004.11.013] [Citation(s) in RCA: 575] [Impact Index Per Article: 28.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2003] [Revised: 11/04/2004] [Accepted: 11/10/2004] [Indexed: 12/27/2022]
Abstract
Resident dendritic cells (DC) within the T cell area of the lymph node take up soluble antigens that enter via the afferent lymphatics before antigen carrying DC arrive from the periphery. The reticular network within the lymph node is a conduit system forming the infrastructure for the fast delivery of soluble substances from the afferent lymph to the lumen of high endothelial venules (HEVs). Using high-resolution light microscopy and 3D reconstruction, we show here that these conduits are unique basement membrane-like structures ensheathed by fibroblastic reticular cells with occasional resident DC embedded within this cell layer. Conduit-associated DC are capable of taking up and processing soluble antigens transported within the conduits, whereas immigrated mature DC occur remote from the reticular fibers. The conduit system is, therefore, not a closed compartment that shuttles substances through the lymph node but represents the morphological equivalent to the filtering function of the lymph node.
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Affiliation(s)
- Michael Sixt
- Department for Experimental Pathology, Lund University, 22185 Lund, Sweden.
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30
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Tran PK, Tran-Lundmark K, Soininen R, Tryggvason K, Thyberg J, Hedin U. Increased Intimal Hyperplasia and Smooth Muscle Cell Proliferation in Transgenic Mice With Heparan Sulfate–Deficient Perlecan. Circ Res 2004; 94:550-8. [PMID: 14739157 DOI: 10.1161/01.res.0000117772.86853.34] [Citation(s) in RCA: 74] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Smooth muscle cell (SMC) proliferation is a critical process in vascular disease. Heparan sulfate (HS) proteoglycans inhibit SMC growth, but the role of endogenous counterparts in the vessel wall in control of SMC function is not known in detail. Perlecan is the major HS proteoglycans in SMC basement membranes and in vessel wall extracellular matrix (ECM). In this study, transgenic mice with HS-deficient perlecan were analyzed with respect to vascular phenotype and intimal lesion formation. Furthermore, SMC cultures were established and characterized with respect to morphology, immunocytochemical features, proteoglycan synthesis, proliferative capacity, and ECM binding of basic fibroblast growth factor (FGF-2). In vitro, mutant SMCs formed basement membranes with perlecan core protein, but with decreased levels of HS, they showed diminished secretion of HS-containing perlecan into the medium and a defective ECM-binding capacity of FGF-2. In vitro, mutant SMCs showed increased proliferation compared with wild-type cells, and in vivo, enhanced SMC proliferation and intimal hyperplasia were observed after flow cessation of the carotid artery in mutant mice. The results indicate that the endogenous HS side-chains of perlecan contribute to SMC growth control both in vitro and during intimal hyperplasia, possibly by sequestering heparin-binding mitogens such as FGF-2.
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Affiliation(s)
- Phan-Kiet Tran
- Department of Surgical Sciences, Karolinska Hospital, SE-17176 Stockholm, Sweden.
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31
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Gomes RR, Farach-Carson MC, Carson DD. Perlecan Functions in Chondrogenesis: Insights from in vitro and in vivo Models. Cells Tissues Organs 2004; 176:79-86. [PMID: 14745237 DOI: 10.1159/000075029] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
Perlecan is a large heparan sulfate proteoglycan that is typically found in basal lamina of adult and embryonic tissues. Recent studies have demonstrated that perlecan accumulates impressively during cartilage development and is maintained as the major heparan sulfate proteoglycan of adult cartilage. In vertebrates, perlecan mutations result in skeletal defects. Moreover, in vitro studies indicate that perlecan can stimulate early stages of cartilage differentiation and cooperate with chondrogenic growth factors to promote this process. This short article will summarize these results and propose a model for perlecan function that incorporates these genetic and cell biological findings.
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Affiliation(s)
- Ronald R Gomes
- Department of Biological Sciences, University of Delaware, Newark, Del., USA.
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32
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Watanabe M, Aoki Y, Kase H, Tanaka K. Heparanase expression and angiogenesis in endometrial cancer. Gynecol Obstet Invest 2004; 56:77-82. [PMID: 12904690 DOI: 10.1159/000072821] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2003] [Accepted: 01/23/2003] [Indexed: 11/19/2022]
Abstract
Human heparanase has been shown to function in tumor progression, metastatic spread, and tumor angiogenesis. The aim of the present study was to assess heparanase expression in endometrial cancer in correlation with neovascularization and clinicopathological factors. Forty endometrial cancers were obtained from previously untreated patients (median age 55.5, range 33-78 years). The expression of heparanase mRNA was evaluated using a semiquantitative reverse transcriptase-polymerase chain reaction. Tumor angiogenesis was assessed using microvessel counting. The Mann-Whitney U test, one-factor ANOVA test, and Spearman's test were used to determine the relationship between heparanase expression, microvessel density, and clinicopathological parameters. The expression of heparanase mRNA was detected in 20 of 40 (50%) endometrial cancers, and was significantly correlated with FIGO stage IIIc (p=0.0075), the presence of lymph-vascular space involvement (p=0.0041), lymph node metastasis (p=0.0049), and histological tumor grade (p=0.0030). Microvessel density was also associated with FIGO stage IIIc (p=0.027), the presence of lymph-vascular space involvement (p=0.001), lymph node metastasis (p=0.038), ovarian metastasis (p=0.030) and histological tumor grade (p=0.0030). Moreover, we found a strong positive correlation between heparanase expression and microvessel density (r2=0.475, p=0.0001). These results suggest that the expression of heparanase may influence different malignant behaviors in endometrial cancer.
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Affiliation(s)
- Minoru Watanabe
- Department of Obstetrics and Gynecology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.
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Abstract
In recent years, the basement membrane (BM)--a specialized form of extracellular matrix (ECM)--has been recognized as an important regulator of cell behaviour, rather than just a structural feature of tissues. The BM mediates tissue compartmentalization and sends signals to epithelial cells about the external microenvironment. The BM is also an important structural and functional component of blood vessels, constituting an extracellular microenvironment sensor for endothelial cells and pericytes. Vascular BM components have recently been found to be involved in the regulation of tumour angiogenesis, making them attractive candidate targets for potential cancer therapies.
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Affiliation(s)
- Raghu Kalluri
- Center for Matrix Biology, Department of Medicine, Dana 514, Beth Israel Deaconess Medical Center and Harvard Medical School, 330 Brookline Avenue, Boston, Massachusetts 02215, USA.
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Mongiat M, Sweeney SM, San Antonio JD, Fu J, Iozzo RV. Endorepellin, a novel inhibitor of angiogenesis derived from the C terminus of perlecan. J Biol Chem 2003; 278:4238-49. [PMID: 12435733 DOI: 10.1074/jbc.m210445200] [Citation(s) in RCA: 262] [Impact Index Per Article: 11.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Perlecan, a ubiquitous basement membrane heparan sulfate proteoglycan, plays key roles in blood vessel growth and structural integrity. We discovered that the C terminus of perlecan potently inhibited four aspects of angiogenesis: endothelial cell migration, collagen-induced endothelial tube morphogenesis, and blood vessel growth in the chorioallantoic membrane and in Matrigel plug assays. The C terminus of perlecan was active at nanomolar concentrations and blocked endothelial cell adhesion to fibronectin and type I collagen, without directly binding to either protein; henceforth we have named it "endorepellin." We also found that endothelial cells possess a significant number of high affinity (K(d) of 11 nm) binding sites for endorepellin and that endorepellin binds endostatin and counteracts its anti-angiogenic effects. Thus, endorepellin represents a novel anti-angiogenic product, which may retard tumor neovascularization and hence tumor growth in vivo.
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Affiliation(s)
- Maurizio Mongiat
- Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA
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35
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Zanchetta P, Guezennec J. Surface thermodynamics of osteoblasts: relation between hydrophobicity and bone active biomaterials. Colloids Surf B Biointerfaces 2001. [DOI: 10.1016/s0927-7765(01)00200-4] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
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Lundmark K, Tran PK, Kinsella MG, Clowes AW, Wight TN, Hedin U. Perlecan inhibits smooth muscle cell adhesion to fibronectin: role of heparan sulfate. J Cell Physiol 2001; 188:67-74. [PMID: 11382923 DOI: 10.1002/jcp.1094] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Smooth muscle cell migration, proliferation, and deposition of extracellular matrix are key events in atherogenesis and restenosis development. To explore the mechanisms that regulate smooth muscle cell function, we have investigated whether perlecan, a basement membrane heparan sulfate proteoglycan, modulates interaction between smooth muscle cells and other matrix components. A combined substrate of fibronectin and perlecan showed a reduced adhesion of rat aortic smooth muscle cells by 70-90% in comparison to fibronectin alone. In contrast, perlecan did not interfere with cell adhesion to laminin. Heparinase treated perlecan lost 60% of its anti-adhesive effect. Furthermore, heparan sulfate as well as heparin reduced smooth muscle cell adhesion when combined with fibronectin whereas neither hyaluronan nor chondroitin sulfate had any anti-adhesive effects. Addition of heparin as a second coating to a preformed fibronectin matrix did not affect cell adhesion. Cell adhesion to the 105- and 120 kDa cell-binding fragments of fibronectin, lacking the main heparin-binding domains, was also inhibited by heparin. In addition, co-coating of fibronectin and (3)H-heparin showed that heparin was not even incorporated in the substrate. Morphologically, smooth muscle cells adhering to a substrate prepared by co-coating of fibronectin and perlecan or heparin were small, rounded, lacked focal contacts, and showed poorly developed stress fibers of actin. The results show that the heparan sulfate chains of perlecan lead to altered interactions between smooth muscle cells and fibronectin, possibly due to conformational changes in the fibronectin molecule. Such interactions may influence smooth muscle cell function in atherogenesis and vascular repair processes.
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Affiliation(s)
- K Lundmark
- Department of Surgical Sciences, Division of Vascular Surgery, Karolinska Hospital, SE-171, 76 Stockholm, Sweden.
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37
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Armeanu S, MÜller CA, Klein G. Involvement of E-cadherin in the Development of Erythroid Cells; Subject Heading. HEMATOLOGY (AMSTERDAM, NETHERLANDS) 2001; 5:307-316. [PMID: 11399629 DOI: 10.1080/10245332.2000.11746524] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/21/2022]
Abstract
The cadherins represent a large family of structurally and functionally related cell adhesion molecules involved in morphogenesis of multicellular organisms and maintenance of solid tissues. In the hematopoietic system, however, almost nothing was known about the involvement of this family. PCR screening of RNA of human bone marrow mononuclear cells with specific primers for different classical cadherins revealed that members of this family are also expressed by bone marrow cells. Here we report that E-cadherin, which is mainly expressed by cells of epithelial origin, plays a critical role in the development of human erythrocytes. FACS analysis with human E-cadherin-specific antibodies and the use of immunoaffinity columns revealed that expression of E-cadherin is restricted to defined maturation stages of the erythropoietic cell lineage. Erythroblasts and normoblasts express E-cadherin, but mature erythrocytes do not. Lymphoid and all the other myeloid cell lineages do not express E-cadherin at any developmental stage. The differentiation of the erythroid lineage in vitro could be influenced by addition of anti-E-cadherin antibodies in a concentration dependent manner indicating a direct involvement of this cell adhesion molecule in the differentiation process. In line with these in vitro data is the finding that E-cadherin is down regulated during erythroleukemia on the developing erythroid cells. Our results suggest an unanticipated function of E-cadherin in the hematopoietic system.
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Affiliation(s)
- Sorin Armeanu
- Medical University Clinic, Section for Transplantation Immunology and Immunohematology, 72072 Tübingen, FRG
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Sixt M, Hallmann R, Wendler O, Scharffetter-Kochanek K, Sorokin LM. Cell adhesion and migration properties of beta 2-integrin negative polymorphonuclear granulocytes on defined extracellular matrix molecules. Relevance for leukocyte extravasation. J Biol Chem 2001; 276:18878-87. [PMID: 11278780 DOI: 10.1074/jbc.m010898200] [Citation(s) in RCA: 93] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023] Open
Abstract
Regulated adhesion of leukocytes to the extracellular matrix is essential for transmigration of blood vessels and subsequent migration into the stroma of inflamed tissues. Although beta(2)-integrins play an indisputable role in adhesion of polymorphonuclear granulocytes (PMN) to endothelium, we show here that beta(1)- and beta(3)-integrins but not beta(2)-integrin are essential for the adhesion to and migration on extracellular matrix molecules of the endothelial cell basement membrane and subjacent interstitial matrix. Mouse wild type and beta(2)-integrin null PMN and the progranulocytic cell line 32DC13 were employed in in vitro adhesion and migration assays using extracellular matrix molecules expressed at sites of extravasation in vivo, in particular the endothelial cell laminins 8 and 10. Wild type and beta(2)-integrin null PMN showed the same pattern of ECM binding, indicating that beta(2)-integrins do not mediate specific adhesion of PMN to the extracellular matrix molecules tested; binding was observed to the interstitial matrix molecules, fibronectin and vitronectin, via integrins alpha(5)beta(1) and alpha(v)beta(3), respectively; to laminin 10 via alpha(6)beta(1); but not to laminins 1, 2, and 8, collagen type I and IV, perlecan, or tenascin-C. PMN binding to laminins 1, 2, and 8 could not be induced despite surface expression of functionally active integrin alpha(6)beta(1), a major laminin receptor, demonstrating that expression of alpha(6)beta(1) alone is insufficient for ligand binding and suggesting the involvement of accessory factors. Nevertheless, laminins 1, 8, and 10 supported PMN migration, indicating that differential cellular signaling via laminins is independent of the extent of adhesion. The data demonstrate that adhesive and nonadhesive interactions with components of the endothelial cell basement membrane and subjacent interstitium play decisive roles in controlling PMN movement into sites of inflammation and illustrate that beta(2)-integrins are not essential for such interactions.
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Affiliation(s)
- M Sixt
- Interdisciplinary Center for Clinical Research and the Institute for Experimental Medicine, Nikolaus Fiebiger Center, University of Erlangen-Nuremberg, 91054 Erlangen, Germany
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Abstract
Laminins are a family of disulfide-linked heterotrimeric proteins consisting of 3 different subunits termed α, β, and γ chains. Combinations of 11 characterized laminin subunits (α1-α5, β1-β3, and γ1-γ3) generate at least 12 laminin isoforms, which can serve different functions. Although expression of laminin in the hematopoietic microenvironment has been known for many years, the nature of the laminin isoforms present in the human bone marrow is poorly characterized. The present study attempts to clarify this issue. Reverse transcriptase–polymerase chain reaction analysis of human bone marrow stromal cells suggested the expression of many laminin isoforms in the marrow. Northern blot and immunoblot analysis, however, showed that laminin-8/9 and laminin-10/11 are the most abundant laminin isoforms synthesized by human bone marrow stromal cells. Other isoforms, if present, certainly play a minor role in the hematopoietic microenvironment. Functionally, laminin-10/11 preparations showed strong adhesive interactions with human CD34+ cell lines. Antibodies against the β1 integrin subunit inhibited these interactions. Other laminin isoforms, especially laminin-1 and laminin-2/4, showed only weak or no adhesive interactions with the hematopoietic cell lines tested, explaining former negative results. In addition to its adhesion-mediating properties, laminin-10/11 preparations also showed a mitogenic activity for human hematopoietic progenitor cells. Taken together, these data suggest that laminin in the bone marrow plays a hitherto unexpected important function in the development of hematopoietic progenitor cells.
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Abstract
AbstractLaminins are a family of disulfide-linked heterotrimeric proteins consisting of 3 different subunits termed α, β, and γ chains. Combinations of 11 characterized laminin subunits (α1-α5, β1-β3, and γ1-γ3) generate at least 12 laminin isoforms, which can serve different functions. Although expression of laminin in the hematopoietic microenvironment has been known for many years, the nature of the laminin isoforms present in the human bone marrow is poorly characterized. The present study attempts to clarify this issue. Reverse transcriptase–polymerase chain reaction analysis of human bone marrow stromal cells suggested the expression of many laminin isoforms in the marrow. Northern blot and immunoblot analysis, however, showed that laminin-8/9 and laminin-10/11 are the most abundant laminin isoforms synthesized by human bone marrow stromal cells. Other isoforms, if present, certainly play a minor role in the hematopoietic microenvironment. Functionally, laminin-10/11 preparations showed strong adhesive interactions with human CD34+ cell lines. Antibodies against the β1 integrin subunit inhibited these interactions. Other laminin isoforms, especially laminin-1 and laminin-2/4, showed only weak or no adhesive interactions with the hematopoietic cell lines tested, explaining former negative results. In addition to its adhesion-mediating properties, laminin-10/11 preparations also showed a mitogenic activity for human hematopoietic progenitor cells. Taken together, these data suggest that laminin in the bone marrow plays a hitherto unexpected important function in the development of hematopoietic progenitor cells.
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41
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Jenney CR, Anderson JM. Adsorbed serum proteins responsible for surface dependent human macrophage behavior. JOURNAL OF BIOMEDICAL MATERIALS RESEARCH 2000; 49:435-47. [PMID: 10602077 DOI: 10.1002/(sici)1097-4636(20000315)49:4<435::aid-jbm2>3.0.co;2-y] [Citation(s) in RCA: 193] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
Substrate specific cellular responses are the result of a complex biological system that includes protein adsorption, receptor-ligand binding, and signal transduction. This investigation attempted to identify specific proteins adsorbed from human serum that may be responsible for the previously reported in vitro surface dependent behavior of human macrophages and foreign body giant cells (FBGCs). The adsorption of human albumin, alpha(2)-macroglobulin, complement factor 3b, fibronectin, IgG, thrombospondin, vitronectin (VN), and von Willebrand factor (vWF) from a 25% serum solution was quantified with (125)I-labeled protein. Adsorption substrates included clean glass, alkyl-silane modified glass, amino-silane modified glass, poly(ethylene oxide) (PEO)-coupled glass, and the reference biomaterials poly(etherurethane urea), Silastic(R), and poly(tetrafluoroethylene) (PTFE). Following quantification of 2-h adsorption, surfaces were treated with sodium dodecyl sulfate (SDS) and the level of adsorbed proteins remaining was quantified. The pre- and post-SDS adsorption were both compared to previously reported surface dependent in vitro macrophage and FBGC behavior on the same surfaces; however, no correlations could be made. Adsorption strength, defined as the percentage of initially adsorbed protein that remained adsorbed after SDS treatment, correlated well with previously reported in vitro cellular behavior indicating that adsorbed vWF, IgG, and VN may be involved in the modulation of adherent macrophage and FBGC behavior. Those surfaces that strongly adsorbed vWF also inhibited long-term macrophage adhesion, while those surfaces that strongly adsorbed IgG promoted long-term macrophage adhesion. In addition, the highest levels of FBGC formation had been observed only on those surfaces that strongly adsorbed VN. Subsequent human monocyte cultures on protein preadsorbed substrates confirmed the inhibitory effect of adsorbed vWF and the promoting effect of IgG on longterm macrophage adhesion as predicted by adsorption strength correlations. However, preadsorbed VN was not observed to modulate FBGC formation, which is in contrast to the conclusions of the adsorption correlations.
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Affiliation(s)
- C R Jenney
- Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio 44106, USA
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42
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Paka L, Goldberg IJ, Obunike JC, Choi SY, Saxena U, Goldberg ID, Pillarisetti S. Perlecan mediates the antiproliferative effect of apolipoprotein E on smooth muscle cells. An underlying mechanism for the modulation of smooth muscle cell growth? J Biol Chem 1999; 274:36403-8. [PMID: 10593935 DOI: 10.1074/jbc.274.51.36403] [Citation(s) in RCA: 40] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Apolipoprotein E (apoE) is known to inhibit cell proliferation; however, the mechanism of this inhibition is not clear. We recently showed that apoE stimulates endothelial production of heparan sulfate (HS) enriched in heparin-like sequences. Because heparin and HS are potent inhibitors of smooth muscle cell (SMC) proliferation, in this study we determined apoE effects on SMC HS production and cell growth. In confluent SMCs, apoE (10 microg/ml) increased (35)SO(4) incorporation into PG in media by 25-30%. The increase in the medium was exclusively due to an increase in HSPGs (2.2-fold), and apoE did not alter chondroitin and dermatan sulfate proteoglycans. In proliferating SMCs, apoE inhibited [(3)H]thymidine incorporation into DNA by 50%; however, despite decreasing cell number, apoE increased the ratio of (35)SO(4) to [(3)H]thymidine from 2 to 3.6, suggesting increased HS per cell. Purified HSPGs from apoE-stimulated cells inhibited cell proliferation in the absence of apoE. ApoE did not inhibit proliferation of endothelial cells, which are resistant to heparin inhibition. Analysis of the conditioned medium from apoE-stimulated cells revealed that the HSPG increase was in perlecan and that apoE also stimulated perlecan mRNA expression by >2-fold. The ability of apoE isoforms to inhibit cell proliferation correlated with their ability to stimulate perlecan expression. An anti-perlecan antibody completely abrogated the antiproliferative effect of apoE. Thus, these data show that perlecan is a potent inhibitor of SMC proliferation and is required to mediate the antiproliferative effect of apoE. Because other growth modulators also regulate perlecan expression, this may be a key pathway in the regulation of SMC growth.
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Affiliation(s)
- L Paka
- Department of Radiation Oncology, North Shore-Long Island Jewish Health System, Manhasset, New York 11030, USA
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43
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Groffen AJ, Veerkamp JH, Monnens LA, van den Heuvel LP. Recent insights into the structure and functions of heparan sulfate proteoglycans in the human glomerular basement membrane. Nephrol Dial Transplant 1999; 14:2119-29. [PMID: 10489220 DOI: 10.1093/ndt/14.9.2119] [Citation(s) in RCA: 69] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023] Open
Abstract
As the first barrier to be crossed on the way to urinary space, the glomerular basement membrane (GBM) plays a key role in renal function. The permeability of the GBM for a given molecule is highly dependent on its size, shape and charge. As early as 1980, the charge-selective permeability was demonstrated to relate to the electrostatic properties of covalently bound heparan sulfates (HS) within the GBM. Since the identification of perlecan as a heparan sulfate proteoglycan (HSPG) of basement membranes, the hypothesis that perlecan could be a crucial determinant of GBM permselectivity received considerable attention. In addition to perlecan, the GBM also contains other HSPG species, one of which was identified as agrin. The high local expression of agrin in the GBM, together with the presence of agrin receptors at the cell matrix interface, suggests that this HSPG contributes to glomerular function in multiple ways. Here, we review the current knowledge regarding the structure and functions of HSPGs in the GBM, and discuss how these molecules could be involved in various glomerular diseases. Possible directions for future investigation are suggested.
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Affiliation(s)
- A J Groffen
- Department of Pediatrics, University of Nijmegen, The Netherlands
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Whitelock JM, Graham LD, Melrose J, Murdoch AD, Iozzo RV, Underwood PA. Human perlecan immunopurified from different endothelial cell sources has different adhesive properties for vascular cells. Matrix Biol 1999; 18:163-78. [PMID: 10372557 DOI: 10.1016/s0945-053x(99)00014-1] [Citation(s) in RCA: 113] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
Perlecan, a major heparan sulfate proteoglycan of vascularized tissues, was immunopurified from media conditioned by human endothelial cells of both arterial and venous origin. The heparan sulfate moiety of perlecan from cultured arterial cells differed in amount and/or composition from that produced by a transformed cell line of venous origin. Both forms of perlecan bound basic fibroblast growth factor with Kd approximately 70 nM. In ELISA experiments, perlecan and its protein core bound to various extracellular matrix components in a manner that was strongly influenced by the format of the assay. Human vascular smooth muscle cells and human endothelial cells adhered to perlecan-coated surfaces, and both cell types adhered better to the venous cell-derived than to the arterial cell-derived perlecan. Removal of the heparan sulfate chains abolished this difference and increased the ability of both types of perlecan to adhere vascular cells. Denaturation of perlecan and its protein core also rendered each of them more adhesive, indicating the presence of conformation-independent adhesion determinants in the polypeptide sequence. Their location was investigated using recombinant perlecan domains. Overall, our results represent the first demonstration of human perlecan acting as an adhesive molecule for human vascular cells and suggest that it may play a role in vascular wound healing.
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Affiliation(s)
- J M Whitelock
- Co-operative Research Center for Cardiac Technology, North Ryde, NSW, Australia.
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Abstract
The proteoglycan superfamily now contains more than 30 full-time molecules that fulfill a variety of biological functions. Proteoglycans act as tissue organizers, influence cell growth and the maturation of specialized tissues, play a role as biological filters and modulate growth-factor activities, regulate collagen fibrillogenesis and skin tensile strength, affect tumor cell growth and invasion, and influence corneal transparency and neurite outgrowth. Additional roles, derived from studies of mutant animals, indicate that certain proteoglycans are essential to life whereas others might be redundant. The review focuses on the most recent genetic and molecular biological studies of the matrix proteoglycans, broadly defined as proteoglycans secreted into the pericellular matrix. Special emphasis is placed on the molecular organization of the protein core, the utilization of protein modules, the gene structure and transcriptional control, and the functional roles of the various proteoglycans. When possible, proteoglycans have been grouped into distinct gene families and subfamilies offering a simplified nomenclature based on their protein core design. The structure-function relationship of some paradigmatic proteoglycans is discussed in depth and novel aspects of their biology are examined.
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Affiliation(s)
- R V Iozzo
- Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107-6799, USA.
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Kibler C, Schermutzki F, Waller HD, Timpl R, Müller CA, Klein G. Adhesive interactions of human multiple myeloma cell lines with different extracellular matrix molecules. CELL ADHESION AND COMMUNICATION 1998; 5:307-23. [PMID: 9762471 DOI: 10.3109/15419069809040300] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
Multiple myeloma represents a human B cell malignancy which is characterized by a predominant localization of the malignant cell clone within the bone marrow. With the exception of the terminal stage of the disease the myeloma tumor cells do not circulate in the peripheral blood. The bone marrow microenvironment is believed to play an important role in homing, proliferation and terminal differentiation of myeloma cells. Here we have studied the expression of several extracellular matrix (ECM) molecules in the bone marrow of multiple myeloma patients and analyzed their adhesive capacities with four different human myeloma-derived cell lines. All ECM molecules analyzed (tenascin, laminin, fibronectin, collagen types I, III, V and VI) could be detected in bone marrow cryostat sections of multiple myeloma patients. Adhesion assays showed that only laminin, the microfibrillar collagen type VI and fibronectin were strong adhesive components for the myeloma cell lines U266, IM-9, OPM-2 and NCI-H929. Tenascin and collagen type I were only weak adhesive substrates for these myeloma cells. Adhesion to laminin and fibronectin was beta 1-integrin-mediated since addition of anti-beta 1-integrin antibodies could inhibit the binding of the four different cell types to both matrix molecules. In contrast, integrins do not seem to be involved in binding of the myeloma cells to collagen type VI. Instead, inhibition of binding by heparin suggested that membrane-bound heparan sulfate proteoglycans are responsible ligands for binding to collagen type VI. Adhesion assays with several B-cell lines resembling earlier differentiation stages revealed only weak interactions with tenascin and no interactions with collagen type VI, laminin or fibronectin. In summary, the interactions of human myeloma cells with the extracellular matrix may explain the specific retention of the plasma cells within the bone marrow.
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Affiliation(s)
- C Kibler
- University Medical Clinic, Dept. II, Tübingen, FRG
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Seiffert M, Beck SC, Schermutzki F, Müller CA, Erickson HP, Klein G. Mitogenic and adhesive effects of tenascin-C on human hematopoietic cells are mediated by various functional domains. Matrix Biol 1998; 17:47-63. [PMID: 9628252 DOI: 10.1016/s0945-053x(98)90124-x] [Citation(s) in RCA: 47] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
In the adult organism, the extracellular matrix molecule tenascin-C is prominently expressed in the bone marrow. Bone marrow mononuclear cells can adhere to plastic-immobilized tenascin-C, and in the present study we have used bacterial expression proteins to map the domains of tenascin-C responsible for binding of hematopoietic cells. A strong binding site was found to be located within the fibrinogen-like domain, and this binding could be inhibited by heparin, suggesting interactions with membrane-bound heparan sulfate proteoglycans. A second strong binding site was identified within the fibronectin type III-like repeats 6-8, and was also inhibitable by heparin. Adhesion to both attachment sites could not be blocked by various anti-integrin antibodies. A third hematopoietic cell binding site is located in the fibronectin type III-like repeats 1-5, which harbor an RGD sequence in the third fibronectin type III-like repeat. Binding to this domain, however, seems to be RGD-independent, since RGD-containing peptides could not inhibit cell binding; the addition of heparin also did not block adhesion to this domain. Since contradictory results had been reported on a proliferative effect of soluble tenascin-C, we also analyzed its activity on hematopoietic cells. The heterogeneous bone marrow mononuclear cells show a striking proliferative response in the presence of tenascin-C which is concentration-dependent. This result indicates a strong mitogenic activity of tenascin-C on primary hematopoietic cells. Using recombinant fragments of human tenascin-C, we identified several mitogenic domains within the tenascin-C molecule. These adhesive and mitogenic effects of tenascin-C suggest a direct functional association with proliferation and differentiation of hematopoietic cells within the bone marrow microenvironment.
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Affiliation(s)
- M Seiffert
- University Medical Clinic, Dept. II, Tübingen, Germany
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Klein G, Kibler C, Schermutzki F, Brown J, Müller CA, Timpl R. Cell binding properties of collagen type XIV for human hematopoietic cells. Matrix Biol 1998; 16:307-17. [PMID: 9503364 DOI: 10.1016/s0945-053x(98)90002-6] [Citation(s) in RCA: 23] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Collagen XIV, which belongs to the subclass of fibril-associated collagens with interrupted triple helices (FACITs), is a homotrimeric molecule consisting of three alpha 1 (XIV) chains. Collagen type XIV is strongly expressed in the native human bone marrow, as shown by immunofluorescence staining and immunoblotting with an affinity-purified antibody. Hematopoietic cell lines of myeloid (KG1a, U937, K562) and lymphoid (U266, IM-9) origin were able to attach firmly to purified human collagen XIV preparations. Attachment of these cells was shown to be concentration-dependent. However, other hematopoietic cell lines tested were unable to adhere to collagen XIV, indicating restriction of this cellular interaction. The cellular receptors involved in cell binding to collagen type XIV are probably membrane-bound heparansulfate proteoglycans, since only the the addition of heparin inhibited attachment of the hematopoietic cells to collagen XIV in a concentration-dependent manner. Antibodies against the beta 1-integrin subunit could not interfere with binding to collagen type XIV. Using purified fragments of collagen XIV, it could be demonstrated that at least two different heparin-sensitive adhesion sites are present in the N-terminal globular domain and in the triple-helical domain. These data indicate that collagen XIV represents another collagen type expressed in human bone marrow with strong cell binding properties for defined populations of hematopoietic cells.
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Affiliation(s)
- G Klein
- University Medical Clinic, Department of Internal Medicine II, Tübingen, Germany
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Abstract
Perlecan is a modular heparan sulfate proteoglycan that is an intrinsic constituent of all basement membranes and extracellular matrices. Because of its strategic position and unique structure, perlecan has been implicated in modulating the activity of various growth factors required for normal development and tissue homeostasis. To gain insights into the potential function of perlecan in vivo, we examined the spatiotemporal distribution of its mRNA and protein core during murine embryogenesis. We utilized a new affinity-purified antibody that recognizes specifically the protein core of perlecan together with an in situ RT-PCR approach to perform a systematic analysis of perlecan expression and deposition during murine ontogeny. Perlecan appeared early (E10.5) in tissues of vasculogenesis including heart, pericardium, and major blood vessels. Its early expression coincided with the development of the cardiovascular system. Subsequently (E11-13), the greatest deposition of perlecan occurred within the developing cartilage, especially the cartilage undergoing endochondral ossification, where it remained elevated throughout all the developmental stages, and up to adulthood. Interestingly, the mRNA levels of perlecan were always higher in all the vascularized tissues, principally within endothelial cells, while chondrocytes displayed relatively low mRNA levels. This suggests a higher biosynthesis and turnover rates in the blood vessels vis-à-vis those of cartilaginous and other mesenchymal tissues. During later stages of development (E13-17.5) perlecan mRNA levels progressively increased and its expression correlated with the onset of tissue differentiation of various parenchymal organs including the developing kidneys, lungs, liver, spleen, and gastrointestinal tract. The central nervous system showed no perlecan expression with the exception of the calvaria and choroid plexus. Collectively, the results indicate that perlecan may play crucial roles not only in vasculogenesis but also in the maturation and maintenance of differentiated tissues, including cartilage.
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Affiliation(s)
- M Handler
- Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA
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Iozzo RV, Pillarisetti J, Sharma B, Murdoch AD, Danielson KG, Uitto J, Mauviel A. Structural and functional characterization of the human perlecan gene promoter. Transcriptional activation by transforming growth factor-beta via a nuclear factor 1-binding element. J Biol Chem 1997; 272:5219-28. [PMID: 9030592 DOI: 10.1074/jbc.272.8.5219] [Citation(s) in RCA: 86] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023] Open
Abstract
Perlecan, a modular heparan sulfate proteoglycan of basement membranes and cell surfaces, plays a crucial role in regulating the assembly of extracellular matrices and the binding of nutrients and growth factors to target cells. To achieve a molecular understanding of perlecan gene regulation, we isolated the 5'-flanking region and investigated its functional promoter activity and its response to cytokines. Transient cell transfection assays, using plasmid constructs harboring the perlecan promoter linked to the chloramphenicol acetyltransferase reporter gene, demonstrated that the largest approximately 2.5-kilobase construct contained maximal promoter activity. This promoter region was functionally active in a variety of cells of diverse histogenetic origin, thus corroborating the widespread expression of this gene product. Stepwise 5' deletion analyses demonstrated that the -461-base pair (bp) proximal promoter retained approximately 90% of the total activity, and internal deletions confirmed that the most proximal sequence was essential for proper promoter activity. Nanomolar amounts of transforming growth factor-beta induced 2-3-fold perlecan mRNA and protein core levels in normal human skin fibroblasts, and this induction was transcriptionally regulated; in contrast, tumor necrosis factor-alpha had no effect and was incapable of counteracting the effects of TGF-beta. Using additional 5' deletions and DNase footprinting analyses, we mapped the TGF-beta responsive region to a sequence of 177 bp contained between -461 and -285. This region harbored a 14-bp element similar to a TGF-beta-responsive element present in the promoters of collagen alpha1(I), alpha2(I), elastin, and growth hormone. Electrophoretic mobility shift assays and mutational analyses demonstrated that the perlecan TGF-beta-responsive element bound specifically to TGF-beta-inducible nuclear proteins with high affinity for NF-1 member(s) of transcription factors.
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Affiliation(s)
- R V Iozzo
- Department of Pathology, Anatomy, and Cell Biology,Jefferson Medical C ollege, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
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