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      Angiopoietin-2 promotes osteogenic differentiation of thoracic ligamentum flavum cells via modulating the Notch signaling pathway

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          Abstract

          Thoracic ossification of the ligamentum flavum (TOLF) is heterotopic ossification of spinal ligaments, which may cause serious thoracic spinal canal stenosis and myelopathy. However, the underlying etiology remains inadequately understood. In this study, the ossification patterns of TOLF were analyzed by micro-computer tomography (micro-CT). The expression profile of genes associated with angiogenesis was analyzed in thoracic ligamentum flavum cells at sites of different patterns of ossification using RNA sequencing. Significant differences in the expression profile of several genes were identified from Gene Ontology (GO) analysis. Angiopoietin-2 (ANGPT2) was significantly up-regulated in primary thoracic ligamentum flavum cells during osteogenic differentiation. To address the effect of ANGPT2 on Notch signaling and osteogenesis, ANGPT2 stimulation increased the expression of Notch2 and osteogenic markers of primary thoracic ligamentum flavum cells of immature ossification, while inhibition of ANGPT2 exhibited opposite effect on Notch pathway and osteogenesis of cells of mature ossification. These findings provide the first evidence for positive regulation of ANGPT2 on osteogenic differentiation in human thoracic ligamentum flavum cells via modulating the Notch signaling pathway.

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          Most cited references33

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          Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone.

          The mammalian skeletal system harbours a hierarchical system of mesenchymal stem cells, osteoprogenitors and osteoblasts sustaining lifelong bone formation. Osteogenesis is indispensable for the homeostatic renewal of bone as well as regenerative fracture healing, but these processes frequently decline in ageing organisms, leading to loss of bone mass and increased fracture incidence. Evidence indicates that the growth of blood vessels in bone and osteogenesis are coupled, but relatively little is known about the underlying cellular and molecular mechanisms. Here we identify a new capillary subtype in the murine skeletal system with distinct morphological, molecular and functional properties. These vessels are found in specific locations, mediate growth of the bone vasculature, generate distinct metabolic and molecular microenvironments, maintain perivascular osteoprogenitors and couple angiogenesis to osteogenesis. The abundance of these vessels and associated osteoprogenitors was strongly reduced in bone from aged animals, and pharmacological reversal of this decline allowed the restoration of bone mass.
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            Endothelial Notch activity promotes angiogenesis and osteogenesis in bone.

            Blood vessel growth in the skeletal system and osteogenesis seem to be coupled, suggesting the existence of molecular crosstalk between endothelial and osteoblastic cells. Understanding the nature of the mechanisms linking angiogenesis and bone formation should be of great relevance for improved fracture healing or prevention of bone mass loss. Here we show that vascular growth in bone involves a specialized, tissue-specific form of angiogenesis. Notch signalling promotes endothelial cell proliferation and vessel growth in postnatal long bone, which is the opposite of the well-established function of Notch and its ligand Dll4 in the endothelium of other organs and tumours. Endothelial-cell-specific and inducible genetic disruption of Notch signalling in mice not only impaired bone vessel morphology and growth, but also led to reduced osteogenesis, shortening of long bones, chondrocyte defects, loss of trabeculae and decreased bone mass. On the basis of a series of genetic experiments, we conclude that skeletal defects in these mutants involved defective angiocrine release of Noggin from endothelial cells, which is positively regulated by Notch. Administration of recombinant Noggin, a secreted antagonist of bone morphogenetic proteins, restored bone growth and mineralization, chondrocyte maturation, the formation of trabeculae and osteoprogenitor numbers in endothelial-cell-specific Notch pathway mutants. These findings establish a molecular framework coupling angiogenesis, angiocrine signals and osteogenesis, which may prove significant for the development of future therapeutic applications.
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              Angiopoietin-2 sensitizes endothelial cells to TNF-alpha and has a crucial role in the induction of inflammation.

              The angiopoietins Ang-1 and Ang-2 have been identified as ligands of the receptor tyrosine kinase Tie-2 (refs. 1,2). Paracrine Ang-1-mediated activation of Tie-2 acts as a regulator of vessel maturation and vascular quiescence. In turn, the antagonistic ligand Ang-2 acts by an autocrine mechanism and is stored in endothelial Weibel-Palade bodies from where it can be rapidly released upon stimulation. The rapid release of Ang-2 implies functions of the angiopoietin-Tie system beyond its established role during vascular morphogenesis as a regulator of rapid vascular responses. Here we show that mice deficient in Ang-2 (encoded by the gene Angpt2) cannot elicit an inflammatory response in thioglycollate-induced or Staphylococcus aureus-induced peritonitis, or in the dorsal skinfold chamber model. Recombinant Ang-2 restores the inflammation defect in Angpt2(-/-) mice. Intravital microscopy showed normal TNF-alpha-induced leukocyte rolling in the vasculature of Angpt2(-/-)mice, but rolling cells did not firmly adhere to activated endothelium. Cellular experiments showed that Ang-2 promotes adhesion by sensitizing endothelial cells toward TNF-alpha and modulating TNF-alpha-induced expression of endothelial cell adhesion molecules. Together, these findings identify Ang-2 as an autocrine regulator of endothelial cell inflammatory responses. Ang-2 thereby acts as a switch of vascular responsiveness exerting a permissive role for the activities of proinflammatory cytokines.
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                Author and article information

                Contributors
                Role: InvestigationRole: MethodologyRole: Writing – original draft
                Role: ConceptualizationRole: Supervision
                Role: Methodology
                Role: Methodology
                Role: Methodology
                Role: Methodology
                Role: Methodology
                Role: Methodology
                Role: Methodology
                Role: ConceptualizationRole: SupervisionRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                17 December 2018
                2018
                : 13
                : 12
                : e0209300
                Affiliations
                [1 ] Department of Orthopedics, Peking University Third Hospital, Beijing, China
                [2 ] Central Laboratory, Peking University International Hospital, Beijing, China
                [3 ] Daobio, Inc. Dallas, Texas, United States of America
                [4 ] Department of Orthopedics, Peking University International Hospital, Beijing, China
                [5 ] Bone Research Laboratory, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America
                Augusta University, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0002-7821-483X
                Article
                PONE-D-18-31944
                10.1371/journal.pone.0209300
                6296551
                30557327
                2c0d4eae-6693-47f0-ad50-f9b8b25f88e7
                © 2018 Yang et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 6 November 2018
                : 3 December 2018
                Page count
                Figures: 4, Tables: 0, Pages: 11
                Funding
                This work was supported by the National Natural Science Foundation of China (ZC, Grant numbers: 81772381 and 81700779) and Beijing Municipal Science&Technology Commission (CZ, Grant number: Z181100001818006). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Physiology
                Physiological Processes
                Bone Remodeling
                Ossification
                Medicine and Health Sciences
                Physiology
                Physiological Processes
                Bone Remodeling
                Ossification
                Biology and Life Sciences
                Cell Biology
                Signal Transduction
                Cell Signaling
                Notch Signaling
                Biology and Life Sciences
                Developmental Biology
                Cell Differentiation
                Biology and life sciences
                Molecular biology
                Molecular biology techniques
                Sequencing techniques
                RNA sequencing
                Research and analysis methods
                Molecular biology techniques
                Sequencing techniques
                RNA sequencing
                Biology and Life Sciences
                Physiology
                Cardiovascular Physiology
                Angiogenesis
                Medicine and Health Sciences
                Physiology
                Cardiovascular Physiology
                Angiogenesis
                Biology and Life Sciences
                Developmental Biology
                Angiogenesis
                Biology and life sciences
                Genetics
                Gene expression
                Gene regulation
                Small interfering RNAs
                Biology and life sciences
                Biochemistry
                Nucleic acids
                RNA
                Non-coding RNA
                Small interfering RNAs
                Biology and Life Sciences
                Developmental Biology
                Cell Differentiation
                Osteoblast Differentiation
                Biology and Life Sciences
                Genetics
                Gene Expression
                Custom metadata
                All relevant data are within the manuscript and its Supporting Information file.

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                Uncategorized

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