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      Determination of the physiological range of oxygen tension in bone marrow monocytes using two-photon phosphorescence lifetime imaging microscopy

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          Abstract

          Oxygen is a key regulator of both development and homeostasis. To study the role of oxygen, a variety of in vitro and ex vivo cell and tissue models have been used in biomedical research. However, because of ambiguity surrounding the level of oxygen that cells experience in vivo, the cellular pathway related to oxygenation state and hypoxia have been inadequately studied in many of these models. Here, we devised a method to determine the oxygen tension in bone marrow monocytes using two-photon phosphorescence lifetime imaging microscopy with the cell-penetrating phosphorescent probe, BTPDM1. Phosphorescence lifetime imaging revealed the physiological level of oxygen tension in monocytes to be 5.3% in live mice exposed to normal air. When the mice inhaled hypoxic air, the level of oxygen tension in bone marrow monocytes decreased to 2.4%. By performing in vitro cell culture experiment within the physiological range of oxygen tension, hypoxia changed the molecular phenotype of monocytes, leading to enhanced the expression of CD169 and CD206, which are markers of a unique subset of macrophages in bone marrow, osteal macrophages. This current study enables the determination of the physiological range of oxygen tension in bone marrow with spatial resolution at a cellular level and application of this information on oxygen tension in vivo to in vitro assays. Quantifying oxygen tension in tissues can provide invaluable information on metabolism under physiological and pathophyisological conditions. This method will open new avenues for research on oxygen biology.

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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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            Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion.

            The seven-transmembrane receptor CX(3)CR1 is a specific receptor for the novel CX(3)C chemokine fractalkine (FKN) (neurotactin). In vitro data suggest that membrane anchoring of FKN, and the existence of a shed, soluble FKN isoform allow for both adhesive and chemoattractive properties. Expression on activated endothelium and neurons defines FKN as a potential target for therapeutic intervention in inflammatory conditions, particularly central nervous system diseases. To investigate the physiological function of CX(3)CR1-FKN interactions, we generated a mouse strain in which the CX(3)CR1 gene was replaced by a green fluorescent protein (GFP) reporter gene. In addition to the creation of a mutant CX(3)CR1 locus, this approach enabled us to assign murine CX(3)CR1 expression to monocytes, subsets of NK and dendritic cells, and the brain microglia. Analysis of CX(3)CR1-deficient mice indicates that CX(3)CR1 is the only murine FKN receptor. Yet, defying anticipated FKN functions, absence of CX(3)CR1 interferes neither with monocyte extravasation in a peritonitis model nor with DC migration and differentiation in response to microbial antigens or contact sensitizers. Furthermore, a prominent response of CX(3)CR1-deficient microglia to peripheral nerve injury indicates unimpaired neuronal-glial cross talk in the absence of CX(3)CR1.
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              Direct measurement of local oxygen concentration in the bone marrow of live animals

              Characterizing how the microenvironment, or niche, regulates stem cell activity is central to understanding stem cell biology and to developing strategies for therapeutic manipulation of stem cells 1 . Low oxygen tension (hypoxia) is commonly thought to be a shared niche characteristic in maintaining quiescence in multiple stem cell types 2–4 . However, support for the existence of a hypoxic niche has largely come from indirect evidence such as proteomic analysis 5 , expression of HIF-1 and related genes 6 , and staining with surrogate hypoxic markers (e.g. pimonidazole) 6–8 . Here we perform direct in vivo measurements of local oxygen tension (pO2) in the bone marrow (BM) of live mice. Using two-photon phosphorescence lifetime microscopy (2PLM), we determined the absolute pO2 of the BM to be quite low (<32 mmHg) despite very high vascular density. We further uncovered heterogeneities in local pO2, with the lowest pO2 (~9.9 mmHg, or 1.3%) found in deeper peri-sinusoidal regions. The endosteal region, by contrast, is less hypoxic as it is perfused with small arteries that are often positive for the marker nestin. These pO2 values change dramatically after radiation and chemotherapy, pointing to the role of stress in altering the stem cell metabolic microenvironment.
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                Author and article information

                Contributors
                kenishik@mail.doshisha.ac.jp
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                10 March 2022
                10 March 2022
                2022
                : 12
                : 3497
                Affiliations
                [1 ]GRID grid.136593.b, ISNI 0000 0004 0373 3971, Graduate School of Medicine/Frontier Biosciences, , Osaka University, ; Yamada-oka 2-2, Suita, Osaka 565-0871 Japan
                [2 ]GRID grid.255178.c, ISNI 0000 0001 2185 2753, Laboratory of Cell Biology and Metabolic Biochemistry, Department of Medical Life Systems, Graduate School of Life and Medical Sciences, , Doshisha University, ; Tatara Miyakodani 1-3, Kyotanabe, Kyoto 610-0394 Japan
                [3 ]GRID grid.256642.1, ISNI 0000 0000 9269 4097, Department of Chemistry and Chemical Biology, , Gunma University, ; Kiryu, Gunma 376-8515 Japan
                [4 ]GRID grid.482562.f, Laboratory of Bioimaging and Drug Discovery, , National Institutes of Biomedical Innovation, Health and Nutrition, ; 7-6-8, Saito-Asagi, Ibaraki, Osaka 567-0085 Japan
                [5 ]GRID grid.136593.b, ISNI 0000 0004 0373 3971, Department of Immunology and Cell Biology, WPI-Immunology Frontier Research Center, , Osaka University, ; Yamada-oka 2-2, Suita, Osaka 565-0871 Japan
                [6 ]GRID grid.258799.8, ISNI 0000 0004 0372 2033, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, , Kyoto University, ; Kyoto, 615-8510 Japan
                [7 ]GRID grid.258799.8, ISNI 0000 0004 0372 2033, WPI-Research Initiative-Institute for Integrated Cell-Material Science, , Kyoto University, ; Kyoto, 606-8501 Japan
                Article
                7521
                10.1038/s41598-022-07521-9
                8913795
                35273210
                394ef6c7-3fa6-40de-a369-4bcd3ba24f87
                © The Author(s) 2022

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 11 November 2021
                : 15 February 2022
                Funding
                Funded by: Grants-in-Aid for Scientific Research on Innovative Areas from the JSPS
                Funded by: CREST, JST
                Funded by: Grants-in-Aid for Scientific Research (S) from the JSPS
                Funded by: FundRef http://dx.doi.org/10.13039/100007449, Takeda Science Foundation;
                Funded by: Grants-in-Aid for Scientific Research (B) from the JSPS
                Funded by: Yamada Science Foundation
                Funded by: Toray Science Foundation
                Funded by: the Nakatani Foundation
                Categories
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                © The Author(s) 2022

                Uncategorized
                differentiation,optical imaging
                Uncategorized
                differentiation, optical imaging

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