Mol. Cells 2021; 44(4): 254-266
Published online April 23, 2021
https://doi.org/10.14348/molcells.2021.2155
© The Korean Society for Molecular and Cellular Biology
Correspondence to : kooksh@jbnu.ac.kr (SHK); oasis@jbnu.ac.kr (ESC); leejc88@jbnu.ac.kr (JCL)
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
Numerous studies highlight the potential benefits potentials of supplemental cartilage oligomeric matrix protein-angiopoietin-1 (COMP-Ang1) through improved angiogenic effects. However, our recent findings show that excessive overexpression of COMP-Ang1 induces an impaired bone marrow (BM) microenvironment and senescence of hematopoietic stem cells (HSCs). Here, we investigated the underlying mechanisms of how excessive COMP-Ang1 affects the function of BM-conserved stem cells and hematopoiesis using K14-Cre;inducible-COMP-Ang1-transgenic mice. Excessive COMP-Ang1 induced peripheral egression and senescence of BM HSCs and mesenchymal stem cells (MSCs). Excessive COMP-Ang1 also caused abnormal hematopoiesis along with skewed differentiation of HSCs toward myeloid lineage rather than lymphoid lineage. Especially, excessive COMP-Ang1 disturbed late-stage erythroblast maturation, followed by decreased expression of stromal cell-derived factor 1 (SDF-1) and globin transcription factor 1 (GATA-1) and increased levels of superoxide anion and p-p38 kinase. However, transplantation with the mutant-derived BM cells or treatment with rhCOMP-Ang1 protein did not alter the frequency or GATA-1 expression of erythroblasts in recipient mice or in cultured BM cells. Together, our findings suggest that excessive COMP-Ang1 impairs the functions of BM HSCs and MSCs and hematopoietic processes, eventually leading to abnormal erythropoiesis via imbalanced SDF-1/CXCR4 axis and GATA-1 expression rather than Ang1/Tie2 signaling axis alterations.
Keywords bone marrow-conserved stem cells, cartilage oligomeric matrix protein-angiopoietin-1, globin transcription factor 1, stromal cell-derived factor 1/CXCR4 signaling axis
Angiogenesis is linked with hematopoiesis. Angiopoietin-1 (Ang1), a dominant ligand for long-term repopulating activity of hematopoietic stem cells (HSCs), plays crucial roles not only in vascular and hematopoietic development but also in the maintenance of HSCs in a quiescent state in the bone marrow (BM) niche (Arai et al., 2004; Joo et al., 2011; Suda et al., 2000; Takakura et al., 2000). Thus, in addition to angiogenesis, the Ang1/Tie2 signaling axis is critical for adhesion of long-term HSCs to the BM niche and maintenance of these HSCs in the BM.
Supplemental cartilage oligomeric matrix protein (COMP)-Ang1 suppresses vascular inflammation, leakage, and ischemic side effects, improves cell survival, and enhances blood vessel remodeling and formation (Koh, 2013; Lee et al., 2014; Youn et al., 2011). Administration of COMP-Ang1 by adenoviral vector induced long-lasting vascular enlargement and increased blood flow better than supplementation with recombinant human (
In this study, we examined the underlying mechanisms of how genetic overexpression of COMP-Ang1 affects BM retention, senescence of HSCs and MSCs, and hematopoietic processes using
This study was carried out in strict accordance with the recommendations in the Guide for Animal Care and Use of Jeonbuk National University. Before experiments, all procedures were approved by the University Committee on Ethics in the Care and Use of Laboratory Animals (CBU2014-00055) according to the ARRIVE guidelines.
All animals were cared for based on the guidelines of the Animal Care Committee of Jeonbuk National University. We generated
Cells from BM, spleen, and peripheral blood were collected from mutants, WT littermates, or B6 mice before treatment with red blood cell (RBC) lysis buffer (Sigma-Aldrich, USA) for 15 min on ice. Here BM cells were harvested by flushing the femur and tibia with phosphate-buffered saline (PBS) using a syringe, without crushing bones or treating with collagenase. After washing with PBS, frequencies of BM- and peripheral blood-conserved cells were analyzed using a flow cytometer (BD Calibur or BD Aria; BD Biosciences, USA) installed in the Center for University-Wide Research Facilities (CURF) at Jeonbuk National University. Populations of these cells were sequentially gated using FlowJo software (FlowJo, USA). In this study, populations of Lin-Sca-1+c-Kit+ (LSK) cells and CD150+CD48-LSK cells were phenotypically identified using the following antibodies: lineage markers PE-Cy7-conjugated anti-CD3 (cat.#552774), anti-B220 (CD45R, cat.#552772), anti-CD11b (cat.#552850), anti-Gr-1 (cat.#552958), or anti-TER-119 (cat.#557853) (all of these markers were from BD Biosciences); FITC-conjugated anti-Sca-1 (cat.#557405; BD Biosciences) or PE-conjugated anti-Sca-1 (cat.#553108; BD Biosciences); APC-conjugated anti-c-Kit (cat.#553356; BD Biosciences); perCP/Cy5.5-conjugated anti-CD150 (cat.#46-1502; eBioscience, USA); and APC-Cy7conjugated anti-CD48 (cat.#561826; BD Biosciences). Populations of Lin-Sca-1+CD29+CD105+ cells were phenotypically identified as BM-derived MSCs using the same PE-Cy7-conjugated lineages as used for identification of hematopoietic cells: APC-Cy7-conjugated Sca-1, PE- or FITC-conjugated CD29, and APC-conjugated CD105. For further characterization of MSCs, PE-CF594-conjugated CD44 (cat.#562464; BD Biosciences) and perCP/Cy5.5-conjugated vascular cell adhesion molecule 1 (VCAM-1/CD106) (cat.#562464; BioLegend, USA) antibodies were also used. Senescence-associated-β-galactosidase (SA-β-gal) activity in LSK, CD150+CD48-LSK, and Lin-Sca-1+CD29+CD105+ cells that had already been incubated with the cell surface markers were analyzed with C12FDG (cat.#I2904; Molecular Probes, USA). Alternatively, hematopoietic progenitor cells (HPCs) including granulocyte-monocyte progenitors, common myeloid progenitors, megakaryocyte-erythroid progenitors, and common lymphoid progenitors were defined using PE-conjugated anti-FcR (BD Biosciences), perCP/Cy5.5-conjugated anti-CD34 (BioLegend), or PE-conjugated anti-IL-7R (BD Biosciences) as a basis for LSK cell markers. Erythroblasts at four stages in the BM, peripheral blood, or spleen were discriminatively gated with PE- or FITC-conjugated anti-CD71 (BD Biosciences) and PE-Cy7-conjugated anti-TER-119 (BD Biosciences) antibodies. Myeloid cells were evaluated with PE-Cy7-conjugated anti-CD11b antibody in BM cells of mutants and WT littermates. Mitochondrial superoxide anion levels were analyzed by flow cytometry after staining cells with MitoSOXTM Red reagent (cat.#M36008; Invitrogen, USA). The levels of GATA-1 and p-p38 kinase were determined with PE-conjugated (Cell Signaling Technology, USA) and Alexa Fluor 488-conjugated (cat.#sc-1661; Santa Cruz Biotechnology, USA) antibodies, respectively, after fixation and permeabilization.
To optimize the contrast between blood vessels and surrounding soft tissue, contrast agent Microfil® MV-122 (FlowTech, USA) was administered into the aorta of mutants and WT littermates prior to imaging. Systemic perfusion was started by anesthetizing these animals and then the left heart chamber was punctured in the apex of the left ventricle after opening the chest. Perfusion was started by flushing the circulatory system with 10 ml of 0.9% NaCl and 200 U/ml heparin at 37°C. Thereafter, the manual perfusion was performed with yellow Microfil® (MV-122) at a rate of 3 ml/min. After the left ventricle and right atrium were ligated, mice were placed at 4°C for 24 h and then the knee joints were excised and fixed in 10% formalin for additional 24 h. Femurs were decalcified for two days in Decalcifying Solution-Lite (Sigma-Aldrich). After washing with PBS, tissue samples were scanned with a Skyscan 1076 (Skyscan; Bruker, Belgium) at a pixel size of 9 µm. Images were reconstructed and analyzed using the CtVox software (Bruker).
BM-derived stem and progenitor cells in mutants and WT littermates were harvested from the tibia and femur by flushing with PBS using a syringe. These cells were treated with RBC lysis buffer before washing with PBS. Cells were treated with antibodies specific to LSK and CD150+CD48-LSK cells, or HPCs. Cells were incubated with aqueous buffered solution of FITC-labeled Annexin V (200 ng/ml) and PI (300 ng/ml) at room temperature for 20 min. The frequency of these cells was analyzed using a flow cytometer and the scatter signals of Annexin V- and/or PI-positive cells were evaluated after sequentially gating cell populations using the FlowJo software.
Peripheral blood samples were isolated from mutants and WT littermates and collected into Vacutainer plastic tubes coated with K2EDTA. An automated complete blood cell counter (Sysmex XE-2100; TOA Medical Electronics, Japan) was used to measure the levels of RBCs (number/μl), hemoglobin (Hb, g/dl), hematocrit (Hct, %), and mean corpuscular volume (MCV, fl). For the pre-B CFC assay, whole BM cells (2 × 105 cells per dish) were divided into 35 mm dishes with MethoCultTM M3630 (Stem Cell Technologies, Canada). After 7 days, the number of colonies formed was counted. For colony-forming unit (CFU) assays, whole BM (3 × 104 cells per dish), peripheral blood (1 × 106 cells per dish), and spleen (0.5 × 106 cells per dish) cells were incubated in 35 mm dishes with MethoCult® GF M3434 (Stem Cell Technologies). After 12 days, the numbers of CFU-granulocyte/macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and CFU-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM) colonies were counted using standard criteria. For colony forming unit-erythroid (CFU-E) assay, whole BM cells (2 × 105 cells per dish) from mutants and WT littermates were plated into 35 mm dishes with MethoCultTM M3334 (Stem Cell Technologies). After 14 days of incubation, colonies formed were counted under optic microscopic observation (Carl Zeiss, Germany).
Immunohistochemistry (IHC) assays were performed using the Histostain Plus Rabbit Primary kit (Zymed Laboratories, USA) according to the manufacturer’s instruction. Hind limbs dissected from mutants or WT littermates were fixed in 4% paraformaldehyde at 4°C for 12 h. After rinsing with PBS, specimens were decalcified in 10% EDTA for 4 weeks, dehydrated, embedded in paraffin solution, and sectioned at a thickness of 5 μm. Dried slides were incubated at 60°C for 15 min before treatment with xylene I and II for 10 min each. Thereafter, slides were hydrated through a descending series of ethanol concentration (70%-100%) followed by treatment with 3% hydrogen peroxide. Slides that included the trabecular zone were incubated with rabbit-anti-SDF-1 (cat.#sc-28876, 1:50; Santa Cruz Biotechnology), mouse-anti-CD31 (cat.#ab9498; Abcam, UK), anti-Ang1 (cat.#ab8451; Abcam), or with anti-vascular endothelial growth factor (VEGF) (cat.#BS2853; Bioworld Technology, USA) according to the manufacturer’s instructions. After counterstaining with Mayer’s hematoxylin (Sigma-Aldrich), slides were observed using a microscope linked with a camera and image processing software (Carl Zeiss).
Equal amounts (20 µg/sample) of protein extract obtained from BM cells of mutants and WT littermates were run on 10% to 12% SDS-PAGE followed by blotting onto polyvinyl difluoride membranes. Blots were probed with primary antibodies specific to rabbit-anti-polyclonal SDF-1 (cat.#sc-28876, 1:200; Santa Cruz Biotechnology), rabbit-anti-osteopontin (cat.#ab8448; Abcam), rabbit-anti-runt-related transcription factor 2 (Runx2) (cat.#BS2831; Bioworld Technology, USA), rabbit-anti-osterix (cat.#ab94744; Abcam), or mouse-anti-monoclonal β-actin (cat.#sc-81178, 1:200; Santa Cruz Biotechnology) at 4°C. Membranes were washed and exposed to horseradish peroxidase-conjugated rabbit-anti IgG or mouse-anti Ig. Immunoreactive bands were visualized by enhanced chemiluminescence (Santa Cruz Biotechnology) before exposure to X-ray film (Eastman Kodak, USA). To evaluate the level of SDF-1 in BM supernatants or cultured BM cells, ELISA was performed using an SDF-1 mouse ELISA kit (Abcam) following the manufacturers’ instructions. The levels of inflammatory cytokines such as interleukin (IL)-1α, IL-6, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α in BM supernatants of mutants and WT littermates were measured using Multi-Analyte ELISArray Kits according to the manufacturer’s instructions (Qiagen Sciences, Germany).
To determine osteogenic capacity of the mutant and WT littermate-derived MSCs, these cells were isolated at 2 weeks of age and seeded onto 24-well culture plates (105 cells/well). The non-adherent cells were removed the next day, and adherent cells were cultured in osteogenic differentiating medium (α-minimum essential medium containing 5% fetal bovine serum, 50 μM ascorbic acid, 100 nM dexamethasone, and 10 mM β-glycerophosphate). Cells were stained at 21 days post-incubation with Alizarin red for 5 min after fixation in 4% paraformaldehyde for 30 min. The degree of mineralization was observed under a light microscope. Alizarin red-stained cells were also treated with 10% acetylpyridinum chloride for 20 min and the amount of red dye was quantified by measuring the absorbance at 405 nm.
To investigate whether senescence of BM HSCs is associated with abnormal erythropoiesis and altered GATA-1 expression, BM cells (2 × 106 cells) isolated from mutants and WT littermates (CD45.2) were transplanted by tail vein injection into conditioned recipients (CD45.1/2) that were lethally irradiated 12 to 24 h before transplantation. The numbers of BM erythroblasts, circulating blood cells, and GATA-1-positive erythroblasts were assessed in the recipient mice at 5 months post-transplantation. The frequencies of HSCs positive to MitoSox, C12FDG, and p-p38 kinase in CD45.2-expressing BM cells in the recipients were also determined using flow cytometry at 5 months post-transplantation.
All data are expressed as the mean ± SD and were analyzed using SPSS (ver. 12.0; SPSS, USA). Differences between two groups were analyzed by a Student’s
Initially, we compared various phenotypes in regard to genetic overexpression of COMP-Ang1 with those of WT littermates. Similar to our previous study (Kook et al., 2018) and with other reports (Suri et al., 1998; Thurston et al., 1999), the
We explored whether genetic overexpression of COMP-Ang1 in
As the
SDF-1 is known to play crucial roles in maintaining HSCs via induction of the SDF-1/CXCR4 signaling axis (Katayama et al., 2006; Zhang et al., 2003). We next investigated how overexpression of COMP-Ang1 in
To examine whether excessive COMP-Ang1-mediated senescence of HSCs is associated with defects in erythroblast maturation, we transplanted LSK cells derived from the
As the Ang1/Tie2 signaling axis plays important roles in maintaining the quiescence and survival of HSCs, we subsequently explored whether the COMP-Ang1/Tie2 signaling is related to the GATA-1 expression in and frequency of erythroblasts using B6-derived BM cells. B6 mouse-derived BM erythroblasts expressed Tie2 at various levels depending on maturation stages (Supplementary Fig. S4). When
As the SDF-1/CXCR4 axis and GATA-1 play important roles in hematopoiesis and erythrocyte maturation, we further examined how excessive COMP-Ang1 affects the expression of CXCR4 and GATA-1 in BM-derived or circulating erythroblasts. Overexpression of COMP-Ang1 in
BM niches play a pivotal role in HSCs by producing various cellular factors, in which the SDF-1/CXCR4 signaling axis exerts important roles for maintenance of the HSC pool, as well as for regulation of HSC mobilization and homing (Katayama et al., 2006; Sugiyama et al., 2006; Zhang et al., 2003). Our results indicate that overexpression of COMP-Ang1 in
Osteoblasts are associated with maintenance of HSCs in the BM microenvironment, where angiogenesis is coupled with osteogenesis, which is essential for homeostatic bone renewal and regenerative fracture healing (Kusumbe et al., 2014). We previously found that mutants with
Erythropoiesis occurs mainly in BM and an abnormal BM microenvironment contributes to hematopoietic dysfunction in Fanconi anemia (Kertesz et al., 2004; Zhou et al., 2017). HSC senescence is connected to erythrocyte defect-induced anemia, and activation of the Ang1/Tie2 signaling axis is crucial for maintaining the quiescence and survival of HSCs (Arai et al., 2004; Zhang et al., 2007). However, our results indicate that the impaired maturation of erythroblasts under excessive COMP-Ang1 is not directly associated with senescence of BM HSCs or the Ang1/Tie2 signaling axis (Brindle et al., 2006). Rather, our results support that in addition to GATA-1, the SDF-1/CXCR4 signaling axis is responsible for impaired BM retention and induction of HSC senescence, as well as for disturbed erythropoiesis. GATA-1 plays essential roles in the differentiation and maturation of erythroid cells into erythrocytes (Ferreira et al., 2005; Pevny et al., 1991; 1995; Yu et al., 2002). The regulatory roles of GATA-1 on erythropoiesis are closely related to its potential to control erythroid differentiation and erythroblast formation by inducing the expression of erythroid genes and by cooperating with erythropoietin (Ferreira et al., 2005; Pevny et al., 1991). GATA-1 is downregulated via an autonomous cell signaling mechanism during the terminal stages of erythropoiesis (Ferreira et al., 2005). Inflammatory responses repress GATA-1 expression via mediation by p38 kinase activation in erythroid cells (Bibikova et al., 2014). As erythroid differentiation involves production of reactive oxygen species, efficient antioxidant defense systems are required to limit persistent and severe oxidative stress in erythropoiesis (Matte and de Franceschi, 2019). Hypoxia is also capable of increasing BM numbers of HSCs and HPCs in mice. This condition decreases oxidative stress and increases GATA-1 expression, resulting in increased erythropoiesis and enhanced HSC engraftment (Chen et al., 2016). Overall, a balanced expression of GATA-1 is critical for terminal erythroid differentiation and sequential formation of erythroblasts; thus its overexpression can inhibit terminal differentiation and maturation of these cells (de Thonel et al., 2010; Ferreira et al., 2005; Pevny et al., 1991; Whyatt et al., 1997; 2000). Collectively, our present findings indicate that defective erythroblast maturation during overexpression of COMP-Ang1 is accompanied by a dysregulation of the SDF-1/CXCR4 signaling axis in the BM along with imbalanced GATA-1 expression in erythroblasts rather than in HPCs. These data also suggest that the impaired erythropoietic differentiation is in part associated with enhanced oxidative stress in erythroblasts.
MSCs play crucial roles as a stem cell niche in BM by expressing higher levels of signaling molecules involved in HSC maintenance (Boulais and Frenette, 2015; Ehninger and Trumpp, 2011). Macrophages are part of BM niches, and loss of macrophages is associated with egression of BM HSCs into the peripheral blood and spleen. Our current findings indicate that myeloid cells under excessive angiogenesis or COMP-Ang1 contribute to the production of cellular reactive oxygen species and inflammatory cytokines, thereby negatively affecting hematopoietic differentiation. Our findings also indicate that excessive COMP-Ang1 or angiogenesis is associated with impaired function and senescence induction of not only HSCs but also BM MSCs, through which the BM microenvironment, SDF-1 production, and body weight will be also affected. Furthermore, our findings show no hematopoietic changes after the transplantation of mutant-derived cells, indicating that excessive angiogenesis or COMP-Ang1 expression impairs the functions of BM-conserved stem cells in developmental stages and hampers normal growth and bone mass accrual.
In summary, our previous and current findings highlight that excessive angiogenesis or COMP-Ang1 expression induces senescence and peripheral egression of BM HSCs and MSCs, thereby leading to abnormal hematopoiesis and bone mass accrual. This study also indicates that impairment of the SDF-1/CXCR4 signaling axis and imbalanced GATA-1 expression are associated with excessive COMP-Ang1-mediated disturbance of erythroblast maturation and their peripheral circulation. Although our experimental system is artificial due to the fact that Ang1 from the skin does not prominently regulate hematopoiesis and because Ang1 secreted from BM HSCs and HPCs is critical for the regeneration of their niche (Zhou et al., 2015), our findings suggest that appropriate activation of Ang1/Tie2 signaling and/or a suitable level of Ang1 is required not only for vasculogenesis, angiogenesis, and hematopoiesis, but also for the functions of BM-conserved stem cells.
This research was supported by Basic Science Research Program through the National Research Foundation (NRF) funded by the Ministry of Science, Information and Communications Technology and Future Planning (2018R1A2A3074639, 2019R1A2C2084453, 2020R1C1C1004968, and 2021R1A2C2006032) and by Ministry of Education (2018R1D1A1B07047162), South Korea. We also thank Dr. G.Y. Koh for providing
S.H.K., E.S.C., and J.C.L. conceived and designed the experiments. H.J.S., M.H.K., G.B., J.W.H., S.B.P., S.H.K., and H.S.S. performed the experiments. J.W.H., M.H.K., and E.S.C. generated transgenic mice. H.J.S., M.H.K., G.B., S.H.K., E.S.C., and J.C.L. analyzed the data. H.J.S., G.B., S.H.K., E.S.C., and J.C.L. contributed reagents/materials/analysis tools. H.J.S., M.H.K., G.B., E.S.C., S.H.K., and J.C.L. wrote the manuscript.
The authors have no potential conflicts of interest to disclose.
Mol. Cells 2021; 44(4): 254-266
Published online April 30, 2021 https://doi.org/10.14348/molcells.2021.2155
Copyright © The Korean Society for Molecular and Cellular Biology.
Hyun-Jaung Sim1,2,4 , Min-Hye Kim2,4
, Govinda Bhattarai1,4
, Jae-Won Hwang1
, Han-Sol So2
, Sher Bahadur Poudel3
, Eui-Sic Cho1,*
, Sung-Ho Kook1,2,*
, and Jeong-Chae Lee1,2, *
1Cluster for Craniofacial Development and Regeneration Research, Institute of Oral Biosciences and School of Dentistry, Jeonbuk National University, Jeonju 54896, Korea, 2Department of Bioactive Material Sciences, Research Center of Bioactive Materials, Jeonbuk National University, Jeonju 54896, Korea, 3Department of Basic Science & Craniofacial Biology, College of Dentistry, New York University, New York, NY 10010, USA, 4These authors contributed equally to this work.
Correspondence to:kooksh@jbnu.ac.kr (SHK); oasis@jbnu.ac.kr (ESC); leejc88@jbnu.ac.kr (JCL)
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
Numerous studies highlight the potential benefits potentials of supplemental cartilage oligomeric matrix protein-angiopoietin-1 (COMP-Ang1) through improved angiogenic effects. However, our recent findings show that excessive overexpression of COMP-Ang1 induces an impaired bone marrow (BM) microenvironment and senescence of hematopoietic stem cells (HSCs). Here, we investigated the underlying mechanisms of how excessive COMP-Ang1 affects the function of BM-conserved stem cells and hematopoiesis using K14-Cre;inducible-COMP-Ang1-transgenic mice. Excessive COMP-Ang1 induced peripheral egression and senescence of BM HSCs and mesenchymal stem cells (MSCs). Excessive COMP-Ang1 also caused abnormal hematopoiesis along with skewed differentiation of HSCs toward myeloid lineage rather than lymphoid lineage. Especially, excessive COMP-Ang1 disturbed late-stage erythroblast maturation, followed by decreased expression of stromal cell-derived factor 1 (SDF-1) and globin transcription factor 1 (GATA-1) and increased levels of superoxide anion and p-p38 kinase. However, transplantation with the mutant-derived BM cells or treatment with rhCOMP-Ang1 protein did not alter the frequency or GATA-1 expression of erythroblasts in recipient mice or in cultured BM cells. Together, our findings suggest that excessive COMP-Ang1 impairs the functions of BM HSCs and MSCs and hematopoietic processes, eventually leading to abnormal erythropoiesis via imbalanced SDF-1/CXCR4 axis and GATA-1 expression rather than Ang1/Tie2 signaling axis alterations.
Keywords: bone marrow-conserved stem cells, cartilage oligomeric matrix protein-angiopoietin-1, globin transcription factor 1, stromal cell-derived factor 1/CXCR4 signaling axis
Angiogenesis is linked with hematopoiesis. Angiopoietin-1 (Ang1), a dominant ligand for long-term repopulating activity of hematopoietic stem cells (HSCs), plays crucial roles not only in vascular and hematopoietic development but also in the maintenance of HSCs in a quiescent state in the bone marrow (BM) niche (Arai et al., 2004; Joo et al., 2011; Suda et al., 2000; Takakura et al., 2000). Thus, in addition to angiogenesis, the Ang1/Tie2 signaling axis is critical for adhesion of long-term HSCs to the BM niche and maintenance of these HSCs in the BM.
Supplemental cartilage oligomeric matrix protein (COMP)-Ang1 suppresses vascular inflammation, leakage, and ischemic side effects, improves cell survival, and enhances blood vessel remodeling and formation (Koh, 2013; Lee et al., 2014; Youn et al., 2011). Administration of COMP-Ang1 by adenoviral vector induced long-lasting vascular enlargement and increased blood flow better than supplementation with recombinant human (
In this study, we examined the underlying mechanisms of how genetic overexpression of COMP-Ang1 affects BM retention, senescence of HSCs and MSCs, and hematopoietic processes using
This study was carried out in strict accordance with the recommendations in the Guide for Animal Care and Use of Jeonbuk National University. Before experiments, all procedures were approved by the University Committee on Ethics in the Care and Use of Laboratory Animals (CBU2014-00055) according to the ARRIVE guidelines.
All animals were cared for based on the guidelines of the Animal Care Committee of Jeonbuk National University. We generated
Cells from BM, spleen, and peripheral blood were collected from mutants, WT littermates, or B6 mice before treatment with red blood cell (RBC) lysis buffer (Sigma-Aldrich, USA) for 15 min on ice. Here BM cells were harvested by flushing the femur and tibia with phosphate-buffered saline (PBS) using a syringe, without crushing bones or treating with collagenase. After washing with PBS, frequencies of BM- and peripheral blood-conserved cells were analyzed using a flow cytometer (BD Calibur or BD Aria; BD Biosciences, USA) installed in the Center for University-Wide Research Facilities (CURF) at Jeonbuk National University. Populations of these cells were sequentially gated using FlowJo software (FlowJo, USA). In this study, populations of Lin-Sca-1+c-Kit+ (LSK) cells and CD150+CD48-LSK cells were phenotypically identified using the following antibodies: lineage markers PE-Cy7-conjugated anti-CD3 (cat.#552774), anti-B220 (CD45R, cat.#552772), anti-CD11b (cat.#552850), anti-Gr-1 (cat.#552958), or anti-TER-119 (cat.#557853) (all of these markers were from BD Biosciences); FITC-conjugated anti-Sca-1 (cat.#557405; BD Biosciences) or PE-conjugated anti-Sca-1 (cat.#553108; BD Biosciences); APC-conjugated anti-c-Kit (cat.#553356; BD Biosciences); perCP/Cy5.5-conjugated anti-CD150 (cat.#46-1502; eBioscience, USA); and APC-Cy7conjugated anti-CD48 (cat.#561826; BD Biosciences). Populations of Lin-Sca-1+CD29+CD105+ cells were phenotypically identified as BM-derived MSCs using the same PE-Cy7-conjugated lineages as used for identification of hematopoietic cells: APC-Cy7-conjugated Sca-1, PE- or FITC-conjugated CD29, and APC-conjugated CD105. For further characterization of MSCs, PE-CF594-conjugated CD44 (cat.#562464; BD Biosciences) and perCP/Cy5.5-conjugated vascular cell adhesion molecule 1 (VCAM-1/CD106) (cat.#562464; BioLegend, USA) antibodies were also used. Senescence-associated-β-galactosidase (SA-β-gal) activity in LSK, CD150+CD48-LSK, and Lin-Sca-1+CD29+CD105+ cells that had already been incubated with the cell surface markers were analyzed with C12FDG (cat.#I2904; Molecular Probes, USA). Alternatively, hematopoietic progenitor cells (HPCs) including granulocyte-monocyte progenitors, common myeloid progenitors, megakaryocyte-erythroid progenitors, and common lymphoid progenitors were defined using PE-conjugated anti-FcR (BD Biosciences), perCP/Cy5.5-conjugated anti-CD34 (BioLegend), or PE-conjugated anti-IL-7R (BD Biosciences) as a basis for LSK cell markers. Erythroblasts at four stages in the BM, peripheral blood, or spleen were discriminatively gated with PE- or FITC-conjugated anti-CD71 (BD Biosciences) and PE-Cy7-conjugated anti-TER-119 (BD Biosciences) antibodies. Myeloid cells were evaluated with PE-Cy7-conjugated anti-CD11b antibody in BM cells of mutants and WT littermates. Mitochondrial superoxide anion levels were analyzed by flow cytometry after staining cells with MitoSOXTM Red reagent (cat.#M36008; Invitrogen, USA). The levels of GATA-1 and p-p38 kinase were determined with PE-conjugated (Cell Signaling Technology, USA) and Alexa Fluor 488-conjugated (cat.#sc-1661; Santa Cruz Biotechnology, USA) antibodies, respectively, after fixation and permeabilization.
To optimize the contrast between blood vessels and surrounding soft tissue, contrast agent Microfil® MV-122 (FlowTech, USA) was administered into the aorta of mutants and WT littermates prior to imaging. Systemic perfusion was started by anesthetizing these animals and then the left heart chamber was punctured in the apex of the left ventricle after opening the chest. Perfusion was started by flushing the circulatory system with 10 ml of 0.9% NaCl and 200 U/ml heparin at 37°C. Thereafter, the manual perfusion was performed with yellow Microfil® (MV-122) at a rate of 3 ml/min. After the left ventricle and right atrium were ligated, mice were placed at 4°C for 24 h and then the knee joints were excised and fixed in 10% formalin for additional 24 h. Femurs were decalcified for two days in Decalcifying Solution-Lite (Sigma-Aldrich). After washing with PBS, tissue samples were scanned with a Skyscan 1076 (Skyscan; Bruker, Belgium) at a pixel size of 9 µm. Images were reconstructed and analyzed using the CtVox software (Bruker).
BM-derived stem and progenitor cells in mutants and WT littermates were harvested from the tibia and femur by flushing with PBS using a syringe. These cells were treated with RBC lysis buffer before washing with PBS. Cells were treated with antibodies specific to LSK and CD150+CD48-LSK cells, or HPCs. Cells were incubated with aqueous buffered solution of FITC-labeled Annexin V (200 ng/ml) and PI (300 ng/ml) at room temperature for 20 min. The frequency of these cells was analyzed using a flow cytometer and the scatter signals of Annexin V- and/or PI-positive cells were evaluated after sequentially gating cell populations using the FlowJo software.
Peripheral blood samples were isolated from mutants and WT littermates and collected into Vacutainer plastic tubes coated with K2EDTA. An automated complete blood cell counter (Sysmex XE-2100; TOA Medical Electronics, Japan) was used to measure the levels of RBCs (number/μl), hemoglobin (Hb, g/dl), hematocrit (Hct, %), and mean corpuscular volume (MCV, fl). For the pre-B CFC assay, whole BM cells (2 × 105 cells per dish) were divided into 35 mm dishes with MethoCultTM M3630 (Stem Cell Technologies, Canada). After 7 days, the number of colonies formed was counted. For colony-forming unit (CFU) assays, whole BM (3 × 104 cells per dish), peripheral blood (1 × 106 cells per dish), and spleen (0.5 × 106 cells per dish) cells were incubated in 35 mm dishes with MethoCult® GF M3434 (Stem Cell Technologies). After 12 days, the numbers of CFU-granulocyte/macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and CFU-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM) colonies were counted using standard criteria. For colony forming unit-erythroid (CFU-E) assay, whole BM cells (2 × 105 cells per dish) from mutants and WT littermates were plated into 35 mm dishes with MethoCultTM M3334 (Stem Cell Technologies). After 14 days of incubation, colonies formed were counted under optic microscopic observation (Carl Zeiss, Germany).
Immunohistochemistry (IHC) assays were performed using the Histostain Plus Rabbit Primary kit (Zymed Laboratories, USA) according to the manufacturer’s instruction. Hind limbs dissected from mutants or WT littermates were fixed in 4% paraformaldehyde at 4°C for 12 h. After rinsing with PBS, specimens were decalcified in 10% EDTA for 4 weeks, dehydrated, embedded in paraffin solution, and sectioned at a thickness of 5 μm. Dried slides were incubated at 60°C for 15 min before treatment with xylene I and II for 10 min each. Thereafter, slides were hydrated through a descending series of ethanol concentration (70%-100%) followed by treatment with 3% hydrogen peroxide. Slides that included the trabecular zone were incubated with rabbit-anti-SDF-1 (cat.#sc-28876, 1:50; Santa Cruz Biotechnology), mouse-anti-CD31 (cat.#ab9498; Abcam, UK), anti-Ang1 (cat.#ab8451; Abcam), or with anti-vascular endothelial growth factor (VEGF) (cat.#BS2853; Bioworld Technology, USA) according to the manufacturer’s instructions. After counterstaining with Mayer’s hematoxylin (Sigma-Aldrich), slides were observed using a microscope linked with a camera and image processing software (Carl Zeiss).
Equal amounts (20 µg/sample) of protein extract obtained from BM cells of mutants and WT littermates were run on 10% to 12% SDS-PAGE followed by blotting onto polyvinyl difluoride membranes. Blots were probed with primary antibodies specific to rabbit-anti-polyclonal SDF-1 (cat.#sc-28876, 1:200; Santa Cruz Biotechnology), rabbit-anti-osteopontin (cat.#ab8448; Abcam), rabbit-anti-runt-related transcription factor 2 (Runx2) (cat.#BS2831; Bioworld Technology, USA), rabbit-anti-osterix (cat.#ab94744; Abcam), or mouse-anti-monoclonal β-actin (cat.#sc-81178, 1:200; Santa Cruz Biotechnology) at 4°C. Membranes were washed and exposed to horseradish peroxidase-conjugated rabbit-anti IgG or mouse-anti Ig. Immunoreactive bands were visualized by enhanced chemiluminescence (Santa Cruz Biotechnology) before exposure to X-ray film (Eastman Kodak, USA). To evaluate the level of SDF-1 in BM supernatants or cultured BM cells, ELISA was performed using an SDF-1 mouse ELISA kit (Abcam) following the manufacturers’ instructions. The levels of inflammatory cytokines such as interleukin (IL)-1α, IL-6, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α in BM supernatants of mutants and WT littermates were measured using Multi-Analyte ELISArray Kits according to the manufacturer’s instructions (Qiagen Sciences, Germany).
To determine osteogenic capacity of the mutant and WT littermate-derived MSCs, these cells were isolated at 2 weeks of age and seeded onto 24-well culture plates (105 cells/well). The non-adherent cells were removed the next day, and adherent cells were cultured in osteogenic differentiating medium (α-minimum essential medium containing 5% fetal bovine serum, 50 μM ascorbic acid, 100 nM dexamethasone, and 10 mM β-glycerophosphate). Cells were stained at 21 days post-incubation with Alizarin red for 5 min after fixation in 4% paraformaldehyde for 30 min. The degree of mineralization was observed under a light microscope. Alizarin red-stained cells were also treated with 10% acetylpyridinum chloride for 20 min and the amount of red dye was quantified by measuring the absorbance at 405 nm.
To investigate whether senescence of BM HSCs is associated with abnormal erythropoiesis and altered GATA-1 expression, BM cells (2 × 106 cells) isolated from mutants and WT littermates (CD45.2) were transplanted by tail vein injection into conditioned recipients (CD45.1/2) that were lethally irradiated 12 to 24 h before transplantation. The numbers of BM erythroblasts, circulating blood cells, and GATA-1-positive erythroblasts were assessed in the recipient mice at 5 months post-transplantation. The frequencies of HSCs positive to MitoSox, C12FDG, and p-p38 kinase in CD45.2-expressing BM cells in the recipients were also determined using flow cytometry at 5 months post-transplantation.
All data are expressed as the mean ± SD and were analyzed using SPSS (ver. 12.0; SPSS, USA). Differences between two groups were analyzed by a Student’s
Initially, we compared various phenotypes in regard to genetic overexpression of COMP-Ang1 with those of WT littermates. Similar to our previous study (Kook et al., 2018) and with other reports (Suri et al., 1998; Thurston et al., 1999), the
We explored whether genetic overexpression of COMP-Ang1 in
As the
SDF-1 is known to play crucial roles in maintaining HSCs via induction of the SDF-1/CXCR4 signaling axis (Katayama et al., 2006; Zhang et al., 2003). We next investigated how overexpression of COMP-Ang1 in
To examine whether excessive COMP-Ang1-mediated senescence of HSCs is associated with defects in erythroblast maturation, we transplanted LSK cells derived from the
As the Ang1/Tie2 signaling axis plays important roles in maintaining the quiescence and survival of HSCs, we subsequently explored whether the COMP-Ang1/Tie2 signaling is related to the GATA-1 expression in and frequency of erythroblasts using B6-derived BM cells. B6 mouse-derived BM erythroblasts expressed Tie2 at various levels depending on maturation stages (Supplementary Fig. S4). When
As the SDF-1/CXCR4 axis and GATA-1 play important roles in hematopoiesis and erythrocyte maturation, we further examined how excessive COMP-Ang1 affects the expression of CXCR4 and GATA-1 in BM-derived or circulating erythroblasts. Overexpression of COMP-Ang1 in
BM niches play a pivotal role in HSCs by producing various cellular factors, in which the SDF-1/CXCR4 signaling axis exerts important roles for maintenance of the HSC pool, as well as for regulation of HSC mobilization and homing (Katayama et al., 2006; Sugiyama et al., 2006; Zhang et al., 2003). Our results indicate that overexpression of COMP-Ang1 in
Osteoblasts are associated with maintenance of HSCs in the BM microenvironment, where angiogenesis is coupled with osteogenesis, which is essential for homeostatic bone renewal and regenerative fracture healing (Kusumbe et al., 2014). We previously found that mutants with
Erythropoiesis occurs mainly in BM and an abnormal BM microenvironment contributes to hematopoietic dysfunction in Fanconi anemia (Kertesz et al., 2004; Zhou et al., 2017). HSC senescence is connected to erythrocyte defect-induced anemia, and activation of the Ang1/Tie2 signaling axis is crucial for maintaining the quiescence and survival of HSCs (Arai et al., 2004; Zhang et al., 2007). However, our results indicate that the impaired maturation of erythroblasts under excessive COMP-Ang1 is not directly associated with senescence of BM HSCs or the Ang1/Tie2 signaling axis (Brindle et al., 2006). Rather, our results support that in addition to GATA-1, the SDF-1/CXCR4 signaling axis is responsible for impaired BM retention and induction of HSC senescence, as well as for disturbed erythropoiesis. GATA-1 plays essential roles in the differentiation and maturation of erythroid cells into erythrocytes (Ferreira et al., 2005; Pevny et al., 1991; 1995; Yu et al., 2002). The regulatory roles of GATA-1 on erythropoiesis are closely related to its potential to control erythroid differentiation and erythroblast formation by inducing the expression of erythroid genes and by cooperating with erythropoietin (Ferreira et al., 2005; Pevny et al., 1991). GATA-1 is downregulated via an autonomous cell signaling mechanism during the terminal stages of erythropoiesis (Ferreira et al., 2005). Inflammatory responses repress GATA-1 expression via mediation by p38 kinase activation in erythroid cells (Bibikova et al., 2014). As erythroid differentiation involves production of reactive oxygen species, efficient antioxidant defense systems are required to limit persistent and severe oxidative stress in erythropoiesis (Matte and de Franceschi, 2019). Hypoxia is also capable of increasing BM numbers of HSCs and HPCs in mice. This condition decreases oxidative stress and increases GATA-1 expression, resulting in increased erythropoiesis and enhanced HSC engraftment (Chen et al., 2016). Overall, a balanced expression of GATA-1 is critical for terminal erythroid differentiation and sequential formation of erythroblasts; thus its overexpression can inhibit terminal differentiation and maturation of these cells (de Thonel et al., 2010; Ferreira et al., 2005; Pevny et al., 1991; Whyatt et al., 1997; 2000). Collectively, our present findings indicate that defective erythroblast maturation during overexpression of COMP-Ang1 is accompanied by a dysregulation of the SDF-1/CXCR4 signaling axis in the BM along with imbalanced GATA-1 expression in erythroblasts rather than in HPCs. These data also suggest that the impaired erythropoietic differentiation is in part associated with enhanced oxidative stress in erythroblasts.
MSCs play crucial roles as a stem cell niche in BM by expressing higher levels of signaling molecules involved in HSC maintenance (Boulais and Frenette, 2015; Ehninger and Trumpp, 2011). Macrophages are part of BM niches, and loss of macrophages is associated with egression of BM HSCs into the peripheral blood and spleen. Our current findings indicate that myeloid cells under excessive angiogenesis or COMP-Ang1 contribute to the production of cellular reactive oxygen species and inflammatory cytokines, thereby negatively affecting hematopoietic differentiation. Our findings also indicate that excessive COMP-Ang1 or angiogenesis is associated with impaired function and senescence induction of not only HSCs but also BM MSCs, through which the BM microenvironment, SDF-1 production, and body weight will be also affected. Furthermore, our findings show no hematopoietic changes after the transplantation of mutant-derived cells, indicating that excessive angiogenesis or COMP-Ang1 expression impairs the functions of BM-conserved stem cells in developmental stages and hampers normal growth and bone mass accrual.
In summary, our previous and current findings highlight that excessive angiogenesis or COMP-Ang1 expression induces senescence and peripheral egression of BM HSCs and MSCs, thereby leading to abnormal hematopoiesis and bone mass accrual. This study also indicates that impairment of the SDF-1/CXCR4 signaling axis and imbalanced GATA-1 expression are associated with excessive COMP-Ang1-mediated disturbance of erythroblast maturation and their peripheral circulation. Although our experimental system is artificial due to the fact that Ang1 from the skin does not prominently regulate hematopoiesis and because Ang1 secreted from BM HSCs and HPCs is critical for the regeneration of their niche (Zhou et al., 2015), our findings suggest that appropriate activation of Ang1/Tie2 signaling and/or a suitable level of Ang1 is required not only for vasculogenesis, angiogenesis, and hematopoiesis, but also for the functions of BM-conserved stem cells.
This research was supported by Basic Science Research Program through the National Research Foundation (NRF) funded by the Ministry of Science, Information and Communications Technology and Future Planning (2018R1A2A3074639, 2019R1A2C2084453, 2020R1C1C1004968, and 2021R1A2C2006032) and by Ministry of Education (2018R1D1A1B07047162), South Korea. We also thank Dr. G.Y. Koh for providing
S.H.K., E.S.C., and J.C.L. conceived and designed the experiments. H.J.S., M.H.K., G.B., J.W.H., S.B.P., S.H.K., and H.S.S. performed the experiments. J.W.H., M.H.K., and E.S.C. generated transgenic mice. H.J.S., M.H.K., G.B., S.H.K., E.S.C., and J.C.L. analyzed the data. H.J.S., G.B., S.H.K., E.S.C., and J.C.L. contributed reagents/materials/analysis tools. H.J.S., M.H.K., G.B., E.S.C., S.H.K., and J.C.L. wrote the manuscript.
The authors have no potential conflicts of interest to disclose.