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Osteoblast-specific Angiopoietin 1 overexpression increases bone mass
Biochemical and Biophysical Research Communications 362 (2007) 1019–1025 www.elsevier.com/locate/ybbrc
Osteoblast-specific Angiopoietin 1 overexpression increases bone mass Toru Suzuki a
a,b,1
, Takeshi Miyamoto a,b,c,*,1, Nobuyuki Fujita a,b, Ken Ninomiya Ryotaro Iwasaki a,d, Yoshiaki Toyama b, Toshio Suda a,*
a,b
,
Department of Cell Differentiation, The Sakaguchi Laboratory, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan b Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan c Department of Musculoskeletal Reconstruction and Regeneration Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan d Department of Dentistry and Oral Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan Received 16 August 2007 Available online 28 August 2007
Abstract Although osteoblasts express the angiogenic protein Angiopoietin 1 (Ang1), the role of Ang1 in bone formation remains largely unknown. Here we report that Ang1 overexpression in osteoblasts driven by the osteoblast-specific 2.3 kb alpha 1 type 1 collagen promoter results in increased bone mass in vivo. In Ang1-transgenic mice (Ang1-Tg), bone volume and bone parameters increased significantly compared with wild-type littermates, although the Ang1 receptor, Tie2 was not expressed in osteoblasts. Tie2 is primarily expressed in vascular endothelial cells, and Ang1-Tie2 signaling is reportedly crucial for angiogenesis. We found that the number of vascular endothelial cells was significantly elevated in Ang1-Tg mice compared with that of wild-type littermates, an increase accompanied by increased alkaline-phosphatase activity, a marker of osteoblast activation. The number of osteoclasts in the bone of Ang1-Tg mice did not differ from wild-type littermates. These results indicate that angiogenesis induced by Ang1 expressed in osteoblasts is coupled with osteogenesis. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Angiopoietin 1; Osteoblast; Coupling; Alpha 1 type 1 collagen promoter; Transgenic
Endochondral ossification is a sequential process in which apoptotic hypertrophic chondrocytes are eliminated by chondroclasts and replaced by osteoblasts, accompanied by vascular endothelial cell invasion [1,2]. Longitudinal bone growth is mainly mediated by proliferation of chondrocytes in growth plates, and bone formation is a result of a well-controlled balance between bone-resorption by osteoclasts and bone formation by osteoblasts [1–3]. The role of vascular endothelial growth factor (VEGF) has
*
Corresponding authors. Address: Department of Cell Differentiation, The Sakaguchi Laboratory, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Fax: +81 3 5363 3475. E-mail addresses: [email protected] (T. Miyamoto), sudato@ sc.itc.keio.ac.jp (T. Suda). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.08.099
been demonstrated in bone development [4,5]; however, the role of the angiogenic factor Angiopoietin 1 (Ang1) in regulating bone volume has not well been characterized. Ang1 binds to and phosphorylates a receptor tyrosine kinase exhibiting an Ig and epidermal growth factor homology domain 2 (Tie2) [6–9]. Although Ang1 is secreted, its low solubility has made it difficult to purify and maintain as a stable protein, thus preventing study of its biological effects. However, gene ablation and transgenic studies of Ang1 have revealed its indispensable role in development and maintenance of the blood and lymphatic vascular systems, as well as the hematopoietic systems [10– 15]. Ang1 is expressed in osteoblasts [16–19]; however, its role in bone formation is largely unknown. Here, in order to examine the effects of Ang1 expressed in osteoblasts on the bone formation, we established Ang1transgenic mice (Ang1-Tg) in which Ang1 expression was
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driven by the osteoblast-specific 2.3 kb alpha 1 type 1 collagen (Col1a1) promoter. Ang1 expression was detected in the bone of Ang1-Tg mice, and both bone volume and alkaline-phosphatase (ALP) activity increased, as did numbers of vascular endothelial cells in Ang1-Tg bone. By contrast, the number of osteoclasts in bone did not differ between Ang1-Tg and wild-type littermates. These results indicate that Ang1 expressed in osteoblasts plays a role in control of bone volume through angiogenesis in the bone. Materials and methods Generation of Col1a1-Ang1 transgenic mice. Animals were cared for in accordance with guidelines of Keio University School of Medicine. The mouse Ang1 open reading frame beginning with a suitable Kozak sequence (Ang1 fragment) was PCR amplified. An NcoI- and SalI-digested Ang1 fragment was cloned downstream of the mouse osteoblast-specific Col1a1 promoter in the pBS vector. The Col1a1-Ang1 fragment was digested by NheI and SalI and cloned into the pIRES2-EGFP vector (Clontech) to yield pCol1a1-Ang1-IRES-EGFP-PolyA. Col1a1-Ang1IRES-EGFP-PolyA was digested by NheI and AflII to purify the Ang1 transgene. Transgenic mice were generated in a Slc:BDF1 background by transgene microinjection (Japan SLC, Shizuoka, Japan), and injected embryos were implanted into pseudopregnant Slc:ICR females. The presence of the transgene was evaluated by PCR of tail DNA using the following primer set: forward primer, 5 0 0 -AAGCTGACCCTGAAGTT CATCTGC-3 0 0 ; reverse primer, 5 0 0 -CTGCTTGTCGGCCATGATATA GAC-3 0 0 . RT-PCR analysis. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) from femurs of mouse neonates. To prevent DNA contamination, total RNA was purified using RNeasy Total RNA Isolation Kit using RNase free Dnase (Qiagen GmbH, Hilden, Germany). RNA was quantified by an Ultro spec 3300 pro (GE Healthcare, Buckinghamshire, UK) to select samples with an absorbance ratio of (A260/ A280) of 2.0. Randomly primed complementary DNA (cDNA) was synthesized from 1 lg total RNA using Advantage RT-for- PCR kit (Clontech). RT-PCR analysis of Ang1 and b-actin expression was performed using the following primers: Ang1, forward primer, 5 0 0 -ACT AGTAGTACAATGACAGTTTTCCTTTCC-3 0 0 and reverse primer, 5 0 0 AGATCTTCAAAAGTCCAAGGGCCGGATCAT-3 0 0 ; b-actin, forward primer, 5 0 0 -GTGATGGTGGGAATGGGTCAGAAGGAC-3 0 0 and reverse primer, 5 0 0 -GAAGGAAGGCTGGAAAAGAGCCTCAGG -3 0 0 . Quantitative real-time PCR analysis of Tie2 expression relative to b-actin was analyzed using TaqMan assays (7500 Fast Real-Time PCR system, Applied Biosystems, Foster City, CA) using the following probes: Tie2, Mm00443242_ml; b-actin, Mm00607939_sl. Western blot analysis. Mouse calvarias were cut into chips and immediately placed into liquid nitrogen. Frozen chips were crushed to particles using a Cool Mill (Tokken, Chiba, Japan) and solubilized with lysis buffer (1% Triton X-100, 150 mM sodium chloride, 1 mM EDTA, 50 mM tris–hydroxymethyl aminomethane). Cell lysates mixed with loading buffer [tris–Glycine SDS Sample Buffer (Invitrogen, Carlsbad, CA) with 5% 2-mercaptoethanol (2-ME)] were incubated at 80 °C for 3 min, loaded onto 10% polyacrylamide gels, and electrophoresed in SDS– PAGE. Proteins were transferred to PVDF membranes (Nihon Millipore Ltd., Yonezawa, Japan) and blotted with goat anti-Ang1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) followed by HRP-conjugated rabbit anti-goat IgG (Invitrogen). Proteins were visualized using ECL Western Blotting Detection Reagents (GE Healthcare). Membranes were stripped by stripping buffer (2% SDS, 62.5 mM tris–hydroxymethyl aminomethane, pH 6.7, with 0.7% 2-ME) and re-blotted with rabbit anti-actin antibody (Sigma–Aldrich, St. Louis, MO) followed by goat anti-rabbit HRP (Invitrogen). Microcomputed tomography (CT) analysis. Femurs of 8-week-old mice were fixed with 70% ethanol and imaged by lCT (eXplore lCT; GE Healthcare). The following parameters were determined by advance bone
analysis (GE Healthcare), bone mineral density (BMD; mg/cc), bone volume/tissue volume (BV/TV; %), trabecular bone thickness (Tb.Th; lm), trabecular number (Tb. N; per mm), and trabecular separation (Tb.Sp; lm). Three-dimensional images were reconstructed by ResolveRT (Mercury Computer Systems, Inc., Chelmsford, MA). Histological and immunohistochemical analysis. Femurs and tibias were dissected from 6- to 8-week-old mice, fixed with 4% paraformaldehyde, and embedded in paraffin. Sections of 5 lm thickness were stained with hematoxylin and eosin (H/E) or mouse anti-Cathepsin K monoclonal antibody (BD PharMingen, San Diego, CA) followed by Alexa 488-conjugated anti-mouse IgG (Invitrogen). For CD31 and ALP staining, frozen sections of hindlimb that had not been decalcified were cut from 6- to 8week-old femurs and tibias embedded in mouse liver paste, as described [20,21]. Sections were incubated as follows: rat anti-CD31 monoclonal antibody (BD PharMingen) followed by Alexa 488-conjugated anti-rat IgG (Invitrogen), mouse anti-ALP monoclonal antibody (BD PharMingen) followed by Alexa 488-conjugated anti-mouse IgG (Invitrogen). TOTO3 (Invitrogen) was used for nuclear staining. Immunoreactivity was detected by confocal microscopy (Olympus, Tokyo, Japan). Quantification of immunoreactive cells or fluorescence intensity was determined using Scion Image Analysis Program (National Institutes of Health, USA), a public domain image processing and analysis program.
Results Generation of Ang1-transgenic (Ang1-Tg) mice To understand the role of Ang1 expressed in osteoblasts in bone formation in vivo, we generated a transgenic mouse overexpressing Ang1 in osteoblasts under control of the osteoblast-specific Col1a1 promoter. The Ang1-Tg transgene consisted of murine Ang1, IRES-EGFP, and a polyA sequence (Fig. 1A). Osteoblast specificity of the Col1a1 promoter has been reported [22,23]. Fifty-one lines of offspring were generated by embryo microinjection, and the presence of the transgene tested by PCR of tail DNA using transgene-specific primer sets (Fig. 1B and data not shown). Sixteen founder lines were established by mating founders with C57/BL6 wild-type mice. Ang1 expression has been reported in osteoblasts [16–19]; however, increased Ang1 mRNA expression was detected in long bones from transgenic mice by RT-PCR and by Western blotting of lysates from calvaria compared with controls (Fig. 1C and D and data not shown). Osteoblast-specific Ang1 overexpression results in increased bone mass in vivo Since the role of Ang1 in bone formation is not well known, we analyzed bone volume in Ang1-Tg mice. Ang1-Tg mice showed significantly increased bone mineral density (BMD) as analyzed by microcomputed tomography (lCT) compared with littermate controls (Fig. 2A). Bone volume/tissue volume (BV/TV) and trabecular thickness (Tb.Th) also significantly increased, while trabecular separation (Tb.Sp) significantly decreased in 8-week-old Ang1-Tg mice compared with control littermates (Fig. 2A). Furthermore, the bone marrow cavity of the metaphysics region was filled with bone in Ang1-Tg mice (Fig. 2B). These results overall indicate that increased
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Fig. 1. Generation of Ang1-transgenic (Ang1-Tg) mice. (A) Schematic construct of the Ang1 transgene containing the Col1a1 promoter followed by Ang1, IRES-EGFP, and a polyA sequence. (B) Tail DNA was extracted from F0 founders, and genomic PCR to detect the Ang1 transgene was performed. Representative data are shown. Ang1 transgenes were detected in lanes 2 and 4 but not in 1 and 3. (C) Total RNA was extracted from whole bone of an Ang1-Tg mouse (TG) or a wild-type littermate (WT), and RT-PCR analysis of Ang1 and the internal control, b-actin, was undertaken. (D) Total lysates were extracted from calvaria of an Ang1-Tg mouse (TG) or a wild-type littermate (WT), and Western blot analysis of Ang1 and the internal control, actin, was undertaken.
Fig. 2. Osteoblast-specific Ang1 overexpression results in increased bone mass in vivo. (A) Femoral bones were dissected from 8-week-old Ang1-Tg (TG) or wild-type littermates (WT), and BMD (bone mineral density), BV/TV (bone volume/tissue volume), Tb.Th (trabecular thickness), and Tb.Sp (trabecular separation) were analyzed by microcomputed tomography (lCT) (*P < 0.05). (B) Femoral bones were dissected from 8-week-old Ang1-Tg (TG) or control littermates (WT), and microradiographic analysis of femurs was undertaken by lCT. The bone cavity of the femoral metaphysic indicated by a bar was filled with increased bone in Ang1-Tg mice.
Ang1 expression in osteoblasts upregulates bone mass in vivo. Increased trabecular bone mass and osteoblast activity by elevated Ang1 expression in osteoblasts To confirm the observed increase in bone mass in Ang1Tg mice, bone sections were analyzed histologically.
Increased length and thickness of trabecular bones were observed in Ang1-Tg mice compared with wild-type littermates (Fig. 3A). Immunohistochemical analysis of alkaline phosphatase (ALP), a marker of osteoblasts, revealed increased ALP activity in the trabecular area of Ang1-Tg mice compared with wild-type controls (Fig. 3B). Thus Ang1-Tg mice show increased trabecular bone mass and osteoblastic activity.
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Fig. 3. Increased trabecular bone mass and osteoblast activity by elevated Ang1 expression in osteoblasts. (A) Tibial sections of 8-week-old Ang1-Tg (TG) or wild-type littermates (WT) were stained by H/E and observed microscopically. Arrows indicate trabecular bone. Higher magnification images are shown in boxes and trabecular thickness is indicated by a bar. Trabecular bone length and thickness increased in Ang1-Tg compared with wild-type littermates. (B) Tibial sections of 8-week-old Ang1-Tg (TG) or control littermates (WT) were stained by mouse anti-ALP antibody followed by Alexa 488conjugated anti-mouse IgG. TOTO3 was used as a nuclear counter-stain. Sections were then examined under a confocal microscope (left panel) and ALP fluorescence intensity was quantified (right panel) (*P < 0.05). Significantly elevated ALP expression was detected in Ang1-Tg mice compared with control littermates.
CD31-positive endothelial cells are increased while osteoclast activity is not modulated in bone by osteoblast-specific Ang1 overexpression To determine whether the effects of Ang1 in osteoblasts are direct or indirect we asked whether the Ang1 receptor, Tie2, is expressed in osteoblasts. Tie2 expression was not detected in primary osteoblasts derived from calvaria or the osteoblastic cell line MC3T3-E1 by both real-time PCR and flow cytometry (Fig. 4A and data not shown). These results suggest that the effect of Ang1 on bone formation may be indirect. Interestingly, a significantly elevated number of CD31-positive vascular endothelial cells was detected in the trabecular region of Ang1-Tg mice compared with wild-type littermates (Fig. 4B), suggesting that increased angiogenesis mediated by Ang1 is partially responsible for increased bone mass. Bone homeostasis requires a delicate balance between activities of bone-resorbing osteoclasts and bone-forming osteoblasts [3], and reduced osteoclast activity causes increased bone mass in vivo. To evaluate potential reduction in osteoclast activity in Ang1-Tg mice, the presence of osteoclasts was analyzed by staining with the osteoclast-specific maturation marker, Cathepsin K (Fig. 4C). The number of Cathepsin K-positive mature osteoclasts was not significantly changed in transgenic versus control mice, suggesting that Ang1 upregulates bone mass possibly through increased angiogenesis.
Discussion Bone volume is regulated by a balance between bone formation by osteoblasts and bone-resorption by osteoclasts. Osteoblasts are derived from mesenchymal stem cells, whereas osteoclasts are from hematopoietic stem cells. During differentiation, osteoblasts express osteoblastic markers such as Col1a1 and ALP, while osteoclasts express TRAP, Cathepsin K and DC-STAMP [24–27]. These two cell types cooperatively regulate bone mass [3]. Here we demonstrate the role of the Ang1 angiogenic factor expressed in one of these cell types, osteoblasts, on bone formation. Our transgenic strategy, which upregulates transgene expression only in the appropriate tissue, reflects the physiological function of the misexpressed gene. Using that approach, we show that increased Ang1 expression in osteoblasts resulted in upregulated bone mass and ALP activity coupled with an elevated number of vascular endothelial cells. Vascular invasion induced by VEGF has been reported to occur in bone development [1,2,4,5]. During long bone development, avascular proliferating chondrocytes give rise to hypertrophic chondrocytes and undergo apoptosis, which in turn induces vascular invasion and ossification in developing bones. Thus vascular invasion may stimulate bone development. Angiogenesis has been described as involved in tumor growth, and such tumor angiogenesis
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Fig. 4. CD31-positive endothelial cells are increased while osteoclast activity is not modulated in bone by osteoblast-specific Ang1 overexpression. (A) Total RNA was extracted from primary osteoblasts and the endothelial cell line, bEnd3, and real-time PCR analysis of Tie2 relative to the b-actin internal control was undertaken. Tie2 was not expressed in osteoblasts. (B) Tibial sections of 8-week-old Ang1-Tg (TG) or control littermates (WT) were stained by rat anti-CD31 antibody followed by Alexa 488-conjugated anti-rat IgG. TOTO3 was used as a nuclear counter-stain. Sections were examined under a confocal microscope (left panel), and the number of CD31-positive cells determined (right panel) (*P < 0.05). The number of CD31-positive cells significantly increased in Ang1-Tg mice compared with controls. (C) Tibial sections of 8-week-old Ang1-Tg or control littermates were stained by mouse anti-Cathepsin K antibody followed by Alexa 488-conjugated anti-mouse IgG, and observed under a confocal microscope. TOTO3 was used to stain nuclei. Left, representative data. Right, data are mean relative numbers ± SD of Cathepsin K-positive osteoclasts in Ang1-Tg mice compared with controls.
may supply oxygen and various factors required for tumor development. Ang1 overexpression was observed in various malignant tumors [28] and resulted in increased vascular network and tumor growth [29], and that angiogenesis by Ang1 supports the bone formation. It has been reported that reduced ALP activity after skeletal unloading is closely correlated with reduced expression of endothelial cell marker PECAM-1 (CD31) in bone marrow cells [30]. Thus, angiogenesis promoted by Ang1 may play a key role in bone homeostasis. In contrast, several tissues remain avascular by a blocking vascular invasion of those tissues [31–33]. For example, cardiac valves are known avascular tissues, and loss of avascularity causes induction of ossification in valvular tissues, which in turn causes valvular heart disease [33]. Thus angiogenesis and ossification are closely related, and control of angiogenesis may be useful to regulate bone formation, control tumor development, and maintain avascular tissue homeostasis. VEGF functions physiologically in bone repair or that it is used in treatment of fractures [34,35]. Our present study suggests a potential use of Ang1 as a therapeutic agent for bone repair through angiogenesis. However, Ang1 production is hindered by protein aggregation and insolubility due to formation of disulfide-linked higher-order structures. Recently, some of these difficulties have been resolved by
replacing the Ang-1 N-terminus with the short coiled-coil domain of cartilage oligomeric matrix protein (COMP), resulting in a chimeric protein designated COMP-Ang1 [36]. Since COMP-Ang1 treatment has been shown to promote improved wound healing, which is correlated with increased angiogenesis [37], COMP-Ang1 represents a potentially effective alternative treatment to native Ang1, and its use may lead to beneficial therapies for bone repair by increasing angiogenesis. Acknowledgments We thank Y. Sato and A. Kumakubo for technical support. T. Miyamoto was supported by a grant-in-aid for Young Scientists (B), Japan. T. Suda was supported by a grant-in-aid from Specially Promoted Research of the Ministry of Education, Science, Sports and Culture, Japan. T. Suzuki was supported by a grant-in-aid from the 21st century COE Program of the Ministry of Education, Culture, Sports, Science and Technology, Japan, to Keio University. The authors have no conflicting financial interests. References [1] H.M. Kronenberg, Developmental regulation of the growth plate, Nature 15 (2003) 332–336.
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[2] N. Ortega, D.J. Behonick, Z. Werb, Matrix remodeling during endochondral ossification, Trends Cell Biol. 14 (2004) 86–93. [3] C. Zhao, N. Irie, Y. Takada, K. Shimoda, T. Miyamoto, T. Nishiwaki, T. Suda, K. Matsuo, Bidirectional ephrinB2-EphB4 signaling controls bone homeostasis, Cell Metab. 4 (2006) 111–121. [4] H.P. Gerber, T.H. Vu, A.M. Ryan, J. Kowalski, Z. Werb, N. Ferrara, VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation, Nat. Med. 5 (1999) 623–628. [5] Y. Wang, C. Wan, L. Deng, X. Liu, X. Cao, S.R. Gilbert, M.L. Bouxsein, M.C. Faugere, R.E. Guldberg, L.C. Gerstenfeld, V.H. Haase, R. S Johnson, E. Schipani, T.L. Clemens, The hypoxiainducible factor alpha pathway couples angiogenesis to osteogenesis during skeletal development, J. Clin. Invest. 117 (2007) 1616–1626. [6] N.W. Gale, G.D. Yancopoulos, Growth factors acting via endothelial cell-specific receptor tyrosine kinases: VEGFs, angiopoietins, and ephrins in vascular development, Genes Dev. 13 (1999) 1055–1066. [7] I. Kim, H.G. Kim, J.N. So, J.H. Kim, H.J. Kwak, G.Y. Koh, Angiopoietin-1 regulates endothelial cell survival through the phosphatidylinositol 3 0 -Kinase/Akt signal transduction pathway, Circ. Res. 86 (2000) 24–29. [8] K.T. Kim, H.H. Choi, M.O. Steinmetz, B. Maco, R.A. Kammerer, S.Y. Ahn, H.Z. Kim, G.M. Lee, G.Y. Koh, Oligomerization and multimerization are critical for angiopoietin-1 to bind and phosphorylate Tie2, J. Biol. Chem. 280 (2005) 20126–20131. [9] S. Davis, T.H. Aldrich, P.F. Jones, A. Acheson, D.L. Compton, V. Jain, T.E. Ryan, J. Bruno, C. Radziejewski, P.C. Maisonpierre, G.D. Yancopoulos, Isolation of angiopoietin-1, a ligand for the TIE2 receptor, by secretion-trap expression cloning, Cell 87 (1996) 1161– 1169. [10] G. Thurston, C. Suri, K. Smith, J. McClain, T.N. Sato, G.D. Yancopoulos, D.M. McDonald, Leakage-resistant blood vessels in mice transgenically overexpressing angiopoietin-1, Science 286 (1999) 2511–2514. [11] C. Suri, J. McClain, G. Thurston, D.M. McDonald, H. Zhou, E.H. Oldmixon, T.N. Sato, G.D. Yancopoulos, Increased vascularization in mice overexpressing angiopoietin-1, Science 282 (1998) 468–471. [12] A.L. Haninec, D. Voskas, A. Needles, A.S. Brown, F.S. Foster, D.J. Dumont, Transgenic expression of Angiopoietin 1 in the liver leads to changes in lymphatic and blood vessel architecture, Biochem. Biophys. Res. Commun. 345 (2006) 1299–1307. [13] N.W. Gale, G. Thurston, S.F. Hackett, R. Renard, Q. Wang, J. McClain, C. Martin, C. Witte, M.H. Witte, D. Jackson, C. Suri, P.A. Campochiaro, S.J. Wiegand, G.D. Yancopoulos, Angiopoietin-2 is required for postnatal angiogenesis and lymphatic patterning, and only the latter role is rescued by Angiopoietin-1, Dev. Cell 3 (2002) 411–423. [14] N. Takakura, T. Watanabe, S. Suenobu, Y. Yamada, T. Noda, Y. Ito, M. Satake, T. Suda, A role for hematopoietic stem cells in promoting angiogenesis, Cell 102 (2000) 199–209. [15] C. Suri, P.F. Jones, S. Patan, S. Bartunkova, P.C. Maisonpierre, S. Davis, T.N. Sato, G.D. Yancopoulos, Requisite role of angiopoietin1, a ligand for the TIE2 receptor, during embryonic angiogenesis, Cell 87 (1996) 1171–1180. [16] F. Arai, A. Hirao, M. Ohmura, H. Sato, S. Matsuoka, K. Takubo, K. Ito, G.Y. Koh, T. Suda, Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche, Cell 118 (2004) 149–161. [17] T. Kasama, T. Isozaki, T. Odai, M. Matsunawa, K. Wakabayashi, H.T. Takeuchi, S. Matsukura, M. Adachi, M. Tezuka, K. Kobayashi, Expression of angiopoietin-1 in osteoblasts and its inhibition by tumor necrosis factor-alpha and interferon-gamma, Transl. Res. 149 (2007) 265–273. [18] J.H. Park, H.I. Song, J.M. Rho, M.R. Kim, J.R. Kim, B.H. Park, T.S. Park, H.S. Baek, Parathyroid hormone (1–34) augments angiopoietin-1 expression in human osteoblast-like cells, Exp. Clin. Endocrinol. Diabetes 114 (2006) 438–443.
[19] A. Horner, S. Bord, A.W. Kelsall, N. Coleman, J.E. Compston, Tie2 ligands angiopoietin-1 and angiopoietin-2 are coexpressed with vascular endothelial cell growth factor in growing human bone, Bone 28 (2001) 65–71. [20] N. Fujita, T. Miyamoto, J. Imai, N. Hosogane, T. Suzuki, M. Yagi, K. Morita, K. Ninomiya, K. Miyamoto, H. Takaishi, M. Matsumoto, H. Morioka, H. Yabe, K. Chiba, S. Watanabe, Y. Toyama, T. Suda, CD24 is expressed specifically in the nucleus pulposus of intervertebral discs, Biochem. Biophys. Res. Commun. 338 (2005) 1890–1896. [21] K. Morita, T. Miyamoto, N. Fujita, Y. Kubota, K. Ito, K. Takubo, K. Miyamoto, K. Ninomiya, T. Suzuki, R. Iwasaki, M. Yagi, H. Takaishi, Y. Toyama, T. Suda, Reactive oxygen species induce chondrocyte hypertrophy in endochondral ossification, J. Exp. Med. 204 (2007) 1613–1623. [22] R.J.H. Eberspaecher, B.D. Crombrugghe, Separate cis-acting DNA elements of the mouse pro-1(I) collagen promoter direct expression of reporter genes to different type I collagen-producing cells in transgenic mice, J. Cell Biol. 129 (1995) 1421–1432. [23] W. Liu, S. Toyosawa, T. Furuichi, N. Kanatani, C. Yoshida, Y. Liu, M. Himeno, S. Narai, A. Yamaguchi, T. Komori, Overexpression of Cbfa1 in osteoblasts inhibits osteoblast maturation and causes osteopenia with multiple fractures, J. Cell Biol. 155 (2001) 157–166. [24] G.S. Stein, J.B. Lian, J.L. Stein, A.J. Van Wijnen, M. Montecino, Transcriptional control of osteoblast growth and differentiation, Physiol. Rev. 76 (1996) 593–629. [25] T. Miyamoto, T. Suda, Differentiation and function of osteoclasts, Keio J. Med. 52 (2003) 1–7. [26] M. Yagi, T. Miyamoto, Y. Sawatani, K. Iwamoto, N. Hosogane, N. Fujita, K. Morita, K. Ninomiya, T. Suzuki, K. Miyamoto, Y. Oike, M. Takeya, Y. Toyama, T. Suda, DC-STAMP is essential for cell–cell fusion in osteoclasts and foreign body giant cells, J. Exp. Med. 202 (2005) 345–351. [27] T. Miyamoto, The dendritic cell-specific transmembrane protein DCSTAMP is essential for osteoclast fusion and osteoclast boneresorbing activity, Mod. Rheumatol. 16 (2006) 341–342. [28] G. Zadeh, R. Reti, K. Koushan, Q. Baoping, P. Shannon, A. Guha, Regulation of the pathological vasculature of malignant astrocytomas by angiopoietin-1, Neoplasia 7 (2005) 1081–1090. [29] MR. Machein, A. Knedla, R. Knoth, S. Wagner, E. Neuschl, K.H. Plate, Angiopoietin-1 promotes tumor angiogenesis in a rat glioma model, Am. J. Pathol. 165 (2004) 1557–1570. [30] M. Sakuma-Zenke, A. Sakai, S. Nakayamada, N. Kunugita, T. Tabata, S. Uchida, S. Tanaka, T. Mori, K. Nakai, Y. Tanaka, T. Nakamura, Reduced expression of platelet endothelial cell adhesion molecule-1 in bone marrow cells in mice after skeletal unloading, J. Bone Miner. Res. 20 (2005) 1002–1010. [31] C. Shukunami, Y. Oshima, Y. Hiraki, Chondromodulin-I and tenomodul: a new class of tissue-specific angiogenesis inhibitors found in hypovascular connective tissues, Biochem. Biophys. Res. Commun. 333 (2005) 299–307. [32] B.K. Ambati, M. Nozaki, N. Singh, A. Takeda, P.D. Jani, T. Suthar, R.J. Albuquerque, E. Richter, E. Sakurai, M.T. Newcomb, M.E. Kleinman, R.B. Caldwell, Q. Lin, Y. Ogura, A. Orecchia, D.A. Samuelson, D.W. Agnew, J. St Leger, W.R. Green, P.J. Mahasreshti, D.T. Curiel, D. Kwan, H. Marsh, S. Ikeda, L.J. Leiper, J.M. Collinson, S. Bogdanovich, T.S. Khurana, M. Shibuya, M.E. Baldwin, N. Ferrara, H.P. Gerber, S. De Falco, J. Witta, J.Z. Baffi, B.J. Raisler, J. Ambati, Corneal avascularity is due to soluble VEGF receptor-1, Nature 443 (2006) 993–997. [33] M. Yoshioka, S. Yuasa, K. Matsumura, K. Kimura, T. Shiomi, N. Kimura, C. Shukunami, Y. Okada, M. Mukai, H. Shin, R. Yozu, M. Sata, S. Ogawa, Y. Hiraki, K. Fukuda, Chondromodulin-I maintains cardiac valvular function by preventing angiogenesis, Nat. Med. 12 (2006) 1151–1159. [34] J. Street, M. Bao, L. deGuzman, S. Bunting, F.V. Peale Jr., N. Ferrara, H. Steinmetz, J. Hoeffel, J.L. Cleland, A. Daugherty, N.V.
T. Suzuki et al. / Biochemical and Biophysical Research Communications 362 (2007) 1019–1025 Bruggen, H.P. Redmond, R.A. Carano, E.H. Filvaroff, Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover, Proc. Natl. Acad. Sci. USA 99 (2002) 9656–9661. [35] F. Geiger, H. Bertram, I. Berger, H. Lorenz, O. Wall, C. Eckhardt, H.G. Simank, W. Richter, Vascular endothelial growth factor geneactivated matrix (VEGF165-GAM) enhances osteogenesis and angiogenesis in large segmental bone defects, J. Bone Miner. Res. 20 (2005) 2028–2035.
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[36] C.H. Cho, R.A. Kammerer, H.J. Lee, M.O. Steinmetz, Y.S. Ryu, S.H. Lee, K. Yasunaga, K.T. Kim, I. Kim, H.H. Choi, W. Kim, S.H. Kim, S.K. Park, G.M. Lee, G.Y. Koh, COMP-Ang1: a designed angiopoietin-1 variant with nonleaky angiogenic activity, Proc. Natl. Acad. Sci. USA 101 (2004) 5547–5552. [37] C.H. Cho, H.K. Sung, K.T. Kim, H.G. Cheon, G.T. Oh, H.J. Hong, O.J. Yoo, G.Y. Koh, COMP-angiopoietin-1 promotes wound healing through enhanced angiogenesis, lymphangiogenesis, and blood flow in a diabetic mouse model, Proc. Natl. Acad. Sci. USA 103 (2006) 4946–4951.
Osteoblast-specific Angiopoietin 1 overexpression increases bone mass Toru Suzuki a
a,b,1
, Takeshi Miyamoto a,b,c,*,1, Nobuyuki Fujita a,b, Ken Ninomiya Ryotaro Iwasaki a,d, Yoshiaki Toyama b, Toshio Suda a,*
a,b
,
Department of Cell Differentiation, The Sakaguchi Laboratory, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan b Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan c Department of Musculoskeletal Reconstruction and Regeneration Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan d Department of Dentistry and Oral Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan Received 16 August 2007 Available online 28 August 2007
Abstract Although osteoblasts express the angiogenic protein Angiopoietin 1 (Ang1), the role of Ang1 in bone formation remains largely unknown. Here we report that Ang1 overexpression in osteoblasts driven by the osteoblast-specific 2.3 kb alpha 1 type 1 collagen promoter results in increased bone mass in vivo. In Ang1-transgenic mice (Ang1-Tg), bone volume and bone parameters increased significantly compared with wild-type littermates, although the Ang1 receptor, Tie2 was not expressed in osteoblasts. Tie2 is primarily expressed in vascular endothelial cells, and Ang1-Tie2 signaling is reportedly crucial for angiogenesis. We found that the number of vascular endothelial cells was significantly elevated in Ang1-Tg mice compared with that of wild-type littermates, an increase accompanied by increased alkaline-phosphatase activity, a marker of osteoblast activation. The number of osteoclasts in the bone of Ang1-Tg mice did not differ from wild-type littermates. These results indicate that angiogenesis induced by Ang1 expressed in osteoblasts is coupled with osteogenesis. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Angiopoietin 1; Osteoblast; Coupling; Alpha 1 type 1 collagen promoter; Transgenic
Endochondral ossification is a sequential process in which apoptotic hypertrophic chondrocytes are eliminated by chondroclasts and replaced by osteoblasts, accompanied by vascular endothelial cell invasion [1,2]. Longitudinal bone growth is mainly mediated by proliferation of chondrocytes in growth plates, and bone formation is a result of a well-controlled balance between bone-resorption by osteoclasts and bone formation by osteoblasts [1–3]. The role of vascular endothelial growth factor (VEGF) has
*
Corresponding authors. Address: Department of Cell Differentiation, The Sakaguchi Laboratory, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Fax: +81 3 5363 3475. E-mail addresses: [email protected] (T. Miyamoto), sudato@ sc.itc.keio.ac.jp (T. Suda). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.08.099
been demonstrated in bone development [4,5]; however, the role of the angiogenic factor Angiopoietin 1 (Ang1) in regulating bone volume has not well been characterized. Ang1 binds to and phosphorylates a receptor tyrosine kinase exhibiting an Ig and epidermal growth factor homology domain 2 (Tie2) [6–9]. Although Ang1 is secreted, its low solubility has made it difficult to purify and maintain as a stable protein, thus preventing study of its biological effects. However, gene ablation and transgenic studies of Ang1 have revealed its indispensable role in development and maintenance of the blood and lymphatic vascular systems, as well as the hematopoietic systems [10– 15]. Ang1 is expressed in osteoblasts [16–19]; however, its role in bone formation is largely unknown. Here, in order to examine the effects of Ang1 expressed in osteoblasts on the bone formation, we established Ang1transgenic mice (Ang1-Tg) in which Ang1 expression was
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driven by the osteoblast-specific 2.3 kb alpha 1 type 1 collagen (Col1a1) promoter. Ang1 expression was detected in the bone of Ang1-Tg mice, and both bone volume and alkaline-phosphatase (ALP) activity increased, as did numbers of vascular endothelial cells in Ang1-Tg bone. By contrast, the number of osteoclasts in bone did not differ between Ang1-Tg and wild-type littermates. These results indicate that Ang1 expressed in osteoblasts plays a role in control of bone volume through angiogenesis in the bone. Materials and methods Generation of Col1a1-Ang1 transgenic mice. Animals were cared for in accordance with guidelines of Keio University School of Medicine. The mouse Ang1 open reading frame beginning with a suitable Kozak sequence (Ang1 fragment) was PCR amplified. An NcoI- and SalI-digested Ang1 fragment was cloned downstream of the mouse osteoblast-specific Col1a1 promoter in the pBS vector. The Col1a1-Ang1 fragment was digested by NheI and SalI and cloned into the pIRES2-EGFP vector (Clontech) to yield pCol1a1-Ang1-IRES-EGFP-PolyA. Col1a1-Ang1IRES-EGFP-PolyA was digested by NheI and AflII to purify the Ang1 transgene. Transgenic mice were generated in a Slc:BDF1 background by transgene microinjection (Japan SLC, Shizuoka, Japan), and injected embryos were implanted into pseudopregnant Slc:ICR females. The presence of the transgene was evaluated by PCR of tail DNA using the following primer set: forward primer, 5 0 0 -AAGCTGACCCTGAAGTT CATCTGC-3 0 0 ; reverse primer, 5 0 0 -CTGCTTGTCGGCCATGATATA GAC-3 0 0 . RT-PCR analysis. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) from femurs of mouse neonates. To prevent DNA contamination, total RNA was purified using RNeasy Total RNA Isolation Kit using RNase free Dnase (Qiagen GmbH, Hilden, Germany). RNA was quantified by an Ultro spec 3300 pro (GE Healthcare, Buckinghamshire, UK) to select samples with an absorbance ratio of (A260/ A280) of 2.0. Randomly primed complementary DNA (cDNA) was synthesized from 1 lg total RNA using Advantage RT-for- PCR kit (Clontech). RT-PCR analysis of Ang1 and b-actin expression was performed using the following primers: Ang1, forward primer, 5 0 0 -ACT AGTAGTACAATGACAGTTTTCCTTTCC-3 0 0 and reverse primer, 5 0 0 AGATCTTCAAAAGTCCAAGGGCCGGATCAT-3 0 0 ; b-actin, forward primer, 5 0 0 -GTGATGGTGGGAATGGGTCAGAAGGAC-3 0 0 and reverse primer, 5 0 0 -GAAGGAAGGCTGGAAAAGAGCCTCAGG -3 0 0 . Quantitative real-time PCR analysis of Tie2 expression relative to b-actin was analyzed using TaqMan assays (7500 Fast Real-Time PCR system, Applied Biosystems, Foster City, CA) using the following probes: Tie2, Mm00443242_ml; b-actin, Mm00607939_sl. Western blot analysis. Mouse calvarias were cut into chips and immediately placed into liquid nitrogen. Frozen chips were crushed to particles using a Cool Mill (Tokken, Chiba, Japan) and solubilized with lysis buffer (1% Triton X-100, 150 mM sodium chloride, 1 mM EDTA, 50 mM tris–hydroxymethyl aminomethane). Cell lysates mixed with loading buffer [tris–Glycine SDS Sample Buffer (Invitrogen, Carlsbad, CA) with 5% 2-mercaptoethanol (2-ME)] were incubated at 80 °C for 3 min, loaded onto 10% polyacrylamide gels, and electrophoresed in SDS– PAGE. Proteins were transferred to PVDF membranes (Nihon Millipore Ltd., Yonezawa, Japan) and blotted with goat anti-Ang1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) followed by HRP-conjugated rabbit anti-goat IgG (Invitrogen). Proteins were visualized using ECL Western Blotting Detection Reagents (GE Healthcare). Membranes were stripped by stripping buffer (2% SDS, 62.5 mM tris–hydroxymethyl aminomethane, pH 6.7, with 0.7% 2-ME) and re-blotted with rabbit anti-actin antibody (Sigma–Aldrich, St. Louis, MO) followed by goat anti-rabbit HRP (Invitrogen). Microcomputed tomography (CT) analysis. Femurs of 8-week-old mice were fixed with 70% ethanol and imaged by lCT (eXplore lCT; GE Healthcare). The following parameters were determined by advance bone
analysis (GE Healthcare), bone mineral density (BMD; mg/cc), bone volume/tissue volume (BV/TV; %), trabecular bone thickness (Tb.Th; lm), trabecular number (Tb. N; per mm), and trabecular separation (Tb.Sp; lm). Three-dimensional images were reconstructed by ResolveRT (Mercury Computer Systems, Inc., Chelmsford, MA). Histological and immunohistochemical analysis. Femurs and tibias were dissected from 6- to 8-week-old mice, fixed with 4% paraformaldehyde, and embedded in paraffin. Sections of 5 lm thickness were stained with hematoxylin and eosin (H/E) or mouse anti-Cathepsin K monoclonal antibody (BD PharMingen, San Diego, CA) followed by Alexa 488-conjugated anti-mouse IgG (Invitrogen). For CD31 and ALP staining, frozen sections of hindlimb that had not been decalcified were cut from 6- to 8week-old femurs and tibias embedded in mouse liver paste, as described [20,21]. Sections were incubated as follows: rat anti-CD31 monoclonal antibody (BD PharMingen) followed by Alexa 488-conjugated anti-rat IgG (Invitrogen), mouse anti-ALP monoclonal antibody (BD PharMingen) followed by Alexa 488-conjugated anti-mouse IgG (Invitrogen). TOTO3 (Invitrogen) was used for nuclear staining. Immunoreactivity was detected by confocal microscopy (Olympus, Tokyo, Japan). Quantification of immunoreactive cells or fluorescence intensity was determined using Scion Image Analysis Program (National Institutes of Health, USA), a public domain image processing and analysis program.
Results Generation of Ang1-transgenic (Ang1-Tg) mice To understand the role of Ang1 expressed in osteoblasts in bone formation in vivo, we generated a transgenic mouse overexpressing Ang1 in osteoblasts under control of the osteoblast-specific Col1a1 promoter. The Ang1-Tg transgene consisted of murine Ang1, IRES-EGFP, and a polyA sequence (Fig. 1A). Osteoblast specificity of the Col1a1 promoter has been reported [22,23]. Fifty-one lines of offspring were generated by embryo microinjection, and the presence of the transgene tested by PCR of tail DNA using transgene-specific primer sets (Fig. 1B and data not shown). Sixteen founder lines were established by mating founders with C57/BL6 wild-type mice. Ang1 expression has been reported in osteoblasts [16–19]; however, increased Ang1 mRNA expression was detected in long bones from transgenic mice by RT-PCR and by Western blotting of lysates from calvaria compared with controls (Fig. 1C and D and data not shown). Osteoblast-specific Ang1 overexpression results in increased bone mass in vivo Since the role of Ang1 in bone formation is not well known, we analyzed bone volume in Ang1-Tg mice. Ang1-Tg mice showed significantly increased bone mineral density (BMD) as analyzed by microcomputed tomography (lCT) compared with littermate controls (Fig. 2A). Bone volume/tissue volume (BV/TV) and trabecular thickness (Tb.Th) also significantly increased, while trabecular separation (Tb.Sp) significantly decreased in 8-week-old Ang1-Tg mice compared with control littermates (Fig. 2A). Furthermore, the bone marrow cavity of the metaphysics region was filled with bone in Ang1-Tg mice (Fig. 2B). These results overall indicate that increased
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Fig. 1. Generation of Ang1-transgenic (Ang1-Tg) mice. (A) Schematic construct of the Ang1 transgene containing the Col1a1 promoter followed by Ang1, IRES-EGFP, and a polyA sequence. (B) Tail DNA was extracted from F0 founders, and genomic PCR to detect the Ang1 transgene was performed. Representative data are shown. Ang1 transgenes were detected in lanes 2 and 4 but not in 1 and 3. (C) Total RNA was extracted from whole bone of an Ang1-Tg mouse (TG) or a wild-type littermate (WT), and RT-PCR analysis of Ang1 and the internal control, b-actin, was undertaken. (D) Total lysates were extracted from calvaria of an Ang1-Tg mouse (TG) or a wild-type littermate (WT), and Western blot analysis of Ang1 and the internal control, actin, was undertaken.
Fig. 2. Osteoblast-specific Ang1 overexpression results in increased bone mass in vivo. (A) Femoral bones were dissected from 8-week-old Ang1-Tg (TG) or wild-type littermates (WT), and BMD (bone mineral density), BV/TV (bone volume/tissue volume), Tb.Th (trabecular thickness), and Tb.Sp (trabecular separation) were analyzed by microcomputed tomography (lCT) (*P < 0.05). (B) Femoral bones were dissected from 8-week-old Ang1-Tg (TG) or control littermates (WT), and microradiographic analysis of femurs was undertaken by lCT. The bone cavity of the femoral metaphysic indicated by a bar was filled with increased bone in Ang1-Tg mice.
Ang1 expression in osteoblasts upregulates bone mass in vivo. Increased trabecular bone mass and osteoblast activity by elevated Ang1 expression in osteoblasts To confirm the observed increase in bone mass in Ang1Tg mice, bone sections were analyzed histologically.
Increased length and thickness of trabecular bones were observed in Ang1-Tg mice compared with wild-type littermates (Fig. 3A). Immunohistochemical analysis of alkaline phosphatase (ALP), a marker of osteoblasts, revealed increased ALP activity in the trabecular area of Ang1-Tg mice compared with wild-type controls (Fig. 3B). Thus Ang1-Tg mice show increased trabecular bone mass and osteoblastic activity.
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Fig. 3. Increased trabecular bone mass and osteoblast activity by elevated Ang1 expression in osteoblasts. (A) Tibial sections of 8-week-old Ang1-Tg (TG) or wild-type littermates (WT) were stained by H/E and observed microscopically. Arrows indicate trabecular bone. Higher magnification images are shown in boxes and trabecular thickness is indicated by a bar. Trabecular bone length and thickness increased in Ang1-Tg compared with wild-type littermates. (B) Tibial sections of 8-week-old Ang1-Tg (TG) or control littermates (WT) were stained by mouse anti-ALP antibody followed by Alexa 488conjugated anti-mouse IgG. TOTO3 was used as a nuclear counter-stain. Sections were then examined under a confocal microscope (left panel) and ALP fluorescence intensity was quantified (right panel) (*P < 0.05). Significantly elevated ALP expression was detected in Ang1-Tg mice compared with control littermates.
CD31-positive endothelial cells are increased while osteoclast activity is not modulated in bone by osteoblast-specific Ang1 overexpression To determine whether the effects of Ang1 in osteoblasts are direct or indirect we asked whether the Ang1 receptor, Tie2, is expressed in osteoblasts. Tie2 expression was not detected in primary osteoblasts derived from calvaria or the osteoblastic cell line MC3T3-E1 by both real-time PCR and flow cytometry (Fig. 4A and data not shown). These results suggest that the effect of Ang1 on bone formation may be indirect. Interestingly, a significantly elevated number of CD31-positive vascular endothelial cells was detected in the trabecular region of Ang1-Tg mice compared with wild-type littermates (Fig. 4B), suggesting that increased angiogenesis mediated by Ang1 is partially responsible for increased bone mass. Bone homeostasis requires a delicate balance between activities of bone-resorbing osteoclasts and bone-forming osteoblasts [3], and reduced osteoclast activity causes increased bone mass in vivo. To evaluate potential reduction in osteoclast activity in Ang1-Tg mice, the presence of osteoclasts was analyzed by staining with the osteoclast-specific maturation marker, Cathepsin K (Fig. 4C). The number of Cathepsin K-positive mature osteoclasts was not significantly changed in transgenic versus control mice, suggesting that Ang1 upregulates bone mass possibly through increased angiogenesis.
Discussion Bone volume is regulated by a balance between bone formation by osteoblasts and bone-resorption by osteoclasts. Osteoblasts are derived from mesenchymal stem cells, whereas osteoclasts are from hematopoietic stem cells. During differentiation, osteoblasts express osteoblastic markers such as Col1a1 and ALP, while osteoclasts express TRAP, Cathepsin K and DC-STAMP [24–27]. These two cell types cooperatively regulate bone mass [3]. Here we demonstrate the role of the Ang1 angiogenic factor expressed in one of these cell types, osteoblasts, on bone formation. Our transgenic strategy, which upregulates transgene expression only in the appropriate tissue, reflects the physiological function of the misexpressed gene. Using that approach, we show that increased Ang1 expression in osteoblasts resulted in upregulated bone mass and ALP activity coupled with an elevated number of vascular endothelial cells. Vascular invasion induced by VEGF has been reported to occur in bone development [1,2,4,5]. During long bone development, avascular proliferating chondrocytes give rise to hypertrophic chondrocytes and undergo apoptosis, which in turn induces vascular invasion and ossification in developing bones. Thus vascular invasion may stimulate bone development. Angiogenesis has been described as involved in tumor growth, and such tumor angiogenesis
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Fig. 4. CD31-positive endothelial cells are increased while osteoclast activity is not modulated in bone by osteoblast-specific Ang1 overexpression. (A) Total RNA was extracted from primary osteoblasts and the endothelial cell line, bEnd3, and real-time PCR analysis of Tie2 relative to the b-actin internal control was undertaken. Tie2 was not expressed in osteoblasts. (B) Tibial sections of 8-week-old Ang1-Tg (TG) or control littermates (WT) were stained by rat anti-CD31 antibody followed by Alexa 488-conjugated anti-rat IgG. TOTO3 was used as a nuclear counter-stain. Sections were examined under a confocal microscope (left panel), and the number of CD31-positive cells determined (right panel) (*P < 0.05). The number of CD31-positive cells significantly increased in Ang1-Tg mice compared with controls. (C) Tibial sections of 8-week-old Ang1-Tg or control littermates were stained by mouse anti-Cathepsin K antibody followed by Alexa 488-conjugated anti-mouse IgG, and observed under a confocal microscope. TOTO3 was used to stain nuclei. Left, representative data. Right, data are mean relative numbers ± SD of Cathepsin K-positive osteoclasts in Ang1-Tg mice compared with controls.
may supply oxygen and various factors required for tumor development. Ang1 overexpression was observed in various malignant tumors [28] and resulted in increased vascular network and tumor growth [29], and that angiogenesis by Ang1 supports the bone formation. It has been reported that reduced ALP activity after skeletal unloading is closely correlated with reduced expression of endothelial cell marker PECAM-1 (CD31) in bone marrow cells [30]. Thus, angiogenesis promoted by Ang1 may play a key role in bone homeostasis. In contrast, several tissues remain avascular by a blocking vascular invasion of those tissues [31–33]. For example, cardiac valves are known avascular tissues, and loss of avascularity causes induction of ossification in valvular tissues, which in turn causes valvular heart disease [33]. Thus angiogenesis and ossification are closely related, and control of angiogenesis may be useful to regulate bone formation, control tumor development, and maintain avascular tissue homeostasis. VEGF functions physiologically in bone repair or that it is used in treatment of fractures [34,35]. Our present study suggests a potential use of Ang1 as a therapeutic agent for bone repair through angiogenesis. However, Ang1 production is hindered by protein aggregation and insolubility due to formation of disulfide-linked higher-order structures. Recently, some of these difficulties have been resolved by
replacing the Ang-1 N-terminus with the short coiled-coil domain of cartilage oligomeric matrix protein (COMP), resulting in a chimeric protein designated COMP-Ang1 [36]. Since COMP-Ang1 treatment has been shown to promote improved wound healing, which is correlated with increased angiogenesis [37], COMP-Ang1 represents a potentially effective alternative treatment to native Ang1, and its use may lead to beneficial therapies for bone repair by increasing angiogenesis. Acknowledgments We thank Y. Sato and A. Kumakubo for technical support. T. Miyamoto was supported by a grant-in-aid for Young Scientists (B), Japan. T. Suda was supported by a grant-in-aid from Specially Promoted Research of the Ministry of Education, Science, Sports and Culture, Japan. T. Suzuki was supported by a grant-in-aid from the 21st century COE Program of the Ministry of Education, Culture, Sports, Science and Technology, Japan, to Keio University. The authors have no conflicting financial interests. References [1] H.M. Kronenberg, Developmental regulation of the growth plate, Nature 15 (2003) 332–336.
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[2] N. Ortega, D.J. Behonick, Z. Werb, Matrix remodeling during endochondral ossification, Trends Cell Biol. 14 (2004) 86–93. [3] C. Zhao, N. Irie, Y. Takada, K. Shimoda, T. Miyamoto, T. Nishiwaki, T. Suda, K. Matsuo, Bidirectional ephrinB2-EphB4 signaling controls bone homeostasis, Cell Metab. 4 (2006) 111–121. [4] H.P. Gerber, T.H. Vu, A.M. Ryan, J. Kowalski, Z. Werb, N. Ferrara, VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation, Nat. Med. 5 (1999) 623–628. [5] Y. Wang, C. Wan, L. Deng, X. Liu, X. Cao, S.R. Gilbert, M.L. Bouxsein, M.C. Faugere, R.E. Guldberg, L.C. Gerstenfeld, V.H. Haase, R. S Johnson, E. Schipani, T.L. Clemens, The hypoxiainducible factor alpha pathway couples angiogenesis to osteogenesis during skeletal development, J. Clin. Invest. 117 (2007) 1616–1626. [6] N.W. Gale, G.D. Yancopoulos, Growth factors acting via endothelial cell-specific receptor tyrosine kinases: VEGFs, angiopoietins, and ephrins in vascular development, Genes Dev. 13 (1999) 1055–1066. [7] I. Kim, H.G. Kim, J.N. So, J.H. Kim, H.J. Kwak, G.Y. Koh, Angiopoietin-1 regulates endothelial cell survival through the phosphatidylinositol 3 0 -Kinase/Akt signal transduction pathway, Circ. Res. 86 (2000) 24–29. [8] K.T. Kim, H.H. Choi, M.O. Steinmetz, B. Maco, R.A. Kammerer, S.Y. Ahn, H.Z. Kim, G.M. Lee, G.Y. Koh, Oligomerization and multimerization are critical for angiopoietin-1 to bind and phosphorylate Tie2, J. Biol. Chem. 280 (2005) 20126–20131. [9] S. Davis, T.H. Aldrich, P.F. Jones, A. Acheson, D.L. Compton, V. Jain, T.E. Ryan, J. Bruno, C. Radziejewski, P.C. Maisonpierre, G.D. Yancopoulos, Isolation of angiopoietin-1, a ligand for the TIE2 receptor, by secretion-trap expression cloning, Cell 87 (1996) 1161– 1169. [10] G. Thurston, C. Suri, K. Smith, J. McClain, T.N. Sato, G.D. Yancopoulos, D.M. McDonald, Leakage-resistant blood vessels in mice transgenically overexpressing angiopoietin-1, Science 286 (1999) 2511–2514. [11] C. Suri, J. McClain, G. Thurston, D.M. McDonald, H. Zhou, E.H. Oldmixon, T.N. Sato, G.D. Yancopoulos, Increased vascularization in mice overexpressing angiopoietin-1, Science 282 (1998) 468–471. [12] A.L. Haninec, D. Voskas, A. Needles, A.S. Brown, F.S. Foster, D.J. Dumont, Transgenic expression of Angiopoietin 1 in the liver leads to changes in lymphatic and blood vessel architecture, Biochem. Biophys. Res. Commun. 345 (2006) 1299–1307. [13] N.W. Gale, G. Thurston, S.F. Hackett, R. Renard, Q. Wang, J. McClain, C. Martin, C. Witte, M.H. Witte, D. Jackson, C. Suri, P.A. Campochiaro, S.J. Wiegand, G.D. Yancopoulos, Angiopoietin-2 is required for postnatal angiogenesis and lymphatic patterning, and only the latter role is rescued by Angiopoietin-1, Dev. Cell 3 (2002) 411–423. [14] N. Takakura, T. Watanabe, S. Suenobu, Y. Yamada, T. Noda, Y. Ito, M. Satake, T. Suda, A role for hematopoietic stem cells in promoting angiogenesis, Cell 102 (2000) 199–209. [15] C. Suri, P.F. Jones, S. Patan, S. Bartunkova, P.C. Maisonpierre, S. Davis, T.N. Sato, G.D. Yancopoulos, Requisite role of angiopoietin1, a ligand for the TIE2 receptor, during embryonic angiogenesis, Cell 87 (1996) 1171–1180. [16] F. Arai, A. Hirao, M. Ohmura, H. Sato, S. Matsuoka, K. Takubo, K. Ito, G.Y. Koh, T. Suda, Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche, Cell 118 (2004) 149–161. [17] T. Kasama, T. Isozaki, T. Odai, M. Matsunawa, K. Wakabayashi, H.T. Takeuchi, S. Matsukura, M. Adachi, M. Tezuka, K. Kobayashi, Expression of angiopoietin-1 in osteoblasts and its inhibition by tumor necrosis factor-alpha and interferon-gamma, Transl. Res. 149 (2007) 265–273. [18] J.H. Park, H.I. Song, J.M. Rho, M.R. Kim, J.R. Kim, B.H. Park, T.S. Park, H.S. Baek, Parathyroid hormone (1–34) augments angiopoietin-1 expression in human osteoblast-like cells, Exp. Clin. Endocrinol. Diabetes 114 (2006) 438–443.
[19] A. Horner, S. Bord, A.W. Kelsall, N. Coleman, J.E. Compston, Tie2 ligands angiopoietin-1 and angiopoietin-2 are coexpressed with vascular endothelial cell growth factor in growing human bone, Bone 28 (2001) 65–71. [20] N. Fujita, T. Miyamoto, J. Imai, N. Hosogane, T. Suzuki, M. Yagi, K. Morita, K. Ninomiya, K. Miyamoto, H. Takaishi, M. Matsumoto, H. Morioka, H. Yabe, K. Chiba, S. Watanabe, Y. Toyama, T. Suda, CD24 is expressed specifically in the nucleus pulposus of intervertebral discs, Biochem. Biophys. Res. Commun. 338 (2005) 1890–1896. [21] K. Morita, T. Miyamoto, N. Fujita, Y. Kubota, K. Ito, K. Takubo, K. Miyamoto, K. Ninomiya, T. Suzuki, R. Iwasaki, M. Yagi, H. Takaishi, Y. Toyama, T. Suda, Reactive oxygen species induce chondrocyte hypertrophy in endochondral ossification, J. Exp. Med. 204 (2007) 1613–1623. [22] R.J.H. Eberspaecher, B.D. Crombrugghe, Separate cis-acting DNA elements of the mouse pro-1(I) collagen promoter direct expression of reporter genes to different type I collagen-producing cells in transgenic mice, J. Cell Biol. 129 (1995) 1421–1432. [23] W. Liu, S. Toyosawa, T. Furuichi, N. Kanatani, C. Yoshida, Y. Liu, M. Himeno, S. Narai, A. Yamaguchi, T. Komori, Overexpression of Cbfa1 in osteoblasts inhibits osteoblast maturation and causes osteopenia with multiple fractures, J. Cell Biol. 155 (2001) 157–166. [24] G.S. Stein, J.B. Lian, J.L. Stein, A.J. Van Wijnen, M. Montecino, Transcriptional control of osteoblast growth and differentiation, Physiol. Rev. 76 (1996) 593–629. [25] T. Miyamoto, T. Suda, Differentiation and function of osteoclasts, Keio J. Med. 52 (2003) 1–7. [26] M. Yagi, T. Miyamoto, Y. Sawatani, K. Iwamoto, N. Hosogane, N. Fujita, K. Morita, K. Ninomiya, T. Suzuki, K. Miyamoto, Y. Oike, M. Takeya, Y. Toyama, T. Suda, DC-STAMP is essential for cell–cell fusion in osteoclasts and foreign body giant cells, J. Exp. Med. 202 (2005) 345–351. [27] T. Miyamoto, The dendritic cell-specific transmembrane protein DCSTAMP is essential for osteoclast fusion and osteoclast boneresorbing activity, Mod. Rheumatol. 16 (2006) 341–342. [28] G. Zadeh, R. Reti, K. Koushan, Q. Baoping, P. Shannon, A. Guha, Regulation of the pathological vasculature of malignant astrocytomas by angiopoietin-1, Neoplasia 7 (2005) 1081–1090. [29] MR. Machein, A. Knedla, R. Knoth, S. Wagner, E. Neuschl, K.H. Plate, Angiopoietin-1 promotes tumor angiogenesis in a rat glioma model, Am. J. Pathol. 165 (2004) 1557–1570. [30] M. Sakuma-Zenke, A. Sakai, S. Nakayamada, N. Kunugita, T. Tabata, S. Uchida, S. Tanaka, T. Mori, K. Nakai, Y. Tanaka, T. Nakamura, Reduced expression of platelet endothelial cell adhesion molecule-1 in bone marrow cells in mice after skeletal unloading, J. Bone Miner. Res. 20 (2005) 1002–1010. [31] C. Shukunami, Y. Oshima, Y. Hiraki, Chondromodulin-I and tenomodul: a new class of tissue-specific angiogenesis inhibitors found in hypovascular connective tissues, Biochem. Biophys. Res. Commun. 333 (2005) 299–307. [32] B.K. Ambati, M. Nozaki, N. Singh, A. Takeda, P.D. Jani, T. Suthar, R.J. Albuquerque, E. Richter, E. Sakurai, M.T. Newcomb, M.E. Kleinman, R.B. Caldwell, Q. Lin, Y. Ogura, A. Orecchia, D.A. Samuelson, D.W. Agnew, J. St Leger, W.R. Green, P.J. Mahasreshti, D.T. Curiel, D. Kwan, H. Marsh, S. Ikeda, L.J. Leiper, J.M. Collinson, S. Bogdanovich, T.S. Khurana, M. Shibuya, M.E. Baldwin, N. Ferrara, H.P. Gerber, S. De Falco, J. Witta, J.Z. Baffi, B.J. Raisler, J. Ambati, Corneal avascularity is due to soluble VEGF receptor-1, Nature 443 (2006) 993–997. [33] M. Yoshioka, S. Yuasa, K. Matsumura, K. Kimura, T. Shiomi, N. Kimura, C. Shukunami, Y. Okada, M. Mukai, H. Shin, R. Yozu, M. Sata, S. Ogawa, Y. Hiraki, K. Fukuda, Chondromodulin-I maintains cardiac valvular function by preventing angiogenesis, Nat. Med. 12 (2006) 1151–1159. [34] J. Street, M. Bao, L. deGuzman, S. Bunting, F.V. Peale Jr., N. Ferrara, H. Steinmetz, J. Hoeffel, J.L. Cleland, A. Daugherty, N.V.
T. Suzuki et al. / Biochemical and Biophysical Research Communications 362 (2007) 1019–1025 Bruggen, H.P. Redmond, R.A. Carano, E.H. Filvaroff, Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover, Proc. Natl. Acad. Sci. USA 99 (2002) 9656–9661. [35] F. Geiger, H. Bertram, I. Berger, H. Lorenz, O. Wall, C. Eckhardt, H.G. Simank, W. Richter, Vascular endothelial growth factor geneactivated matrix (VEGF165-GAM) enhances osteogenesis and angiogenesis in large segmental bone defects, J. Bone Miner. Res. 20 (2005) 2028–2035.
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[36] C.H. Cho, R.A. Kammerer, H.J. Lee, M.O. Steinmetz, Y.S. Ryu, S.H. Lee, K. Yasunaga, K.T. Kim, I. Kim, H.H. Choi, W. Kim, S.H. Kim, S.K. Park, G.M. Lee, G.Y. Koh, COMP-Ang1: a designed angiopoietin-1 variant with nonleaky angiogenic activity, Proc. Natl. Acad. Sci. USA 101 (2004) 5547–5552. [37] C.H. Cho, H.K. Sung, K.T. Kim, H.G. Cheon, G.T. Oh, H.J. Hong, O.J. Yoo, G.Y. Koh, COMP-angiopoietin-1 promotes wound healing through enhanced angiogenesis, lymphangiogenesis, and blood flow in a diabetic mouse model, Proc. Natl. Acad. Sci. USA 103 (2006) 4946–4951.