Osteoblast-specific Angiopoietin 1 overexpression increases bone mass
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.
Available from: Yoshitaka Hishikawa
- "In vitro experiments showed that Ang1 has the ability to bind with components of the basement membrane as well as to type I collagen [7, 29]. Moreover, several reports described that osteoblasts expressed Ang1 but not Tie2 [24, 28, 31], and that Ang1 induced bone formation by facilitating angiogenesis , whereas others reported that Ang1 synergistically enhanced BMP-induced bone formation . However, the expression of Ang1 and Tie2 in tooth has yet to be examined. "
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ABSTRACT: Angiopoietin-1 regulates vascular angiogenesis and stabilization, and is reported to promote bone formation by facilitating angiogenesis. To estimate the role of Ang1 in odontogenesis, we explored the distribution of Ang1 and the receptor, Tie2 in the mouse developing and mature first molar of the mandible. At embryonic day 18, when differentiation of odontoblasts begins, immunosignals for Ang1 were intensely detected in the basement membrane and the distal side, which faced the basement membrane of odontoblasts. In situ hybridization revealed that Ang1 was expressed in odontoblasts and ameloblasts facing the basement membrane. Tie2 was localized in the distal side of odontoblasts. After birth, Ang1 was detected in the predentin, whereas both Ang1 and Tie2 were colocalized in odontoblasts and odontoblast processes. These distributions were retained up to 8 weeks. In contrast to odontoblasts, ameloblasts, cementoblasts and osteoblasts expressed Ang1 but did not express Tie2. Colocalization of Ang1 and Tie2 in odontoblasts and selective expression of Tie2 in odontoblasts among cells responsible for calcified tissue formation suggested the involvement of autocrine signals of Ang1-Tie2 in dentinogenesis.
Available from: Eui-Sic Cho
- "The importance of Ang1 in these processes is supported by the observation that adding excessive Ang1 promotes bone formation, whereas blocking Tie2, the angiopoietin receptor, inhibits vascular ingrowth and delays or disrupts regeneration. In addition, osteoblast‐mediated overexpression of Ang1 reportedly increases ALP activity and bone mass in vivo, suggesting that Ang1 is also coupled to bone formation [Suzuki et al., 2007]. Furthermore, increased Ang1 expression during angiogenesis‐induced chondrocyte maturation involving endochondral ossification has been demonstrated [Fang et al., 2005; Jeong et al., 2010; Park et al., 2010]. "
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ABSTRACT: Endochondral ossification is essential for new bone formation and remodeling during the distraction stage. Endochondral ossification is attributed to chondrocyte maturation, which is induced by various factors, such as the cellular environment, gene transcription, and growth factor expression. Cartilage oligomeric matrix protein (COMP)-angiopoietin 1 (Ang1) is more soluble, stable, and potent than endogenous Ang1, and COMP-Ang1 treatment has osteogenic and angiogenic effects in an in vivo model of bone fracture healing. Although the osteogenic effects of COMP-Ang1 have been demonstrated, the precise mechanism by which COMP-Ang1 induces chondrocyte maturation and triggers endochondral ossification is not understood. Here, we investigated the possible mechanism by which COMP-Ang1 induces chondrocyte maturation. First, using a WST assay, we found that COMP-Ang1 is nontoxic in rat chondrocytes. Then, we isolated total RNA from COMP-Ang1-treated rat chondrocytes, and analyzed the decrease in chondrogenic gene expression and the increase in osteogenic gene expression using real-time RT-PCR. Gene and protein expression of heme oxygenase-1 (HO-1), which maintains chondrocytes in an immature stage, decreased in a dose-dependent manner upon COMP-Ang1 treatment. To clarify the relationship between HO-1 and COMP-Ang1 in chondrocyte maturation, we used cobalt protoporphyrin IX (CoPP IX), an HO-1 inducer, and tin protoporphyrin IX (SnPP-IX), an HO-1 inhibitor. Treatment with various combinations of CoPP IX, SnPP IX, and COMP-Ang1 confirmed that COMP-Ang1 accelerates chondrocyte maturation by reducing HO-1. In conclusion, our results suggest that COMP-Ang1 accelerates chondrocyte maturation by interacting with HO-1. J. Cell. Biochem. 114: 2513-2521, 2013. © 2013 Wiley Periodicals, Inc.
Available from: Gyun Min Lee
- "VEGF-A aggravates joint destruction by directly increasing the differentiation and activity of osteoclasts [8,29]. In contrast, Ang1 can suppress osteoclast activity indirectly via enhanced osteoblast maturation [30,31]. In this study, MMP-3 and IL-1 levels were lower in the Tie2-Fc-treated group than DAAP or VEGF-Trap, but RANKL levels were lower in the VEGF-Trap-treated group than in the DAAP or Tie2-Fc groups. "
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ABSTRACT: Angiogenesis plays a critical role in synovial inflammation and joint destruction in rheumatoid arthritis (RA). The vascular endothelial growth factor (VEGF)-A and angiopoietins are two important mediators of synovial angiogenesis. We have previously developed a novel chimeric decoy receptor, namely double anti-angiogenic protein (DAAP), which can bind both VEGF-A and angiopoietins, blocking their actions. This study was performed to evaluate the anti-arthritic effect of DAAP and combination effect with the TNF- inhibitor in collagen-induced arthritis (CIA).
Recombinant DAAP, VEGF-Trap, Tie2-Fc, and dimeric-Fc proteins were produced and purified from CHO cells in large-scale bioreactors. CIA was induced in DBA/1 mice with type II collagen. The preventive effect of DAAP was determined and compared with other decoy receptors such as VEGF-Trap or Tie2-Fc, which block VEGF-A or angiopoietins, respectively. The clinical, radiographic, pathologic, and immunohistochemical analyses were performed in CIA mice. The levels of matrix metalloprotease-3 (MMP-3) and interleukin-1 (IL-1) were quantified by ELISA, and receptor activator of nuclear factor-kappa B ligand (RANKL) mRNA levels were measured with polymerase chain reaction (PCR). Finally, we investigated the combination effects of DAAP with low dose of TNF-alpha decoy receptor (etanercept, 10 mg/kg).
DAAP had a much greater inhibitory effect on arthritis severity and bone destruction than VEGF-Trap or Tie2-Fc on clinical and radiographic evaluation. These inhibitory effects were accompanied by significantly diminishing pathologic abnormalities, CD31-positive vasculature, and synovial infiltration by F4/80-positive macrophages. The levels of MMP-3, IL-1, and RANKL were much lower in DAAP-injected group than those of control. Furthermore, DAAP showed therapeutic effect and a combination effect with etanercept when injected after arthritis onset in established CIA.
DAAP has not only potent prophylactic effects on both inflammation and bone destruction, but also therapeutic effects, alone and combination effects with a TNF- inhibitor in CIA mice. These results suggest that DAAP could be used as an effective new therapeutic agent for RA.
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