COMP-Ang1, a chimeric form of Angiopoietin 1, enhances BMP2-induced osteoblast differentiation and bone formation.
BACKGROUND
Angiogenesis is closely associated with bone formation, especially endochondral ossification. Angiopoietin 1 (Ang1) is a specific growth factor functioning to generate a stable and matured vasculature through the Tie2 receptor/PI3K/AKT pathway. Recently cartilage oligomeric matrix protein (COMP)-Ang1, an Ang1 variant which is more potent than native Ang1 in phosphorylating Tie2 receptor and AKT, was developed. This study was designed to examine the effects of angiogenic COMP-Ang1 on BMP2-induced osteoblast differentiation and bone formation.


METHODS
Expression of endogenous Ang-1 and its binding receptor Tie 2 mRNA was examined in osteoblast-like cells and primary mouse calvarial cells by RT-PCR analysis, and was also monitored during osteoblast differentiation induced by BMP-2 and/or ascorbic acid and beta-glycerophosphate. Effects of COMP-Ang-1 on osteoblast differentiation and mineralization were evaluated by alkaline phosphatase (ALP) activity and osteocalcin (OC) production, and Alizarin red stain. For a molecular mechanism, Western blot and OG2 and 6xOSE promoter assays were done. For in vivo evaluation, adenoviral (Ad) vectors containing COMP-Ang-1 or BMP-2 gene were administered into thigh muscle of mice, and after 2 weeks bone formation was analyzed by micro-computed tomography and histology. Angiogenic event of COMP-Ang1 was confirmed by immunofluorescence analysis with anti-CD31 antibody.


RESULTS
Expression of Tie2 receptor was significantly increased in the course of osteoblast differentiation. Treatment or overexpression of COMP-Ang1 enhanced BMP2-induced ALP activity, OC production, and mineral deposition in a dose-dependent manner. In addition, COMP-Ang1 synergistically increased OG2 and 6xOSE promoter activities of BMP2, and sustained p38, Smad and AKT phosphorylation of BMP2. Notably, in vivo intramuscular injection of COMP-Ang1 dose-dependently enhanced BMP2-induced ectopic bone formation with increases in CD31 reactivity.


CONCLUSIONS
These results suggest that COMP-Ang1 synergistically enhanced osteoblast differentiation and bone formation through potentiating BMP2 signaling pathways and angiogenesis. Combination of BMP2 and COMP-Ang1 should be clinically useful for therapeutic application to fracture and destructive bone diseases.
Dental Science Research Institute, BK21 Project, School of Dentistry, Chonnam National University, Gwangju 500-757, Korea.
Byung-Chul
BC
Jeong
Hyun-Joo
HJ
Kim
In-Ho
IH
Bae
Kkot-Nim
KN
Lee
Kwang-Youl
KY
Lee
Won-Mann
WM
Oh
Sun-Hun
SH
Kim
In-Chol
IC
Kang
Shee-Eun
SE
Lee
Gou-Young
GY
Koh
Kyung-Keun
KK
Kim
Jeong-Tae
JT
Koh
D042683:Angiopoietin-1 / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D055396:Bone Morphogenetic Protein 2 / Q000494:pharmacology*; D002113:Calcification, Physiologic / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D010012:Osteogenesis / Q000187:drug effects* / Q000235:genetics; D010766:Phosphorylation / Q000187:drug effects; D042787:Receptor, TIE-2 / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects; D014158:Transcription, Genetic / Q000187:drug effects; D015533:Transcriptional Activation / Q000187:drug effects
D042683:Angiopoietin-1; D055396:Bone Morphogenetic Protein 2; C515576:COMP-Ang1 fusion protein; D011993:Recombinant Fusion Proteins; D042787:Receptor, TIE-2