Role of cartilage-derived anti-angiogenic factor, chondromodulin-I, during endochondral bone formation.
OBJECTIVE
Cartilage is a typical avasclar tissue that exhibits powerful resistance to angiogenesis or vascular invasion. We previously identified a cartilage-specific 25 kDa glycosylated protein, chondromodulin-I (ChM-I), as anti-angiogenic factor. Taking advantage of ectopic bone formation and xenograft tumour model by human chondrosarcoma cell line OUMS-27, we examined how ChM-I is involved in switching of angiogenesis in cartilage.


METHODS
Gene expression pattern of ChM-I was examined in 4-week-old mice and mouse embryos by northern blot analysis and in situ hybridization. To evaluate the effect of ChM-I on ectopic bone formation, guanidine extracts of demineralized bone matrix were mixed with the ChM-I-bound heparin-Sepharose beads and were implanted onto the fasciae of back muscle of 6-week old nude mice. To analyse the effect of ChM-I on tumour angiogenesis, the level of ChM-I mRNA in cartilaginous tumours was assessed by competitive PCR, and compared with that of articular cartilage. Then, human chondrosarcoma OUMS-27 cells were inoculated into the back of nude mice to form a tumour about 45 mm3 in size. Recombinant ChM-I protein was administrated into OUMS-27 xenograft tumours for the initial 5 days to study its effect against tumour-angiogenesis.


RESULTS
ChM-I gene was specifically expressed in cartilage of 4-week-old mice. Eye and thymus were also identified as minor expression sites. However, during endochondral bone development, cartilage changes its character from anti-angiogenic into angiogenic prior to the replacement of calcified cartilage by bone. In embryos, ChM-I mRNA was expressed in proliferative and upper hypertrophic cartilage zones in the developing cartilaginous bone rudiments, but completely abolished in lower hypertrophic and calcified cartilage zones. Purified ChM-I protein apparently inhibited vascular invasion into cartilage induced by the implantation of demineralized bone matrix in nude mice, leading to the inhibition of replacement of cartilage. The level of ChM-I transcripts in the lower-grade chondrosarcomas was substantially reduced to several hundreds or less in the lower-grade chondrosarcomas, compared with that of articular cartilage or other benign cartilage tumours. The local administration of recombinant human ChM-I almost completely blocked tumour angiogenesis and growth in the human chondrosarcoma xenografts in mice.


CONCLUSIONS
ChM-I is involved in the anti-angiogenic property of cartilage and its absence creates a permissive microenvironment for vascular invasion into cartilage under physiological and pathological conditions.
Shukunami
C
C
Department of Molecular Interaction and Tissue Engineering, Institute for Frontier Medical Sciences, Kyoto University, Japan. shukunam@frontier.kyoto-u.ac.jp
Hiraki
Y
Y
D000818:Animals; D015152:Blotting, Northern / Q000379:methods; D016025:Bone Transplantation; D002356:Cartilage / Q000502:physiology*; D006224:Cricetinae; D015870:Gene Expression; D006133:Growth Substances / Q000502:physiology*; D017403:In Situ Hybridization / Q000379:methods; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D051379:Mice; D008819:Mice, Nude; D018919:Neovascularization, Physiologic / Q000502:physiology*; D010012:Osteogenesis / Q000502:physiology*; D016133:Polymerase Chain Reaction / Q000379:methods; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured / Q000637:transplantation
C489408:Cnmd protein, mouse; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins