Shukunami C, Hiraki Y
Department of Molecular Interaction and Tissue Engineering, Institute for Frontier Medical Sciences, Kyoto University, Japan.
Osteoarthritis Cartilage. 2001;9 Suppl A:S91-101. doi: 10.1053/joca.2001.0450.
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.
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.
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.
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.
软骨是一种典型的无血管组织，对血管生成或血管侵袭具有强大的抵抗力。我们之前鉴定出一种软骨特异性的25 kDa糖基化蛋白，软骨调节素-I（ChM-I），作为抗血管生成因子。利用人软骨肉瘤细胞系OUMS-27构建的异位骨形成和异种移植肿瘤模型，我们研究了ChM-I如何参与软骨中血管生成的转变。
通过Northern印迹分析和原位杂交检测4周龄小鼠和小鼠胚胎中ChM-I的基因表达模式。为了评估ChM-I对异位骨形成的影响，将脱矿骨基质的胍提取物与结合ChM-I的肝素-琼脂糖珠混合，并植入6周龄裸鼠背部肌肉的筋膜上。为了分析ChM-I对肿瘤血管生成的影响，通过竞争性PCR评估软骨肿瘤中ChM-I mRNA的水平，并与关节软骨进行比较。然后，将人软骨肉瘤OUMS-27细胞接种到裸鼠背部以形成大小约为45 mm3的肿瘤。在最初的5天内将重组ChM-I蛋白施用于OUMS-27异种移植肿瘤，以研究其对肿瘤血管生成的作用。
ChM-I基因在4周龄小鼠的软骨中特异性表达。眼睛和胸腺也被确定为次要表达部位。然而，在软骨内骨发育过程中，软骨在被骨替代钙化软骨之前，其性质从抗血管生成转变为血管生成。在胚胎中，ChM-I mRNA在发育中的软骨骨原基的增殖和上部肥大软骨区表达，但在下部肥大和钙化软骨区完全消失。纯化的ChM-I蛋白明显抑制脱矿骨基质植入裸鼠后诱导的血管侵入软骨，导致软骨替代受到抑制。与关节软骨或其他良性软骨肿瘤相比，低级别软骨肉瘤中ChM-I转录本的水平大幅降低至数百或更低。在小鼠的人软骨肉瘤异种移植中，局部施用重组人ChM-I几乎完全阻断了肿瘤血管生成和生长。
ChM-I参与软骨的抗血管生成特性，其缺失在生理和病理条件下为血管侵入软骨创造了有利的微环境。
