Bovine chondromodulin‐I (ChM‐I) purified from fetal cartilage stimulated the matrix synthesis of chondrocytes, and inhibited the growth of vascular endothelial cells in vitro. The human counterpart of this bovine growth regulating factor has not been identified. We report here the cloning of human ChM‐I precursor cDNA and its functional expression in Chinese hamster ovary (CHO) cells. We first identified a genomic DNA fragment which encoded the N‐terminus of the ChM‐I precursor, and then isolated human ChM‐I cDNA from chondrosarcoma tissue by PCR. The deduced amino acid sequence revealed that mature human ChM‐I consists of 120 amino acids. In total, 16 amino acid residues were substituted in the human sequence, compared to the bovine counterpart. Almost of all the substitutions were found in the N‐terminal hydrophilic domain. In the C‐terminal hydrophobic domain (from Phe42 to Val120), the amino acid sequence was identical except for Tyr90, indicating a functional significance of the domain. Northern blotting and in situ hybridization indicated a specific expression of ChM‐I mRNA in cartilage. We also successfully determined the cartilage‐specific localization of ChM‐I protein, using a specific antibody against recombinant human ChM‐I. Multiple transfection of the precursor cDNA into CHO cells enabled us to isolate the mature form of human ChM‐I from the culture supernatant. Purified recombinant human ChM‐I stimulated proteoglycan synthesis in cultured chondrocytes. In contrast, it inhibited the tube morphogenesis of cultured vascular endothelial cells in vitro and angiogenesis in chick chorioallantoic membrane in vivo.

Chondromodulin-I (ChM-I) was previously identified as an angiogenesis inhibitor in cartilage. Here, we demonstrated that the level of ChM-I transcripts was substantially reduced to 100 or even less in the lower-grade chondrosarcomas, in articular cartilage or other benign cartilage tumors. We implanted human chondrosarcoma OUMS-27 cells into nude mice that reproducibly produced tumors with cartilaginous matrix. Tumorinduced angiogenesis was evident when the tumors were excised 30 days after implantation. However, the local administration of recombinant human ChM-I almost completely blocked vascular invasion and tumor growth in vivo. Moreover, ChM-I also inhibited the growth of HT-29 colon adenocarcinoma in vivo, implying its therapeutic potential for solid tumors.z 1999 Federation of European Biochemical Societies.

Chondromodulin-I (ChM-I) is a cartilage-derived angiogenesis inhibitor that has been identified as inhibitory to the growth activity of vascular endothelial cells. In our present study, we demonstrate the anti-angiogenic activity of recombinant human ChM-I (rhChM-I) in mouse corneal angiogenesis and examine its action. We focus on the VEGF-A-induced migration of vascular endothelial cells, a critical regulatory step in angiogenesis. In a modified Boyden chamber assay, nanomolar concentrations of rhChM-I inhibited the chemotactic migration of human umbilical vein endothelial cells (HUVECs) induced by VEGF-A as well as by FGF-2 and IGF-I. The ChM-I action was found to be endothelial cell-specific and independent of cell adhesions. Time-lapse analysis further revealed that rhChM-I markedly reduces VEGF-A-stimulated motility of HUVECs and causes frequent alterations of the moving front due to the appearance of multiple transient protrusions. This action involved the inhibition of cell spreading and the disrupted reorganization of the actin cytoskeleton upon VEGF-A stimulation. Consistent with these observations, rhChM-I was found to significantly reduce the activity of Rac1/Cdc42 during cell spreading, and the VEGF-A-induced Rac1 activity but not its basal activity in quiescent cells. Taken together, our present data suggest that ChM-I impairs the VEGF-A-stimulated motility of endothelial cells by destabilizing lamellipodial extensions.

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BackgroundChondromodulin-I (ChM-I) is an anti-angiogenic glycoprotein that is specifically localized at the extracellular matrix of the avascular mesenchyme including cartilage and cardiac valves. In this study, we characterized the expression pattern of ChM-I during early pregnancy in mice in vivo and its effect on invasion of trophoblastic cells into Matrigel in vitro.ResultsNorthern blot analysis clearly indicated that ChM-I transcripts were expressed in the pregnant mouse uterus at 6.5-9.5 days post coitum. In situ hybridization and immunohistochemistry revealed that ChM-I was localized to the mature decidua surrounding the matrix metalloproteinase-9 (MMP-9)-expressing trophoblasts. Consistent with this observation, the expression of ChM-I mRNA was induced in decidualizing endometrial stromal cells in vitro, in response to estradiol and progesterone. Recombinant human ChM-I (rhChM-I) markedly inhibited the invasion through Matrigel as well as the chemotactic migration of rat Rcho-1 trophoblast cells in a manner independent of MMP activation.ConclusionsThis study demonstrates the inhibitory action of ChM-I on trophoblast migration and invasion, implying the potential role of the ChM-I expression in decidual cells for the regulated tissue remodeling and angiogenesis at feto-maternal interface.