INHIBITION OF ENDOGENOUS EXPRESSION OF CONNECTIVE-TISSUE GROWTH-FACTOR BY ITS ANTISENSE OLIGONUCLEOTIDE AND ANTISENSE RNA SUPPRESSES PROLIFERATION AND MIGRATION OF VASCULAR ENDOTHELIAL-CELLS

Previously, we cloned an mRNA predominantly expressed in hypertrophic chondrocytes by differential display-PCR from a human chondrosarcoma-d erived chondrocytic cell line (HCS-2/8) that is identical to that of c onnective tissue growth factor (CTGF), In the present study, we invest igated the roles of CTGF in the proliferation and migration of vascula r endothelial cells using its antisense oligonucleotide and antisense RNA, because angiogenesis into the hypertrophic zone of cartilage occu rs at the final step of endochondral ossification. Immunohistochemical and immunofluorescence techniques revealed that not only hypertrophic chondrocytes but also endothelial cells in the cost-chondral junction s of mouse ribs were stained with an anti-CTGF antibody in vivo. North ern blot analysis revealed that CTGF was strongly expressed in chondro cytic cells as well as bovine aorta endothelial (BAE) cells in culture , but not in other types of cells such as osteoblastic cells. Its expr ession in BAE cells was greater in the growing phase than in the confl uent phase. When one-half of a monolayer of a confluent culture of BAE cells had been peeled off, only the cells proliferating and extending into the vacant area were stained with the anti-CTGF antibody. The ad dition of an antisense oligonucleotide inhibited the proliferation and extension of the BAE cells into the vacant area. The antisense oligon ucleotide also inhibited the proliferation of BAE cells in the rapidly proliferating phase. In a Boyden chamber assay, pretreatment with the antisense oligonucleotide markedly inhibited the migration of BAE cel ls. Furthermore, the abilities to proliferate and migrate of BAE cells , which were stably transfected with expression vectors that generate the antisense RNA of CTGF cDNA, were markedly lower than those of the control. These findings suggest that endogenous CTGF expression is inv olved in the proliferation and migration of BAE cells.