Inhibition of Endothelial Cell Metalloproteinase Gene Expression with Antisense Phosphorothioate Oligonucleotides
Extracellular matrix (ECM) degradation by matrix metalloproteinases (MMPs) plays an important role in angiogenesis and cancer metastasis. Degradation of ECM by human umbilical vein endothelial cells (HUVECs) is blocked by inhibitors of metalloproteinases, but the individual roles of MMPs involved in this process remains unclear. To study the individual roles of MMPs in HUVECs-mediated ECM degradation, we attempted to block expression of individual MMP genes with antisense phosphorothioate oligodeoxynucleotides (S-oligos) directed at interstitial collagenase and gelatinase A mRNAs. It has been demonstrated that phorbol 12-myristate 13 acetate (PMA) induces expression o~ interstitial collagenase and gelatinase A in HUVECs. In the present study, western immunoblot and gelatin substrate activity gel were performed to examine MMP expression. In the first experiment with cells grown on plastic, S-oligos did not affect the level of MMP measured by western blot or gelatin substrate activity gel. However, in the second experiment.with cells grown on 3H-collagen gels, the antisense collagenase S-oligo was found to suppress collagenase expression. These preliminary results indicate an alternative technique for characterizing individual MMPs by regulating gene expression with antisense S-oligos. This approach should distinguish the individual roles of MMPs in the degradation of ECM in vitro by endothelial cells.