Knockdown of caveolin-1 by antisense oligonucleotides impairs angiogenesisin vitro and in vivo

Knock-out of the gene coding for caveolin-1, the main organizer of caveolae , has not yet been performed. We devised a strategy to knock-down caveolin- 1 gene expression using antisense oligodeoxynucleotides (ODNs). Seven ODNs, covering different regions of caveolin-1 mRNA, were screened by Western bl ot analysis of caveolin-1 levels. The most active and specific was found to reduce caveolin-1 protein levels by 70% at 1 mu M concentration and its ac tion, as demonstrated by a marked reduction (about 50%) in caveolin-1 mRNA levels, was due to a true antisense mechanism. In HUVEC treated with the ac tive ODN, caveolae were undetectable by confocal and electron microscopy, w hile their number was not affected when cells were treated with a scrambled ODN. Using the fibrin gel 3 D angiogenesis test we established that the ac tive (but not the scrambled) ODN strongly suppressed capillary-like tube fo rmation. Moreover, an antisense tailored against chicken caveolin-1 mRNA, w hen tested using the chorio-allantoic membrane technique, dramatically redu ced vessel formation at doses (10-20 mu g) under which control ODNs were in effective and devoid of toxicity. Thus, it is likely that caveolin-1 down r egulation, followed by caveolae disruption, impairs angiogenesis in vitro a nd in vivo. (C) 2000 Academic Press.