Knockdown of Caveolin-1 by Antisense Oligonucleotides Impairs Angiogenesis in Vitro and in Vivo

• Published in: Biochemical and biophysical research communications 2000-09, Vol.276 (2), p.756-761
• Main Authors: Griffoni, Cristiana, Spisni, Enzo, Santi, Spartaco, Riccio, Massimo, Guarnieri, Tiziana, Tomasi, Vittorio
• Format: Article
• Language: English
• Subjects: angiogenesis; Angiogenesis Inhibitors - pharmacology; Animals; antisense oligonucleotides; caveolin; Caveolin 1; Caveolins - antagonists & inhibitors; Caveolins - genetics; Caveolins - physiology; Cells, Cultured; Chick Embryo; Endothelial Growth Factors - genetics; Endothelium, Vascular - drug effects; Endothelium, Vascular - physiology; Gene Expression Regulation; Humans; Lymphokines - genetics; Neovascularization, Physiologic - drug effects; Neovascularization, Physiologic - genetics; Oligonucleotides, Antisense - pharmacology; RNA, Messenger - drug effects; RNA, Messenger - metabolism; Signal Transduction; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1006/bbrc.2000.3484

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 blot analysis of caveolin-1 levels. The most active and specific was found to reduce caveolin-1 protein levels by 70% at 1 μM concentration and its action, 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 active ODN, caveolae were undetectable by confocal and electron microscopy, while 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 active (but not the scrambled) ODN strongly suppressed capillary-like tube formation. Moreover, an antisense tailored against chicken caveolin-1 mRNA, when tested using the chorio-allantoic membrane technique, dramatically reduced vessel formation at doses (10–20 μg) under which control ODNs were ineffective and devoid of toxicity. Thus, it is likely that caveolin-1 down regulation, followed by caveolae disruption, impairs angiogenesis in vitro and in vivo.