Knockdown of caveolin-1 by antisense oligonucleotides impairs angiogenesis in 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 blot analysis of caveolin-1 levels. The most active and specific was found to reduce caveolin-1 protein levels by 70% at 1 microM 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 microg) 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.
Griffoni
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C
Department of Experimental Biology, University of Bologna, Bologna, Italy.
Spisni
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E
Santi
S
S
Riccio
M
M
Guarnieri
T
T
Tomasi
V
V
D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D000818:Animals; D051242:Caveolin 1; D022461:Caveolins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D002642:Chick Embryo; D016228:Endothelial Growth Factors / Q000235:genetics; D004730:Endothelium, Vascular / Q000187:drug effects / Q000502:physiology; D005786:Gene Expression Regulation; D006801:Humans; D008222:Lymphokines / Q000235:genetics; D018919:Neovascularization, Physiologic / Q000187:drug effects* / Q000235:genetics; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D015398:Signal Transduction; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D020533:Angiogenesis Inhibitors; C495540:CAV1 protein, human; D051242:Caveolin 1; D022461:Caveolins; D016228:Endothelial Growth Factors; D008222:Lymphokines; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors