Use of RNA interference to inhibit integrin subunit αV-mediated angiogenesis

Thorsten Graef, Ulrich Steidl, Wolfgang Nedbal, Ulrich Rohr, Roland Fenk, Rainer Haas, Ralf Kronenwett

Research output: Contribution to journalArticle

10 Scopus citations

Abstract

One of the central molecules in capillary formation during angiogenesis is the integrin αVβ3. The aim of this study was to inhibit alphaV-mediated angiogenesis in vitro using RNAs (siRNA) as well as antisense oligodeoxyribonucleotides (asON). Five siRNAs, against the alphaV chain of αVβ3, and three asON, which had the respective sequence of the antisense sequence of three of the siRNAs molecules, were examined. Two of the siRNAs and their respective asON were designed on the basis of computer-predicted secondary structure analysis of αV mRNA. The different molecules were transfected into human umbilical vein endothelials cells (HUVEC) using lipofection. Following stimulation by PMA, two siRNAs showed a dose-dependent inhibition of PMA-induced αV mRNA and protein upregulation, as assessed by real-time RT-PCR and flow cytometry. At a concentration of 25 nM a complete inhibition of protein upregulation was found using siRNAs while transfection of the respective asON sequences reduced the protein upregulation only by 44%. To evaluate the anti-angiogenic potential a cell culture model of human angiogenesis based on the co-cultivation of endothelial cells and dermal fibroblasts was used. Transfection of the siRNA sequence (50 nM) resulted in an inhibition of the total length of capillary-like tubules by 40.6% in comparison to 21.1% using the respective asON sequence. In conclusion, siRNA-based downregulation of αV expression showed a stronger inhibition of capillary tube formation in an angiogenesis in vitro assay, than asON having the same sequence as the antisense strand of the siRNAs. Therefore, siRNAs are useful tools for functional αV knock-down experiments and might be a therapeutic alternative for antagonists which bind directly to the integrins αVβ3 or αVβ5.

Original languageEnglish (US)
Pages (from-to)361-372
Number of pages12
JournalAngiogenesis
Volume8
Issue number4
DOIs
StatePublished - Feb 1 2006
Externally publishedYes

Keywords

  • Alpha V integrin
  • Angiogenesis
  • Antagonist
  • Antisense oligonucleotide
  • Local mRNA structures
  • RNAi
  • siRNA

ASJC Scopus subject areas

  • Physiology
  • Clinical Biochemistry
  • Cancer Research

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  • Cite this

    Graef, T., Steidl, U., Nedbal, W., Rohr, U., Fenk, R., Haas, R., & Kronenwett, R. (2006). Use of RNA interference to inhibit integrin subunit αV-mediated angiogenesis. Angiogenesis, 8(4), 361-372. https://doi.org/10.1007/s10456-005-9026-5