Use of RNA Interference to Inhibit Integrin Subunit alphaV-Mediated Angiogenesis.

Inhibition of angiogenesis is a promising approach for the treatment of solid tumors, inflammatory diseases and different hematological malignancies such as multiple myeloma. One of the central molecules in capillary formation during angiogenesis is the integrin alphaVbeta3 (aVb3), therefore aVb3 is a potential target molecule to inhibit angiogenesis. The aim of this study was to inhibit alphaV-mediated angiogenesis in vitro using RNA interference (RNAi) technology as well as antisense oligodeoxyribonucleotides (asON). We used synthetic small interfering RNAs (siRNA) and asON directed against the alphaV chain of aVb3 to inhibit integrin expression. Five siRNAs were selected on the basis of a systematic alignment of computer-predicted secondary structures of target mRNA and on the basis of current recommendations for siRNA oligonucleotide design. In parallel, 3 asON were examined. They had the sequence of the antisense sequence of 3 of the siRNAs molecules, respectively. SiRNAs, asON and respective control sequences were transfected into human umbilical vein endothelials cells (HUVEC) using lipofection. Following stimulation by phorbol 12-myristate 13-acetate (PMA), two siRNAs showed a dose- and time dependent inhibition of PMA-induced aV-mRNA and -protein upregulation as assessed by real-time RT-PCR and flow cytometry. At a concentration of 25 nM a 100% (SD: 4.9%) inhibition of aV upregulation was observed, whereas transfection of the respective asON sequences resulted in a 63% (SD: 6.1%) inhibition of aV upregulation at 25nM. To evaluate the anti-angiogenic potential of siRNAs in comparison to asON a cell culture model of human angiogenesis based on the co-cultivation of endothelial cells and dermal fibroblasts was used. Transfection of the most efficient siRNA sequence at a concentration of 50nM resulted in an inhibition of total length of capillary-like tubules by 48.7% (SD: 3.6%) in comparison to 21.8% (SD: 9.8%) by the respective asON sequence treated cultures.

In conclusion, siRNAs can successfully be selected on the basis of computer-predicted secondary structures. In comparison with asON having the same sequence as the antisense strand of the respective siRNA the siRNA-mediated inhibition of aV expression showed a stronger inhibition of capillary tube formation in an angiogenesis in vitro assay. Therefore, siRNAs are useful tools for functional aV knock-down experiments and might be a therapeutic alternative for antagonists which bind directly to the integrins aVb3 or aVb5.