In vivo silencing of aquaporin-1 by RNA interference inhibits angiogenesis in the chick embryo chorioallantoic membrane assay

Aquaporin-1(AQP1) is a water channel protein mainly expressed in endothelial and epithelial cells of many tissues, including the vasculature where it serves to increase cell membrane water permeability. Previous studies in active multiple myeloma patients and in AQP1 KO mice indicated an involvement of AQP1 in physiological and tumor angiogenesis. To understand the physiological role of AQP1 in angiogenesis, we used a 21-nucleotide small interfering RNA duplexes (siRNA) to knockdown AQP1 in the chick embryo chorioallantoic membrane (CAM), a commonly used in vivo assay to study both angiogenic and angiostatic molecules. Chicken AQP1 sequence was identified and utilized to synthesize a siRNA directed to the AQP1 sequence. We then tested the efficiency of the siRNA in vitro, using an AQP1 transfected cell line. The level of AQP1 protein reduction obtained using siRNA was 98 % and 92 % after 1 and 2 day transfection respectively. RNA interference experiments were then performed in vivo by using the CAM assay. Results showed that after 4 days of treatment, AQP1 siRNA was able to strongly inhibit angiogenesis. This is the first study showing the in vivo use of RNA interference technique in the CAM assay. Our results strongly support the hypothesis that AQP1 could have a key role in physiological and pathological angiogenesis.