Suppression of Laser-Induced Choroidal Neovascularization by Nontargeted siRNA

Abstract

PURPOSE. To investigate the effect of nontargeted siRNAs on vascular leakage and vascular endothelial growth factor (VEGF)-A expression in the development of choroidal neovascularization (CNV). METHODS. Nontargeted siRNAs were 21-nt (nucleotides) siRNA-Luc (Luciferase) or 16-nt siRNA-Luc. Targeted 21-nt siRNA-Vegfa or phosphate-buffered saline (PBS) was used for comparison. Laser photocoagulation was used to induce CNV in wild-type C57BL/6J mice; 7 days later, vascular leakage was determined by fluorescein angiography, and CNV volumes were measured by confocal microscopy. Expression of VEGF-A in the retinal pigment epithelium (RPE)/choroid was quantified by ELISA 3 days after photocoagulation. RESULTS. Pathologically significant leakage developed in most of the 16nt-siRNA-Luc- or PBS-injected mice but in significantly fewer 21nt-siRNA-Luc- and 21nt-siRNA-Vegfa-injected mice (P = 0.0004, P = 0.0001, respectively). CNV volume in 21-nt siRNA-Luc- and 21nt-siRNA-Vegfa-injected eyes was significantly lower than in PBS-injected eyes (P = 0.0124, P = 0.0040, respectively). CNV volume was not suppressed by 16-nt siRNA-Luc injection (P = 0.7700). The mean VEGF protein level decreased significantly in the 21nt-siRNA-Luc- and 21nt-siRNA-Vegfa-injected eyes compared with PBS-injected eyes 3 days after laser photocoagulation (P = 0.0011, P = 0.0063, respectively). The 16nt-siRNA-Luc-injected eyes did not show VEGF-A suppression 3 days after laser photocoagulation (P = 0.3177). Between 21-nt siRNA-Luc- and 21nt-siRNA-Vegfa-injected eyes, there were no significant differences in CNV volume, the VEGF-A level, or pathologic leakage detected by fluorescein. CONCLUSIONS. These data suggest that nontarget 21nt-siRNA can suppress laser-induced choroidal neovascularization anatomically and functionally through VEGF suppression.

Small‐interfering RNAs (siRNAs) as a promising tool for ocular therapy

“…Further studies also confirmed that 21 nt siRNAs, regardless of their targeting sequences, are able to inhibit experimental CNV in mice (Ashikari et al, 2010;Gu et al, 2010). Moreover, a recent study has determined that 21 nt siRNAs can induce caspase-3-mediated apoptotic death of retinal pigmented epithelial cells in mice by activating surface TLR3 and subsequent nuclear translocation of IFN regulatory factor-3 (Kleinman et al, 2012).…”

“…This results in receptor dimerization and NF-κB activation of genes encoding IFN-γ and IL-12, which, it seems, are the effectors of the antiangiogenesis. In this scheme of events, therefore, inhibition of angiogenesis does not seem to occur through the specific action of siRNAs in suppressing the messenger RNA of VEGF or its receptor.Further studies also confirmed that 21 nt siRNAs, regardless of their targeting sequences, are able to inhibit experimental CNV in mice (Ashikari et al, 2010;Gu et al, 2010). Moreover, a recent study has determined that 21 nt siRNAs can induce caspase-3-mediated apoptotic death of retinal pigmented epithelial cells in mice by activating surface TLR3 and subsequent nuclear translocation of IFN regulatory factor-3 (Kleinman et al, 2012).…”

Efficient Delivery of siRNA by Atelocollagen in a Murine Laser-Induced Choroidal Neovascularization Model

“…The siRNAs, PBS, atelocollagen and chloroquine (30 ng) were injected into the vitreous humor of wild-type mice in a total volume of 2 µl with a 33-gauge double caliber needle (Ito Corporation, Tokyo, Japan) immediately after laser injury, as described previously [18,23]. …”

“…The area of CNV-related fluorescence was measured by Image J software. The summation of whole fluorescent areas in each horizontal section was used as an index for the volume of CNV, as described previously [18,23]. The average of the volume obtained from all 4-6 laser spots per eye were generated (n refers to number of eyes).…”

“…Previously, we reported that 21-nt nontargeted siRNAs suppressed not only CNV volume (anatomically), but also fluorescein leakage (functionally). In addition, they suppressed VEGF-A expression, which plays a key role in the development of CNV [18]. …”