A Novel Single-Strand RNAi Therapeutic Agent Targeting the (Pro)renin Receptor Suppresses Ocular Inflammation

Figures

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  Figure 1. Elevated Protein Levels of Prorenin and s(P)RR in Correlation with CCL2/MCP-1 in Human Eyes with Uveitis (A–D) Correlation between vitreous levels of s(P)RR and prorenin (A), s(P)RR and CCL2/MCP-1 (B), prorenin and CCL2/MCP-1 (C), and activated prorenin and CCL2/MCP-1 (D).

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  Figure 2. Structure and In Vitro Characterization of (P)RR-PshRNA (A) Structure of canonical double-strand siRNA and novel single-strand RNAi (PshRNA) agents. Nucleotides in blue indicate the sense strand of the target [(P)RR/ATP6AP2], nucleotides in red are the antisense strand, and nucleotides in yellow are the linker region. P indicates a proline derivative. (B and C) Each RNAi agent was incubated in the presence of micrococcal nuclease and then separated on gel electrophoresis for incubation with 0.5 U enzyme for several durations (B) or with various units of enzyme for 30 min (C). (D and E) (P)RR/ATP6AP2 mRNA expression levels in human RPE cells (D) and mouse endothelial cells (E) exposed to (P)RR-siRNA or (P)RR-PshRNA compared with control-siRNA or control-PshRNA, respectively (n = 8 per group). ***p < 0.0001. (F and G) Immunoblot analysis for (P)RR in human RPE cells (F) and mouse endothelial cells (G) transfected with control- or (P)RR-PshRNA.

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  Figure 3. In Vivo Structural and Functional Safety of (P)RR-PshRNA (A–D) Normal H&E staining of mouse retinas obtained at 24 and 48 hr after (P)RR-PshRNA or PBS treatment. Scale bar, 20 μm. (E–L) Scotopic ERG recordings at 24 and 48 hr after (P)RR-PshRNA or PBS treatment. There were no differences in the amplitudes of the a-wave and b-wave between eyes treated with PBS and (P)RR-PshRNA (n = 5–8 per group). GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; NS, not significant; RPE, retinal pigment epithelial.

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  Figure 4. Suppression of Cellular Responses by (P)RR-PshRNA in Acute Inflammation (A–D) Flatmounted retinas from EIU mice treated with control-PshRNA (A and B) or (P)RR-PshRNA (C and D). Arrows indicate firmly adhering leukocytes to the inflamed retinal vasculature. Scale bar, 100 μm. (E) Quantification of the number of retinal adherent leukocytes. **p < 0.01 (n = 20–27 per group). (F and G) Representative micrographs of leukocyte infiltration into the vitreous fluid of EIU mice treated with control-PshRNA (F) and (P)RR-PshRNA (G). Arrows indicate infiltrating leukocytes. Scale bar, 30 μm. (H) Quantification of the number of leukocytes in the vitreous fluid. **p < 0.01 (n = 10–14 per group).

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  Figure 5. Suppression of Molecular Responses by (P)RR-PshRNA in Acute Inflammation (A–E) Gene expression levels of inflammatory mediators Il-6 (A), Ccl2/Mcp-1 (B), Icam-1 (C), and Tnf-α (D) and (P)RR/Atp6ap2 (E) in retinas from untreated normal mice (control) and EIU mice treated with PBS, control-PshRNA, or (P)RR-PshRNA. *p < 0.05; **p < 0.01 (n = 10 per group).

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  Figure 6. Suppression of Molecular Responses by (P)RR-PshRNA in Chronic Inflammation (A–D) Flatmounted retinas from diabetic mice treated with control-PshRNA (A and B) or (P)RR-PshRNA (C and D). Scale bar, 100 μm. (E) The number of retinal adherent leukocytes. **p < 0.01 (n = 13–16 per group). (F–J) Gene expression levels of Il-6 (F), Ccl2/Mcp-1 (G), Icam-1 (H), Tnf-α (I), and (P)RR/Atp6ap2 (J) in retinas from untreated normal mice (control) and STZ-induced diabetes treated with PBS, control-PshRNA, or (P)RR-PshRNA. *p < 0.05; **p < 0.01 (n = 10–12 per group).