Suppression of Choroidal Neovascularization and Fibrosis by a Novel RNAi Therapeutic Agent against (Pro)renin Receptor

Figures

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  Figure 1. Suppression of Laser-Induced CNV Formation by (P)RR-PshRNA (A–E) Representative micrographs of CNV lesions (isolectin B4, green) in the RPE-choroid flat mounts at post-laser day 7 from mice treated with (A) PBS, (B) 100 pmol control-PshRNA, or (C) 30, (D) 100, and (E) 300 pmol (P)RR-PshRNA, respectively. Scale bar, 50 μm. (F) Quantification analysis of the size of CNV [PBS = 8,966 ± 2,040 μm2, 100 pmol control-PshRNA = 8,660 ± 1,819 μm2, 30 pmol (P)RR-PshRNA = 6,234 ± 1,424 μm2, 100 pmol (P)RR-PshRNA = 5,526 ± 898 μm2, 300 pmol (P)RR-PshRNA = 4,245 ± 1,185 μm2]. **p < 0.01 (n = 5).

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  Figure 2. Inhibition of CNV-Related Inflammatory Molecule Expression, Macrophage Infiltration, and ERK1/2 Activation by (P)RR-PshRNA (A–F) Gene expression levels of inflammatory molecules (P)RR/Atp6ap2 (A), Ccl2 (B), Icam1 (C), Il6 (D), Tnfa (E), and Emr1 (F) in the RPE-choroid complex of untreated normal mice (control) and CNV mice treated with 100 pmol control-PshRNA or (P)RR-PshRNA. *p < 0.05, **p < 0.01 (n = 6–8). (G–L) Representative micrographs of F4/80-positive macrophages (red) (G and J) in CNV lesions (isolectin B4, green) (H and K) from CNV mice treated with 100 pmol control-PshRNA or (P)RR-PshRNA. (I and L) Merged images. Scale bar, 50 μm. (M) Quantification of CNV area-adjusted number of F4/80-positive macrophages [control-PshRNA = 10.83 ± 2.48/1,000 μm2, (P)RR-PshRNA = 3.17 ± 0.75/1,000 μm2]. **p < 0.01 (n = 4). (N) Immunoblotting analysis for phosphorylated and total ERK1/2 in the RPE-choroid complex of control and CNV mice treated with 100 pmol control-PshRNA or (P)RR-PshRNA at day 3 after laser injury. (O) Densitometry values of phosphorylated ERK1/2 normalized to total ERK1/2. **p < 0.01 (n = 4).

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  Figure 3. Suppression of Subretinal Fibrosis and Fibrotic Molecules via the ERK1/2-Mediated TGF-β/SMAD2/SNAIL Pathway by (P)RR-PshRNA (A and B) Representative micrographs of subretinal fibrosis lesions (type I collagen, red) in the RPE-choroid flat mounts at post-laser day 7 from mice treated with 100 pmol control-PshRNA (A) or (P)RR-PshRNA (B). Scale bar, 50 μm. (C) Quantification analysis of the size of subretinal fibrosis [control-PshRNA = 11,149 ± 3,204 μm2, (P)RR-PshRNA = 8,264 ± 1,376 μm2]. **p < 0.01 (n = 6–8). (D) Gene expression level of profibrotic cytokine Tgfb1 in the RPE-choroid complex of untreated normal mice (control) and CNV mice treated with 100 pmol control-PshRNA or (P)RR-PshRNA. *p < 0.05, **p < 0.01 (n = 6). (E) Immunoblotting for phosphorylated and total ERK1/2 and (P)RR in human RPE cells stimulated with prorenin at 20 nM for 24 h following pretreatment with DMSO, and ERK1/2 inhibitor (U0126) at 10 μM for 30 min or (P)RR-PshRNA at 1 nM for 24 h. (F) Relative gene expression levels of TGFB1 in prorenin-stimulated human RPE cells pretreated with DMSO, U0126, or (P)RR-PshRNA. *p < 0.05, **p < 0.01 (n = 6). (G–J) Gene expression level of EMT markers Acta2 (G), Col1a1…

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  Figure 4. Blockade of Inflammatory Responses by (P)RR-PshRNA in TNF-α-Stimulated Endothelial Cells and RPE Cells (A–F) Gene expression levels of inflammatory mediators Atp6ap2/ATP6AP2 (A), Ccl2/CCL2 (B), Icam1/ICAM1 (C), Il6/IL6 (D), Tnfa/TNFA (E), and profibrotic cytokine Tgfb1/TGFB1 (F) in 1 nM control-PshRNA or (P)RR-PshRNA transfected murine microvascular endothelial cells stimulated with 2 ng/mL TNF-α for 3 h and 1 nM control-PshRNA or (P)RR-PshRNA transfected human RPE cells stimulated with 10 ng/mL TNF-α for 12 h. *p < 0.05, **p < 0.01 (n = 6). n.s., not significant.

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  Figure 5. Attenuation of CNV and Subretinal Fibrosis Formation in RPE-Specific (P)RR/Atp6ap2 CKO Mice (A–D) Representative micrographs of CNV (isolectin B4, green) and subretinal fibrosis (type I collagen, red) in the RPE-choroid flat mount at day 7 after laser injury in control (A and B) and (P)RR/Atp6ap2-CKO (C and D) mice. Their littermates (e.g., Atp6ap2lox/Y/BEST1-Cre−/−) served as control mice. Scale bar, 50 μm. (E and F) Quantification analysis of the (E) size of CNV [control = 9,277 ± 2,590 μm2, (P)RR/Atp6ap2-CKO = 5,963 ± 1,511 μm2] and (F) subretinal fibrosis [control = 10,556 ± 3,172 μm2, (P)RR/Atp6ap2-CKO = 7,452 ± 1,600 μm2]. **p < 0.01 (n = 6).

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  Figure 6. Comparison of (P)RR-PshRNA and Aflibercept for Suppression of CNV and Subretinal Fibrosis Formation (A–H) Representative micrographs of CNV (isolectin B4, green) and subretinal fibrosis (type I collagen, red) in the RPE-choroid flat mount at day 7 after laser injury in mice treated with intravitreal injection of PBS (A and B), aflibercept (2.5 μg) (C and D), (P)RR-PshRNA (100 pmol) (E and F), and mixture of aflibercept and (P)RR-PshRNA (2.5 μg + 100 pmol) (G and H), respectively. Scale bar, 50 μm. (I and J) Quantification analysis of the (I) sizes of CNV [PBS = 8,253 ± 3,124 μm2, aflibercept = 5,732 ± 2,795 μm2, (P)RR-PshRNA = 5,224 ± 2,691 μm2, aflibercept + (P)RR-PshRNA = 4,439 ± 1,535 μm2] and (J) fibrosis [PBS = 11,746 ± 3,678 μm2, aflibercept = 9,112 ± 3,322 μm2, (P)RR-PshRNA = 7,610 ± 3,407 μm2, aflibercept + (P)RR-PshRNA = 6,754 ± 2,895 μm2]. *p < 0.05, **p < 0.01 (n = 5).

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  Figure 7. Co-localization of (P)RR with Phosphorylated ERK1/2 in AMD Patient Specimens (A–L) Double labeling of (P)RR (green, A), CD34 (red, B), and DAPI (blue) (A–C); (P)RR (green, D), phosphorylated ERK1/2 (red, E), and DAPI (blue) (D–F); (P)RR (green, G), RPE65 (red, H), and DAPI (blue) (G–I); and (P)RR (green, J), phosphorylated ERK1/2 (red, K), and DAPI (blue) (J–L) in the CNV tissue specimens. (C, F, I, and L) Merged images. Scale bar, 20 μm.

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  Figure 8. A Schema Showing the Involvement of (P)RR with the Pathogenesis of CNV and Subretinal Fibrosis