Therapeutic targeting of telomerase ameliorates experimental choroidal neovascularization

Abstract

Choroidal neovascularization (CNV) is the principal driver of blindness in neovascular age-related macular degeneration (nvAMD). Increased activity of telomerase, has been associated with endothelial cell proliferation, survival, migration, and invasion in the context of tumor angiogenesis. Expanding on this knowledge, we investigated the role of telomerase in the development of CNV in mouse model. We observed increased gene expression and activity of telomerase in mouse CNV. Genetic deficiency of the telomerase components, telomerase reverse transcriptase (Tert) and telomerase RNA component (Terc) suppressed laser-induced CNV in mice. Similarly, a small molecule inhibitor of TERT (BIBR 1532), and antisense oligonucleotides (ASOs) targeting Tert and Terc reduced CNV growth. Bone marrow chimera studies suggested that telomerase activity in non-bone marrow-derived cells is crucial for the development of CNV. Comparison of BIBR 1532 with VEGF neutralizing therapeutic strategy in mouse revealed a comparable level of angiosuppressive activity. However, when BIBR and anti-VEGF antibodies were administered as a combination at sub-therapeutic doses, a statistically significant suppression of CNV was observed. These findings underscore the potential benefits of combining sub-therapeutic doses of BIBR and anti-VEGF antibodies for developing newer therapeutic strategies for NV-AMD. Telomerase inhibition with BIBR 1532 suppressed induction of multiple cytokines and growth factors critical for neovascularization. In conclusion, our study identifies telomerase as a promising therapeutic target for treating neovascular disease of the eye and thus provides a proof of principle for further exploration of telomerase inhibition as a novel treatment strategy for nvAMD.

Keywords: AMD; Choroidal neovascularization; Telomerase.

Figure 1.. Telomerase promotes laser induced CNV.

A) Tert mRNA levels in RPE/choroid tissue from control or laser-induced CNV mice 3 days post laser photocoagulation (n = 7 samples) are presented. B) Quantification of the telomerase activity in RPE/choroid tissue from control or laser-induced CNV mice 7 days post laser photocoagulation (n = 3 samples) is presented. C) Representative images of FITC-isolectin B4 stained CNV lesions in RPE/choroid/scleral flat mounts from wild-type (WT) mice and Tert knock-out mice (Tert^−/−) mice after day 7 of laser injury are presented. D) The confocal images of the CNV lesions from WT and Tert^−/− mice were quantified using Nikon analysis software. Relative volumes of the CNV lesions are presented (n = 23 CNV lesions for WT, and 21 CNV lesions for Tert^−/−). E) Representative images of the FITC-isolectin B4 stained CNV lesions in WT mice and Terc^−/− mice 7 days post-laser photocoagulation. F) The confocal images of the CNV lesions from WT and Terc^−/− mice were quantified using ImageJ software. Relative volumes of the CNV lesions are presented (n = 32 CNV lesions for WT, and 28 CNV lesions for Terc^−/−). Data are presented means ± SEM. P-values are obtained by Mann-Whitney in (A), (D) and (F) and unpaired t-test in (B). A p-value of <0.05 was considered statistically significant. (*P<0.05). Scale bar, 100μm.

Figure 2.. Tert and Terc specific antisense oligonucleotides (ASOs) suppress CNV growth.

A) Schematic representation of the ASO treatment in laser-induced CNV mouse model. B) Representative confocal images of FITC- isolectin B4 stained RPE/choroid/scleral flatmount showing CNV lesion (7 day lost laser injury) in mice administered with control (Ctr), Tert and Terc ASOs. Scale bar, 100μm. C) Quantification of CNV volume using Image J is presented (n = 40 CNV lesions for Ctr, 25 CNV lesions for Tert ASO, 27 CNV lesions for Terc ASO). Data are mean ± SEM. Statistical analysis was performed using one-way analysis of variance (ANOVA). A P-value of less than 0.05 indicates statistical significance (*P < 0.05, ns = not significant).

Figure 3.. Telomerase activity in non-bone marrow derived cells contributes to the CNV growth

A) Recipient WT and Tert^−/− mice were transplanted with bone marrow cells derived from either from WT and Tert^−/− mice. The laser-induced CNV was produced in bone marrow chimera mice 45 days post bone marrow transplantation. Representative confocal image of isolectin B4 stained CNV lesions are presented. Scale bar, 100μm. B) Quantification of CNV lesion volume using Nikon analysis software (n = 38 CNV lesions for WT>WT (R), 28 CNV lesions for Tert^−/−>WT (R), 37 CNV lesions for Tert^−/−>Tert^−/− (R), 26 CNV lesions for WT> Tert^−/− (R)) is presented. Where (R) represents the recipient mouse. Data are represented as mean ± SEM. P-values were obtained using Kruskal Wallis test with Dunn’s correction. (*P<0.05, ns=not significant).

Figure 4.. Effect of telomerase inhibitor BIBR 1532 on laser induced CNV in mice.

A) Schematic representation of the BIBR 1532 treatment timeline. B) Telomerase activity in RPE/choroid tissue as determined by TRAP assay at day 7 after laser injury with or without BIBR 1532 (1326pg/eye) is presented (n = 4 samples each group). C) Representative images of the isolecton B4 stained CNV lesions in RPE/choroid/scleral flat mounts (at 7 days post laser injury) after intravitreal vehicle or BIBR 1532 (1326pg/eye) administration. Scale bar, 100μm. (n = 24 CNV lesions for Control, 19 CNV lesions for BIBR 1532). D) Confocal images of the CNV lesions from mice intravitreally administered with vehicle or BIBR 1532 were quantified using ImageJ. Relative CNV lesion volumes are presented. E) Dose-dependent effect of intravitreal BIBR 1532 administration was assessed by quantifying the confocal images of the CNV lesions via ImageJ analysis. Relative CNV lesion volumes are presented (n = 39 lesions for vehicle control, 35 lesions for 166pg BIBR 1532, 37 lesions 331pg BIBR 1532, 43 lesions for 663pg BIBR 1532, and 33 lesions for 1326pg BIBR 1532). F) Representative confocal images of laser-induced CNV lesions are presented from Tert^−/− mice intravitreally administered with BIBR 1532 (1326pg/eye). The images were captured at day 7 post laser. Scale bar, 100μm. G) Quantification of laser-induced CNV lesions from Tert^−/− mice intravitreally administered with BIBR 1532 (1326pg/eye) or vehicle is presented. The images were analyzed using Nikon Elements analysis software. Relative CNV volumes are presented (n = 16 CNV lesions for vehicle, 19 CNV lesions for 1326pg BIBR). Data are mean ± SEM. Statistical analysis was performed using unpaired t-test for (B), (D), (G) and Kruskal-Wallis with Dunn’s correction for (E). A P-value of less than 0.05 indicates statistical significance (*P<0.05, ns = not significant).

Figure 5.. BIBR 1532 action is similar to Anti-VEGF therapy in inhibiting CNV growth.

A) Schematic representation of the BIBR 1532 and ant-VEGF treatment timeline. B-D) Quantification of the laser induced- CNV lesions from mice following intravitreal administration of indicated doses of BIBR 1532 and anti-VEGF antibodies alone or in combination is presented. CNV lesion images were analyzed by Nikon Elements software. Relative CNV volumes are presented. Data are represented as mean ± SEM. Statistical analysis was performed using one-way analysis of variance (ANOVA). A P-value of less than 0.05 indicates statistical significance (*P<0.05, ns = not significant).