C20D3-Vitamin A Prevents Retinal Pigment Epithelium Atrophic Changes in a Mouse Model

Abstract

Purpose: This study aimed to evaluate the contribution of vitamin A dimerization to retinal pigment epithelium (RPE) atrophic changes. Leading causes of irreversible blindness, including Stargardt disease and age-related macular degeneration (AMD), occur as a result of atrophic changes in RPE. The cause of the RPE atrophic changes is not apparent. During the vitamin A cycle, vitamin A dimerizes, leading to vitamin A cycle byproducts, such as vitamin A dimers, in the RPE.

Methods: To study the consequence of vitamin A dimerization to RPE atrophic changes, we used a rodent model with accelerated vitamin A dimerization, Abca4-/-/Rdh8-/- mice, and the vitamin A analog C20D3-vitamin A to selectively ameliorate the accelerated rate of vitamin A dimerization.

Results: We show that ameliorating the rate of vitamin A dimerization with C20D3-vitamin A mitigates pathological changes observed in the prodromal phase of the most prevalent retinal degenerative diseases, including fundus autofluorescence changes, dark adaptation delays, and signature RPE atrophic changes.

Conclusions: Data demonstrate that the dimerization of vitamin A during the vitamin A cycle is sufficient alone to cause the prerequisite RPE atrophic changes thought to be responsible for the leading causes of irreversible blindness and that correcting the dimerization rate with C20D3-vitamin A may be sufficient to prevent the RPE atrophic changes.

Translational relevance: Preventing the dimerization of vitamin A with the vitamin A analog C20D3-vitamin A may be sufficient to alter the clinical course of the most prevalent forms of blindness, including Stargardt disease and age-related macular degeneration (AMD).

Conflict of interest statement

Disclosure: D. Zhang, None; K. Robinson, None; I. Washington, IW is an inventor on patents disclosing methods to prevent retinal degeneration

Figures

Figure 1.

The Dimerization of Vitamin A During the Vitamin A Cycle. (A) Vitamin A is a descriptor for retinoids that exhibit the biological activity of retinol. The retina transforms vitamin A between several congeners, all-trans-retinol, all-trans-retinyl esters, all-trans-retinaldehyde, 11-cis-retinol, 11-cis-retinyl esters, 11-cis-retinaldehyde, and retinoic acid, as it shuttles these congeners throughout its compartments. These translocations, isomerizations, oxidations, and reductions are collectively referred to as the vitamin A cycle. Vitamin A enters or exits the retina through exchanges between the RPE with choroid capillaries and the inner retina with retinal blood vessels (double arrows). Cycles between rod cells and the RPE enable rod vision, while cycles between cone and Muller cells enable cone vision. The recycling of 11-cis-retinaldehyde from phagocytosed photoreceptors and the integration of 11-cis-retinaldehyde into newly synthesized photoreceptors is also part of the vitamin A cycle. (B) The vitamin A cycle is driven by (a) the regeneration of visual pigments after bleaching, (b) photoreceptor disc renewal or synthesis (c), photoreceptor disc shedding, (d) visual pigment bleaching with light, and (e) thermal isomerization of visual pigment. As depicted, the vitamin A cycle regulates calcium homeostasis via opsin signaling and gene transcription via retinoic acid production. Vitamin A cycle byproducts (VAB) form from the reaction of a cis or trans-retinaldehyde-Schiff base with another molecule(s) of cis or trans retinaldehyde. Any primary amine in the retina can participate in Schiff base formation and catalyze the dimerization of vitamin A. Retinal: Retinaldehyde.

Figure 2.

C20D3-Vitamin A Prevents the Dimerization of Vitamin A. Shown, a C20 carbon-hydrogen bound of vitamin A at natural isotopic abundance relative to a C20 carbon-hydrogen bound of C20D3-vitamin A. To dimerize, a C20 carbon-hydrogen bond on vitamin A must be broken. Enriching the C20 carbon-hydrogen bonds (H) with deuterium (D) makes the bonds harder to break and the dimerization of vitamin A more difficult.

Figure 3.

C20D3-Vitamin A Prevents the Prodromal Phase of Retinal Degenerations. (A) Representative quantitative fundus autofluorescence (AF) images of  3-month-old Abca4−/−/Rdh8−/− mice administered a diet containing vitamin A as either retinyl acetate or C20D3-retinyl acetate. (B) Average (with SEM) AF intensity of the retinas of mice described in panel A. Each spot corresponds to the AF intensity of a single eye. Eight eyes of eight different animals per cohort were imaged (n = 8). P-value is from a two-sided, unpaired T-test. (C) The qVAB (A2E, iso-A2E, and oxo-A2E) in mice described in panel A. Each point represents between 5 and 10 pooled eyes. (D) Average (SEM) ERG b-wave recoveries following light exposure in ∼12-month-old Abca4−/−/Rdh8−/− mice administered a diet containing retinyl acetate (n = 13 eyes, blue curve, recovered a mean and SD of 71% ± 0.7% after 30 minutes of dark-adapted maximum b-wave) or C20D3-retinyl acetate (n = 12 eyes, red curve, recovered 53 ± 0.5%, P = 0.01, two-sided F-test). (E and F) ERG dose-response curves (average with SEM) for the cohorts of dark-adapted Abca4−/−/Rdh8−/− mice described in panel A at seven months (E, retinyl acetate: n = 16 eyes; C20D3-retinyl acetate: n = 19 eyes) and 18 months of age (F, retinyl acetate: n = 12 eyes; C20D3-retinyl acetate: n = 12 eyes). (G) Percent change in retinal thickness (RPE and neuroretina) from baseline (3 months of age) in mice described in panel A, measured 1 mm from either side of the optic nerve head. Averages and SEM are shown. C20D3-retinyl acetate: n = 6 at three months, n = 14 at 12 months, and n = 4 at 18 months. Retinyl acetate: n = 5 at 3 months, n = 5 at 12 months, and n = 10 at 18 months. Each eye was from a different animal.

Figure 4.

C20D3-Vitamin A Prevents the Signature RPE Atrophic Changes. (A) Representative hematoxylin and eosin (H&E) cross-section of the C20D3-retinyl acetate cohort at 18 months of age. Histology was performed on n = 12 eyes of 12 mice between 13 and 18 months of age. No RPE pathology was observed in the C20D3-retinyl acetate cohort at any age. (B–E) Representative H&E cross-sections of 18-month-old Abca4−/−/Rdh8−/− animals administered vitamin A as retinyl acetate. Each panel represents a slice from a different eye. RPE migration into the neuroretinal space (arrows), RPE swelling (arrowheads), RPE clumping, or regions of missing or atrophic RPE (black arrows) were observed. Histology was performed on n = 16 eyes of 16 mice between 13 and 18 months of age. All eyes displayed areas of RPE pathology after 13 months of age. (F–I) Representative OTC images of human AMD (F, G) and Stargardt disease (H, I) show similar RPE atrophic changes. Each image is from a different eye.