Abnormal vascularization in mouse retina with dysregulated retinal cholesterol homeostasis

Abstract

Several lines of evidence suggest a link between age-related macular degeneration and retinal cholesterol maintenance. Cytochrome P450 27A1 (CYP27A1) is a ubiquitously expressed mitochondrial sterol 27-hydroxylase that plays an important role in the metabolism of cholesterol and cholesterol-related compounds. We conducted a comprehensive ophthalmic evaluation of mice lacking CYP27A1. We found that the loss of CYP27A1 led to dysregulation of retinal cholesterol homeostasis, including unexpected upregulation of retinal cholesterol biosynthesis. Cyp27a1-/- mice developed retinal lesions characterized by cholesterol deposition beneath the retinal pigment epithelium. Further, Cyp27a1-null mice showed pathological neovascularization, which likely arose from both the retina and the choroid, that led to the formation of retinal-choroidal anastomosis. Blood flow alterations and blood vessel leakage were noted in the areas of pathology. The Cyp27a1-/- retina was hypoxic and had activated Müller cells. We suggest a mechanism whereby abolished sterol 27-hydroxylase activity leads to vascular changes and identify Cyp27a1-/- mice as a model for one of the variants of type 3 retinal neovascularization occurring in some patients with age-related macular degeneration.

Figure 1. Age-dependent progression of retinal pathologies in Cyp27a1^–/– mice.

Representative OCT fundus depth images (50° field of view) at the OPL in Cyp27a1^–/– (A, G, and K) and age- and sex-matched Cyp27a1^+/+ (F, J, and N) animals. Areas of pathology are circled, and those that were further examined by SD-OCT cross sections (B, H, and L) are marked with arrows; arrows of the same color indicate the same animal. Representative cross sections of the corresponding areas in Cyp27a1^+/+ animals (E, I, and M) are also shown. Labeling of retinal layers is based on hyper- and hyporeflective OCT bands. Scale bars: 300 μm (A, F, G, J, K, and N); 60 μm (B–E, H, I, L, and M).

Figure 2. Distinct structures of small and large lesions in Cyp27a1^–/– mice.

(A) Representative SD-OCT cross section and (B–E) serial sections through a small hyperreflective spot. (F) SD-OCT cross section and (G–J) serial sections through a large hyperreflective spot. Dark blue arrows (B–D) indicate dilated structure in the INL; pink arrow (B) points to twisting of the OPL; yellow arrows (E and J) show red blood cells in blood vessels; white and green arrows (F, H, and J) mark the blood vessels growing into the ONL; black arrow (G) indicates fibrosis in the OPL; red arrow (G) points to a representative cystic space and edema of the INL; light blue arrow (G) indicates fibrovascular material above BrM; orange arrows (H and J) indicate RPE debris in the ONL; purple arrows (H and J) indicate merged blood vessel in the photoreceptor layer. Scale bars: 30 μm (A–D, F–I); 10 μm (E and J). Original magnification, ×400.

Figure 3. Focal choroidal NV in Cyp27a1^–/– mice.

Panels are representative of stainings carried out on adjacent sections cut through pathologies in Cyp27a1^–/– mice (A–D, and H) or through a corresponding region in Cyp27a1^+/+ mice (E–G). (A, D, and E) H&E staining. (C, G, and H) Staining with isolectin B4, a marker for blood vessels, conjugated to DyLight 594 fluorophore (in red); nuclei were stained with DAPI (in blue). D and H are enlarged areas of A and C, respectively. B and F are control sections with isolectin B4 omitted. Yellow arrows (C) indicate blood vessels in the GCL. Scale bars: 30 μm. Original magnification, ×400.

Figure 4. Focal intraretinal NV and Müller cell activation in Cyp27a1^–/– mice.

Panels are representative of stainings carried out on adjacent retinal sections in Cyp27a1^–/– mice (A–H) or through corresponding regions in Cyp27a1^+/+ mice (I–M). (A and I) H&E staining. (C, F, and K) Stainings with a marker for blood vessels, tomato lectin (in red), which is a less specific blood-vessel marker than isolectin B4, binding to photoreceptors. (D, G, and L) Localization of GFAP, a marker of activated Müller cells (in yellow). (E, H, and M) Localization of GS expressed constitutively in Müller cells (in green). (B and J) Negative control sections treated with serum from nonimmunized animal. For fluorescent images, nuclei were stained with DAPI (in blue), and immunoreactivity/lectin binding was detected by DyLight 649–conjugated (in yellow), DyLight 549–conjugated (in red), and DyLight 488–conjugated (in green) fluorophores. Light blue arrows (C) indicate retinal blood vessels in different retinal layers; white arrow (D) indicates increased GFAP staining in the OPL and ONL in the area of pathology; gold arrows (E and H) indicate disorganized Müller cell bodies in Cyp27a1^–/– specimen; and pink arrows (M) indicate proper organization of Müller cell bodies in Cyp27a1^+/+ specimen. Scale bars: 30 μm. Original magnification, ×400.

Figure 5. Leakage of blood vessels in the area of pathology in Cyp27a1^–/– mice.

Representative FA (55° field of view) in Cyp27a1^+/+ (A) and Cyp27a1^–/– mice (B, D, and E). SD-OCT fundus images (50° field of view) of Cyp27a1^–/– animals (C and F) are also shown. Yellow and light blue arrows point to the areas of dye leakage (B, D, and E) and the corresponding hyperreflective spots in OCT fundus images (C and F); arrows of the same color indicate the same animal; pink arrow points to blood vessel tortuosity and beading. pi, post injection.

Figure 6. Doppler flow of SD-OCT shows RCA and also reveals altered direction of blood flow.

(A and C) Representative OCT fundus depth images (50° field of view) at the OPL in Cyp27a1^–/– and Cyp27a1^+/+ mice, respectively. (B and D) Doppler flow of SD-OCT cross section through pathology in A (indicated by green arrow) and the matching area in Cyp27a1^+/+ retina in C (indicated by orange line). Red and blue colored pixels correspond to direction and relative magnitude of the vector component of blood flow perpendicular to the plane of the scan (V_C in E). Areas with no flow are transparent and show the underlying SD-OCT imaging data. Scale bars: 300 μm (A and C); 60 μm (B and D).

Figure 7. Retinal hypoxia in Cyp27a1^–/– mice.

Representative H&E (A and F), anti-pimonidazole (in dark purple, C–E), and anti-HIF1A stainings (in green, H–J) of Cyp27a1^–/– and Cyp27a1^+/+ mice. (B and G) Negative controls treated with serum from nonimmunized animal. White arrows (C and D) indicate increased staining intensity relative to Cyp27a1^+/+ (E), and gold arrows (H) indicate intense staining in the blood vessel above BrM. For fluorescence imaging, nuclei were stained with DAPI (in blue), and immunoreactivity was detected by DyLight 649–conjugated fluorophore (in green). For pimonidazole adducts, immunoreactivity was detected by the ImmPACT VIP Peroxidase Substrate (in dark purple) and nuclei were stained with methyl green. Scale bars: 30 μm. Original magnification, ×400.

Figure 8. Lipid and cholesterol deposition beneath the RPE in Cyp27a1^–/– mice.

Panels are representative of stainings carried out on adjacent sections cut through pathologies in Cyp27a1^–/– mice (A–F) or through corresponding regions in Cyp27a1^+/+ mice (G–L). (A and G) H&E staining. (B and H) Localizations of neutral lipids with oil red O (in fuchsia). (C and I) Staining with blood-vessel marker isolectin B4 conjugated to DyLight 594 fluorophore (in yellow); nuclei were stained with DAPI (in blue). (D and J) Merger of B and C and H and I, respectively. White arrows (B–D) indicate colocalized staining. (E and K) Detection of UC with filipin (in cyan); nuclei were stained with propidium iodide (in red). (F and L) Enlarged regions of E and K, respectively. Scale bars: 30 μm. Original magnification, ×400.

Figure 9. Retinal sterol profile is altered in Cyp27a1^–/– mice.

Open and dashed bars show concentrations of sterols in WT and Cyp27a1^–/– animals, respectively; white bars, females (F) and gray bars, males (M). Retinas from 40 animals per group were pooled. 24-OH, 24S-hydroxycholesterol; 27-OH, 27-hydroxycholesterol; 27-COOH, 5-cholestenoic acid; 7-keto, 7-ketocholesterol. *P < 0.05, compared with age- and sex-matched WT littermates.

Figure 10. Putative cascade of events initiated by CYP27A1 deficiency in the retina.

Events within columns may occur in parallel or in different order. Events in gray type are putative and have not yet been confirmed experimentally. CHO, cholesterol.