Regeneration of the zebrafish retinal pigment epithelium after widespread genetic ablation.

The retinal pigment epithelium (RPE) is a specialized monolayer of pigmented cells within the eye that is critical for maintaining visual system function. Diseases affecting the RPE have dire consequences for vision, and the most prevalent of these is atrophic (dry) age-related macular degeneration (AMD), which is thought to result from RPE dysfunction and degeneration. An intriguing possibility for treating RPE degenerative diseases like atrophic AMD is the stimulation of endogenous RPE regeneration; however, very little is known about the mechanisms driving successful RPE regeneration in vivo. Here, we developed a zebrafish transgenic model (rpe65a:nfsB-eGFP) that enabled ablation of large swathes of mature RPE. RPE ablation resulted in rapid RPE degeneration, as well as degeneration of Bruch's membrane and underlying photoreceptors. Using this model, we demonstrate for the first time that zebrafish are capable of regenerating a functional RPE monolayer after RPE ablation. Regenerated RPE cells first appear at the periphery of the RPE, and regeneration proceeds in a peripheral-to-central fashion. RPE ablation elicits a robust proliferative response in the remaining RPE. Subsequently, proliferative cells move into the injury site and differentiate into RPE. BrdU incorporation assays demonstrate that the regenerated RPE is likely derived from remaining peripheral RPE cells. Pharmacological disruption using IWR-1, a Wnt signaling antagonist, significantly reduces cell proliferation in the RPE and impairs overall RPE recovery. These data demonstrate that the zebrafish RPE possesses a robust capacity for regeneration and highlight a potential mechanism through which endogenous RPE regenerate in vivo.

Developmental and age-related changes to the elastic lamina of Bruch's membrane in mice.

BACKGROUND: Fibrillin-1, tropoelastin, fibulin-5, and latent transforming growth factor beta-binding protein-2 and protein-4 (LTBP-2 and LTBP-4) are essential proteins for the elastic lamina (EL). In this study, we analyzed each of these molecules in the EL of Bruch's membrane (BM) through development and aging. METHODS: C57BL/6 mice (embryonic (E) days E12.5, E15.5, and E18.5; postnatal (P) days P1, P4, and P7 and P3, P6, and P75 weeks of age) were used. To investigate localization, immunohistochemical staining (IH) was performed. Transmission electron microscopy (TEM) was used to evaluate the formation of microfibrils and tropoelastin. mRNA expression was determined by quantitative real-time PCR (qRT-PCR). RESULTS: All five proteins were expressed in the EL of BM by IH except in embryonic mice. TEM results showed that tropoelastin co-stained with microfibrils. Between 3 and 6 weeks of age, microfibrils became longer and thicker. It was difficult to evaluate the EL of BM in senile mice at 75 weeks of age because of abundant deposits which correspond to drusen. mRNA levels of each protein increased dramatically from E15.5 to P1 days and plateaued by P3 weeks as shown by qRT-PCR. CONCLUSIONS: In conclusion, these five proteins are possibly involved in elastic fiber assembly in BM. We define the date of full assembly of the EL of BM as 3 weeks of age in mice.

Three-dimensional spheroidal culture visualization of membranogenesis of Bruch's membrane and basolateral functions of the retinal pigment epithelium.

PURPOSE: Aging changes in the RPE involve lipid accumulation and membranous basal deposits onto the underlying Bruch's membrane, which may be related to AMD. Conventional in vitro cell culture is limited in its ability to observe the epithelial functions on the basal side. The purpose of this study was to develop a three-dimensional culture system to observe basolateral functions of the RPE. METHODS: Isolated human RPE cells were cultured in a viscous medium on a rounded-bottom culture dish, resulting in spheroid formation. The appearance and size of the spheroids were assessed by light microscopy. Spheroids were fixed in 4% paraformaldehyde for immunohistochemistry or sampled for Western blotting. For transmission electron microscopy (TEM) and scanning electron microscopy (SEM), spheroids were postfixed in 1% osmium tetroxide. RESULTS: The spheroids had a differentiated RPE monolayer with a thin elastic layer, a main layer of Bruch's membrane, on their surface and showed outward deposition of lipoproteins with apoB-100. TEM revealed widely spaced collagen, which was identified as condensation of collagen fibrils by SEM. SEM showed deposition of membranous debris and lipid particles, which have been observed in human Bruch's membrane. Western blotting showed expression of RPE differentiation markers and components of Bruch's membrane and RPE lipoproteins. CONCLUSIONS: This model provides direct views of epithelialization processes involving elastogenesis and functions at the basolateral side such as lipoprotein deposition and may elucidate not only unknown epithelial behaviors but also the pathogenesis of RPE-related diseases.

Bruch's membrane of the brachymorphic mouse.

Bruch's membrane exists between the retinal pigment epithelium and the choriocapillary endothelium. Its structure is very complicated, having five sublayers containing basement membranes of retinal pigment epithelium and choriocapillary endothelium, outer and inner collagenous layers, and a central elastic layer. In the development of Bruch's membrane in normal mice, both basement membranes are created first. Secondarily, collagen fibers are accumulated in the space between these basement membranes and then form a collagenous layer. Finally, the elastic layer elaborated in the collagenous layer separates this into outer and inner collagenous layers. Brachymorphic mice have a disorder in the sulfation pathway, resulting in undersulfation. Consequently, in Bruch's membrane of brachymorphic mice, the expression of decorin, a small proteoglycan containing chondroitin sulfate and an indispensable component in collagen assembly, is at a very low level. It is clear that hypoplasia of the collagenous layer in Bruch's membrane of brachymorphic mice induces a disorder in the following formation of the elastic layer. These findings suggest that the formation of the collagenous layer, regulated with acidic glycoconjugates such as decorin, is important in the development of Bruch's membrane.

Changes in Bruch's membrane and related structures with age.

Age-related macular disease is a major and growing public health burden in developed Caucasian societies, accounting for about 50% of blind registration. Evidence exists that this is an emerging problem in Eastern Asia, although the phenotype appears to differ from that seen in Western society. It is likely that several genes are involved, and that the genes or allelic variants conferring are common. Environment plays a major role in its pathogenesis, and it is believed that genetic susceptibility becomes apparent only if there are sufficient environmental pressures. There is no therapy currently available that will have an impact on the prevalence of blindness from age-related macular disease. It has been shown that visual loss occurs as a reaction to ageing changes in Bruch's membrane, which is interposed between the choriocapillaris and the retinal pigment epithelium. The age changes in all three structures have been partly characterised, and as a consequence, multiple putative pathogenic mechanisms have been proposed. Cross-sectional studies of populations with different genetic background and life styles would serve to prove the importance of inheritance and environment. Molecular genetic analysis of blood from affected sibling pairs from these sources may indicate the relevant genes, the prevalence of which may differ in different communities. Enquiries as to life styles may determine important environmental influences. Examination of donor eyes from these communities may reveal distinctive features that may reflect the variation in genetic predisposition and environmental pressures. It is hoped that the findings from such studies will lead to novel and potentially successful management strategies.

Decreasing hydraulic conductivity of Bruch's membrane: relevance to photoreceptor survival and lipofuscinoses.

Deterioration of visual performance leading to blindness is particularly severe in the early-onset forms of Batten disease. Metabolic support of the neural retina is critically dependent on the choroidal blood supply and on efficient transport pathways through Bruch's membrane and the retinal pigment epithelium (RPE). Conversely, degradation products and, in particular, damaged membranous components of photoreceptor outer segments must be removed in the opposite direction. Incomplete breakdown of damaged "spent" discs leads to the age-related accumulation of lipofuscin-like pigments in the RPE, and these in turn influence the degenerative changes in Bruch's membrane. The generalized and extensive deposition of lipofuscin-like material in Batten disease is therefore likely to exacerbate the degenerative changes in Bruch's membrane, and thereby compromise local fluid dynamics. The hydrodynamic properties of Bruch's membrane were examined in normal donor eyes and showed a precipitous decline of hydraulic conductivity during early life. In fact, the maximal capacity for fluid transport was halved for every 17 years of life. This finding is therefore highly relevant to the development of ensuing pathology in the neuronal ceroid-lipofuscinoses.