Paucity and dysfunction of blood vessels in tissues leading to ischemia is a well-recognized pathological process that often leads to irreversible injury, particularly in neuronal tissue. At the other extreme, excessive vessels (NV) can also compromise tissue function, usually as a result of leakage, hemorrhage, or scarring caused by the abnormal new vessels. In this study, we provide evidence for yet another mechanism of injury associated with NV, even in the absence of clinically significant leakage or hemorrhage: neuronal cell death as a result of increased oxidative stress caused by proximity to the abnormal vessels.
In several diseases that can cause irreversible vision loss, such as RAP (16
) and MacTel (14
), abnormal vessels extend from the highly vascularized inner retina into the normally avascular outer retina. The retina of the Vldlr–/–
mouse exhibits vascular changes very similar to those observed in patients with MacTel and RAP and thus is a good model for studying the general relationship between NV and neurodegeneration. The new vessels observed in the human disease and the Vldlr–/–
mouse exhibit relatively mild permeability defects and are accompanied by glial activation and disruption of the RPE. Another feature common to RAP, MacTel, and the Vldlr–/–
retina is that the nonuniformly distributed focal vascular lesions are directly associated with neuronal degeneration separated by relatively normal regions. While other retinal degenerative models, such as rd1
mice, have associated vascular abnormalities and neuronal loss, many of these result in uniform degeneration and relate more closely to genetic diseases, such as retinitis pigmentosa, rather than to ocular vascular disease. Taken together, these similarities support the use of the Vldlr–/–
mouse as a compelling model to investigate selected human retinal diseases (e.g., MacTel) as well as other, nonocular neurodegenerative disorders with associated NV.
VEGF plays a critical role in normal retinal vascularization and retinal angiogenic diseases (42
). In the Vldlr–/–
retina, VEGF upregulation is observed in photoreceptors and RPE and participates in the stimulation of subretinal NV. Normal VLDLR expression within the avascular outer retina, and upregulation of VEGF coincident with formation of abnormal subretinal vessels in these areas of VLDLR-deficient mice, suggests a mechanism linking VLDLR with proangiogenic factors. No obvious abnormalities were observed in the choroid, suggesting potential RPE polarity of the VEGF overexpression toward the inner retina. VLDLR is a negative regulator of wnt signaling during retina development; upregulation of the wnt coreceptor LRP5/6 and increased downstream wnt signaling occurs in Vldlr–/–
mouse retinas (27
). VEGF is a well-known product of wnt signaling, and VLDLR may normally participate in turning off wnt signaling once retinal vascular development is complete. In the absence of VLDLR, wnt signaling persists, leading to increased levels of VEGF expression in photoreceptor and RPE layers and the angiogenic extension of abnormal intra- and subretinal vessels. Whether such a mechanism operates in diseases such as MacTel and RAP is uncertain, although anti-VEGF therapy has previously been tested in both conditions (45
). Initial efforts to treat RAP lesions using combination angiostatics are also promising (47
). However, in our experience with the Vldlr–/–
mouse, antiangiogenic therapy is only transiently effective, subretinal NV returns within 2 weeks, and therapy must be initiated prior to NV formation. Thus, the usefulness of antiangiogenic therapy in human ocular diseases with attributes similar to those of Vldlr–/–
mice may be limited.
A major finding in this study is that neuronal abnormalities and impaired retinal function developed in association with NV. We were able to prevent focal degeneration of photoreceptors in the regions of vascular abnormalities through the selective delivery of neurotrophic molecules by taking advantage of cellular changes observed in this condition. Müller glia, with cytoplasmic processes spanning the inner and outer retina, were activated in response to vascular changes such as those observed in the Vldlr–/– retina. Upon activation, these cells upregulate GFAP, and their appearance and location precisely correlate, both temporally and spatially, with subretinal NV and associated neuronal degeneration in the outer retina. By targeting the activated Müller cells and taking advantage of their retina-spanning cytoplasmic processes, NT4 was specifically delivered to sites of vascular abnormalities after intravitreal injection of AAV-GFAP-NT4. This strategy using endogenous cells (e.g., activated Müller cells) to deliver gene therapy products to the outer retina could be useful clinically to avoid the need for subretinal injection of the viral vector, a procedure that can have deleterious effects on already diseased retinas. In addition, we were able to dramatically attenuate retinal degeneration in the Vldlr–/– mouse retina, supporting a link between the presence of subretinal vessels and neuronal degeneration.
retinas exhibit increased levels of oxidative stress (35
). Excessive oxygen levels can generate reactive oxygen species that cause molecular damage to lipids, proteins, and nucleic acids, which can subsequently lead to cell death unless neutralized by the antioxidant defense system (48
). The retina, with its high fat content and its exposure to light, is particularly susceptible to such injury. Cone loss is associated with oxidative damage in other models of primary retinal degeneration, such as the rd1
), and oxidative damage has been linked with AMD (40
). Recent studies using adaptive optics also demonstrate focal photoreceptor loss associated with vascular abnormalities in diseases such as MacTel, traditionally considered a retinal vascular disease (A. Roorda, unpublished observations). We attempted to directly assess the role of abnormal NV in causing subsequent neuronal degeneration by blocking NV in the Vldlr–/–
mice. However, even the most potent angiostatic combinations were only transiently effective, while the neurodegeneration was progressive over the course of several weeks to months after initiation of subretinal NV. Thus, prolonged elimination of subretinal NV was impossible without repeated injections, a process which at best leads to injury and variability in the small mouse eye, and often leads to phthisis. However, based on the strong association between subretinal NV and neurodegeneration, the correlation between subretinal NV and oxidative stress, and the neuroprotective results obtained from treatment with antioxidants, we hypothesize that increased vascularization and abnormal localization of the subretinal NV leads to increased oxidative stress and subsequent neuronal damage. No defects were found in the cellular oxidative stress defense pathways of the Vldlr–/–
mice, further supporting our hypothesis.
These findings demonstrating protective effects of antioxidant treatment on neuronal degeneration associated with abnormal NV support prior epidemiological studies in humans showing that an antioxidant-rich diet protects against AMD (40
) and diminishes its progression (49
). However, it should be noted that only moderate levels of protection were observed in these epidemiological studies, particularly when dietary supplementation is initiated after NV is already present. Such dietary measures may be a simple and effective method of preventing neuronal cell loss associated with certain retinal vascular diseases such as MacTel and RAP. In instances of associated neuronal degeneration with substantial ERG abnormalities, gene therapy using intravitreally injected AAV-2 vectors encoding neurotrophic transgenes may also serve to preserve visual function. These findings using the Vldlr–/–
mouse retina model also support other studies that demonstrate benefits of antioxidant-rich diets in relation to other neurodegenerative disorders of the CNS (50
). Thus, our present findings may also have application to other neurodegenerative disorders, such as Huntington disease, Parkinson disease, and Alzheimer disease. Further studies will be required to better understand the link between vascular abnormalities such as those observed in retinal NV, oxidative stress, and neuronal degeneration.