Bogale Aredo

Transgenic mice expressing variants of complement factor H develop AMD-like retinal findings.

PURPOSE Complement factor H (Cfh) is a key regulator of the alternative complement pathway. A Cfh variant (Y402H) increases the risk for AMD. The purpose of this study was to develop a pathophysiologically relevant animal model of AMD based on this genetic risk factor. METHODS The authors generated chimeric Cfh transgenic mouse lines using two constructs consisting of the human CFH sequence for SCR6-8 (with either 402Y or 402H), flanked by the mouse sequence for SCR1-5 and SCR9-20. They tested the expression of the transgenic mRNA and protein molecules and examined the mice at 12 to 14 months of age for clinical and histologic retinal changes. RESULTS Nuclease protection assay and qRT-PCR analysis demonstrated transgenic mRNA expression in the liver and in the posterior segment of the eye. Western blot analysis showed that the transgenic proteins are present in the circulation at levels comparable to those of mouse Cfh. The chimeric proteins were found to be functional, as demonstrated by their ability to restore physiological serum levels of complement component C3 in Cfh KO mice. Clinical examination showed subretinal drusen-like deposits. Histology demonstrated an accumulation of subretinal cells that stained with a macrophage/microglia marker. Basal laminar deposits, long-spaced collagen, and increased numbers of lipofuscin granules were seen on electron microscopy. Immunohistochemistry showed a thicker sub-RPE band of C3d staining. CONCLUSIONS Chimeric Cfh proteins led to AMD-like characteristics in mice. This may represent a good model for studying the role of complement and other components of the immune system in early AMD.

Differences in the distribution, phenotype and gene expression of subretinal microglia/macrophages in C57BL/6N (Crb1rd8/rd8) versus C57BL6/J (Crb1wt/wt) mice

BackgroundMicroglia/macrophages (MG/MΦ) are found in the subretinal space in both mice and humans. Our goal was to study the spatial and temporal distribution, the phenotype, and gene expression of subretinal MG/MΦ in mice with normal retinas and compare them to mice with known retinal pathology.MethodsWe studied C57BL/6 mice with (C57BL/6N), or without (C57BL/6J) the rd8 mutation in the Crb1 gene (which, in the presence of yet unidentified permissive/modifying genes, leads to a retinal degeneration), and documented their fundus appearance and the change with aging. Immunostaining of retinal pigment epithelium (RPE) flat mounts was done for 1) Ionized calcium binding adaptor (Iba)-1, 2) FcγIII/II Receptor (CD16/CD32, abbreviated as CD16), and 3) Macrophage mannose receptor (MMR). Reverse-transcription quantitative PCR (RT-qPCR) was done for genes involved in oxidative stress, complement activation and inflammation.ResultsThe number of yellow fundus spots correlated highly with subretinal Iba-1+ cells. The total number of subretinal MG/MΦ increased with age in the rd8 mutant mice, but not in the wild-type (WT) mice. There was a centripetal shift in the distribution of the subretinal MG/MΦ with age. Old rd8 mutant mice had a greater number of CD16+ MG/MΦ. CD16+ cells had morphological signs of activation, and this was most prominent in old rd8 mutant mice (P <1×10−8 versus old WT mice). Subretinal MG/MΦ in rd8 mutant mice also expressed iNOS and MHC-II, and had ultrastructural signs of activation. Finally, rd8 mutant mouse RPE/ MG/MΦ RNA isolates showed an upregulation of Ccl2, CFB, C3, NF-kβ, CD200R and TNF-alpha. The retinas of rd8 mutant mice showed upregulation of HO-1, C1q, C4, and Nrf-2.ConclusionsWhen compared to C57BL/6J mice, C57BL/6N mice demonstrate increased accumulation of subretinal MG/MΦ, displaying phenotypical, morphological, and gene-expression characteristics consistent with a pro-inflammatory shift. These changes become more prominent with aging and are likely due to the combination of the rd8 mutation and yet unidentified permissive/modulatory genes in the C57BL/6N mice. In contrast, aging leads to a scavenging phenotype in the C57BL/6J subretinal microglia/macrophages.

A chimeric Cfh transgene leads to increased retinal oxidative stress, inflammation, and accumulation of activated subretinal microglia in mice.

PURPOSE Variants of complement factor H (Cfh) affecting short consensus repeats (SCRs) 6 to 8 increase the risk of age-related macular degeneration. Our aim was to explore the effect of expressing a Cfh variant on the in vivo susceptibility of the retina and RPE to oxidative stress and inflammation, using chimeric Cfh transgenic mice (chCfhTg). METHODS The chCfhTg and age-matched C57BL/6J (B6) mice were subjected to oxidative stress by either normal aging, or by exposure to a combination of oral hydroquinone (0.8% HQ) and increased light. Eyes were collected for immunohistochemistry of RPE-choroid flat mounts and of retinal sections, ELISA, electron microscopy, and RPE/microglia gene expression analysis. RESULTS Aging mice to 2 years led to an increased accumulation of basal laminar deposits, subretinal microglia/macrophages (MG/MΦ) staining for CD16 and for malondialdehyde (MDA), and MDA-modified proteins in the retina in chCfhTg compared to B6 mice. The chCfhTg mice maintained on HQ diet and increased light showed greater deposition of basal laminar deposits, more accumulation of fundus spots suggestive of MG/MΦ, and increased deposition of C3d in the sub-RPE space, compared to controls. In addition, chCfhTg mice demonstrated upregulation of NLRP3, IP-10, CD68, and TREM-2 in the RNA isolates from RPE/MG/MΦ. CONCLUSIONS Expression of a Cfh transgene introducing a variant in SCRs 6 to 8 was sufficient to lead to increased retinal/RPE susceptibility to oxidative stress, a proinflammatory MG/MΦ phenotype, and a proinflammatory RPE/MG/MΦ gene expression profile in a transgenic mouse model. Our data suggest that altered interactions of Cfh with MDA-modified proteins may be relevant in explaining the effects of the Cfh variant.

Phosphatidylserine (PS) is exposed in choroidal neovascular endothelium: PS-targeting antibodies inhibit choroidal angiogenesis in vivo and ex vivo

PURPOSE Choroidal neovascularization (CNV) accounts for 90% of cases of severe vision loss in patients with advanced age-related macular degeneration. Identifying new therapeutic targets for CNV may lead to novel combination therapies to improve outcomes and reduce treatment burden. Our goal was to test whether phosphatidylserine (PS) becomes exposed in the outer membrane of choroidal neovascular endothelium, and whether this could provide a new therapeutic target for CNV. METHODS Choroidal neovascularization was induced in C57BL/6J mice using laser photocoagulation. Choroidal neovascularization lesions costained for exposed PS and for intercellular adhesion molecule 2 (or isolectin B4) were imaged in flat mounts and in cross sections. The laser CNV model and a choroidal sprouting assay were used to test the effect of PS-targeting antibodies on choroidal angiogenesis. Choroidal neovascularization lesion size was determined by intercellular adhesion molecule 2 (ICAM-2) staining of flat mounts. RESULTS We found that PS was exposed in CNV lesions and colocalized with vascular endothelial staining. Treatment with PS-targeting antibodies led to a 40% to 80% reduction in CNV lesion area when compared to treatment with a control antibody. The effect was the same as that seen using an equal dose of an anti-VEGF antibody. Results were confirmed using the choroid sprouting assay, an ex vivo model of choroidal angiogenesis. CONCLUSIONS We demonstrated that PS is exposed in choroidal neovascular endothelium. Furthermore, targeting this exposed PS with antibodies may be of therapeutic value in CNV.

Increased susceptibility to fundus camera-delivered light-induced retinal degeneration in mice deficient in oxidative stress response proteins

Abstract Oxidative stress is an important contributor to the pathogenesis of many retinal diseases including age‐related macular degeneration and retinal dystrophies. Light‐induced retinal degeneration (LIRD) can serve as a model in which to study the response of the retina to stress. Of note, many genetic mutant mice are in a C57BL/6 J background and are thus resistant to the usual LIRD models. We recently developed a new model of fundus camera‐delivered light‐induced retinal degeneration (FCD‐LIRD) which is effective in strains of mice expressing the light‐resistant variant of RPE65 (450Met), including C57BL/6 J. In this work we investigated whether FCD‐LIRD would be useful as a model in which to test the effect of genetic mutations on the response of the retina to stress. Furthermore, we tested whether oxidative stress plays an important role in the setting of this new FCD‐LIRD model. FCD‐LIRD was applied to C57BL/6 J mice and to mice simultaneously deficient in three proteins that are important in the response of the retina to oxidative stress (SOD1, DJ‐1 and Parkin). Using fundus photography, we found that retinal damage was dramatically increased in the SOD1/DJ‐1/Parkin deficient mice compared to C57BL/6 J. Outer retinal OCT volume and RPE cell morphology analysis in ZO‐1‐stained flat mounts added support to these findings. Gene expression analysis confirmed a strong oxidative stress response after FCD‐LIRD, which was differentially altered in the SOD1/DJ1/Parkin deficient mice. We conclude that FCD‐LIRD is useful to study the effect of genetic mutations on the response of the retina to light stress in light‐resistant strains of mice. Furthermore, oxidative stress seems to be an important component of FCD‐LIRD. Finally, we have established protocols to quantify the effect of FCD‐LIRD on the retina and RPE which will be useful for future studies. Further dissection of the mechanisms by which the retina responds to light‐induced oxidative stress may result in new strategies to modulate this response, which could lead to a reduction in retinal and RPE damage. HighlightsNovel retinal degeneration model (FCD‐LIRD) is effective in light‐resistant mice.Helps study effect of genetic mutations on retinal response to light stress.Oxidative stress is an important component of FCD‐LIRD.We established protocols to quantify the effect of FCD‐LIRD on the retina and RPE.

Fundus Camera-Delivered Light-Induced Retinal Degeneration in Mice With the RPE65 Leu450Met Variant is Associated With Oxidative Stress and Apoptosis

Purpose Oxidative stress, partly due to light, has an important role in many retinal diseases, including macular degeneration and retinal dystrophies. The Leu450Met variant of RPE65 is expressed in C57BL/6 and in many genetically modified mice. It confers significant resistance to light induced retinal degeneration (LIRD). Our goal was to develop an effective and efficient method to induce LIRD in resistant mice that would recapitulate mechanisms seen in known models of LIRD. Methods The retinas of C57BL/6J mice were exposed to light using a murine fundus camera. Two protocols (with and without intraperitoneal fluorescein) were used. Optical coherence tomography (OCT) helped determine the location and extent of retinal damage. Histology, TUNEL assay, quantitative (q) PCR, and immunohistochemistry were performed. Results Both protocols consistently generated LIRD in C57BL/6J mice. Optical coherence tomography and histology demonstrated that retinal damage starts at the level of the photoreceptor/outer retina and is more prominent in the superior retina. Fundus camera-delivered light-induced retinal degeneration (FCD-LIRD) is associated with apoptosis, subretinal microglia/macrophages, increased expression of oxidative stress response genes, and C3d deposition. Conclusions We characterize two new models of light-induced retinal degeneration that are effective in C57BL/6J mice, and can be modulated in terms of severity. We expect FCD-LIRD to be useful in exploring mechanisms of LIRD in resistant mice, which will be important in increasing our understanding of the retinal response to light damage and oxidative stress.