Early Vascular and Neuronal Changes in a VEGF Transgenic Mouse Model of Retinal Neovascularization

PURPOSE. To investigate early retinal changes in a vascular endothelial growth factor (VEGF) transgenic mouse (tr029VEGF; rhodopsin promoter) with long-term damage that mimics nonproliferative diabetic retinopathy (NPDR) and mild proliferative diabetic retinopathy (PDR). METHODS. Rhodopsin and VEGF expression was assessed up to postnatal day (P)28. Vascular and retinal changes were charted at P7 and P28 using sections and wholemounts stained with hematoxylin and eosin or isolectin IB4 Griffonia simplicifolia Samples were examined using light, fluorescence, and confocal microscopy. RESULTS. Rhodopsin was detected at P5 and reached mature levels by P15; VEGF protein expression was transient, peaking at P10 to P15. In wild-type (wt) mice at P7, vessels had formed in the nerve fiber/retinal ganglion cell layer and showed a centroperipheral maturational gradient; some capillaries had formed a second bed on the vitread side of the inner nuclear layer (INL). By P28, the retinal vasculature had three mature capillary beds, the third abutting the sclerad aspect of the INL. In tr029VEGF mice, capillary bed formation was accelerated compared with that in wt, with abnormal vessels extending to the sclerad side of the INL by P7 and abnormally penetrating the photoreceptors by P28. Compared with P7, vascular lesions were more numerous at P28 when capillary dropout was also evident. At both stages, retinal layers were thinned most where abnormal vessel growth was greatest. CONCLUSIONS. Concomitant damage to the vasculature and neural retina at early stages in tr029VEGF suggest that both tissues are affected, providing opportunities to examine early cellular events that lead to long-term disease. (Invest Ophthalmol Vis Sci. 2006;47:4638 -4645

rAAV.sFlt-1 Gene Therapy Achieves Lasting Reversal of Retinal Neovascularization in the Absence of a Strong Immune Response to the Viral Vector

PURPOSE. To determine the efficacy of rAAV.sFlt-1-mediated gene therapy in a transgenic mouse model of retinal neovascularization (trVEGF029) and to assess whether rAAV.sFlt-1 administration generated any deleterious, long-lasting immune response that could affect efficacy. METHODS. trVEGF029 mice were injected subretinally with rAAV.sFlt-1 or phosphate-buffered saline. Fluorescein angiography and electroretinography were used to compare the extent of fluorescein leakage from retinal vessels and retinal function, respectively. A group of eyes was enucleated, and the retinal vasculature and morphology were studied by confocal and light microscopy. Cells were isolated from the posterior eyecups and spleens of a further group, and immune cell subset populations were investigated by flow cytometry. sFlt-1 protein levels in the eyes were evaluated by ELISA. RESULTS. After a single rAAV.sFlt-1 injection, sFlt-1 protein levels were upregulated, and there was a reduction in fluorescein leakage from the retinal vessels and an improvement in retinal function. Confocal microscopy of isolectin-IB4-labeled retinal wholemounts showed more normal-appearing capillary beds in rAAV.sFlt-1-injected than in PBS-injected trVEGF029 mouse eyes. Light microscopy demonstrated retinal morphology preservation, with fewer aberrant vessels invading the outer nuclear layer of rAAV.sFlt-1-injected eyes. Furthermore, the immune response to subretinal injection of rAAV.sFlt-1 was limited to a transient increase in CD45 ϩ leukocytes that disappeared by 4 weeks after injection. This transient increase was localized to the eye and did not affect long-term therapeutic efficacy. CONCLUSIONS. The data support the notion that rAAV.sFlt-1 gene therapy is safe and effective for the long-term inhibition of deleterious blood vessel growth in the eye. (Invest Ophthalmol Vis Sci. 2009;50:4279 -4287

Analysis of disease-linked rhodopsin mutations based on structure, function, and protein stability calculations

Retinitis pigmentosa (RP) refers to a heterogeneous group of inherited diseases that result in progressive retinal degeneration, characterized by visual field constriction and night blindness. A total of 103 mutations in rhodopsin are linked to RP to date, and the phenotypes range from severe to asymptomatic. To study the relation between phenotype and rhodopsin stability in disease mutants, we used a structure-based approach. For 12 of the mutants located at the protein lipid interphase, we used the von Heijne water membrane transfer scale, and we find that 9 of the mutations could affect membrane insertion. For 91 mutants, we used the protein design algorithm FoldX. The 3 asymptomatic mutations had no significant reduced stability, 2 were unsuitable for FoldX analysis since the structure was incorrect in this region, 63 mutations had a significant change in protein stability (>1.6 kcal/mol), and 23 mutations had energy change values under the prediction error threshold (<1.6 kcal/mol). Out of these 23, the disease-causing effect could be explained by the involvement in other functions (e.g., glycosylation motifs, the interface with arrestin and transducin, and the cilia-binding motif) for 19 mutants. The remaining 4 mutants were probably incorrectly associated with RP or have functionalities not discovered yet. For destabilizing mutations where clinical data were available, we found a highly significant correlation between FoldX energy changes and the average age of night blindness and between FoldX energy changes and daytime vision loss onset. Our detailed structural, functional, and energetic analysis provides a complete picture of the rhodopsin mutations and can guide mutation-specific therapies. (C) 2010 Elsevier Ltd. All rights reserved

Biomarkers for Diabetic Retinopathy – Could Endothelin 2 Be Part of the Answer? - Fig 1

<p><b>Edn2 (A) and Ednrb (B) fold changes in mRNA expression in young and mature retinae of wt, Akita, Kimba and Akimba mice</b>. Data represented are fold changes in expression normalised against Ppia as the housekeeping gene. Differential expression was determined using delta delta Ct according to Livak and Schmittgen [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160442#pone.0160442.ref023" target="_blank">23</a>]. Fold changes in expression in Akita, Kimba and Akimba retinae represent increases or decreases in mRNA expression levels compared to wt retinae. N = 4 per group; *p<0.05 compared to wt and Akita</p

Recombinant adeno-associated virus type 2-mediated gene delivery into the Rpe65-/- knockout mouse eye results in limited rescue

Leber's congenital amaurosis (LCA) is a severe form of retinal dystrophy. Mutations in the RPE65 gene, which is abundantly expressed in retinal pigment epithelial (RPE) cells, account for approximately 10-15% of LCA cases. In this study we used the high turnover, and rapid breeding and maturation time of the knockout mice to assess the efficacy of using rAAV-mediated gene therapy to replace the disrupted RPE65 gene. The potential for rAAV-mediated gene treatment of LCA was then analyzed by determining the pattern of RPE65 expression, the physiological and histological effects that it produced, and any improvement in visual function.rAAV.RPE65 was injected into the subretinal space of knockout mice and control mice. Histological and immunohistological analyses were performed to evaluate any rescue of photoreceptors and to determine longevity and pattern of transgene expression. Electron microscopy was used to examine ultrastructural changes, and electroretinography was used to measure changes in visual function following rAAV.RPE65 injection.rAAV-mediated RPE65 expression was detected for up to 18 months post injection. The delivery of rAAV.RPE65 to mouse retinas resulted in a transient improvement in the maximum b-wave amplitude under both scotopic and photopic conditions (76% and 59% increase above uninjected controls, respectively) but no changes were observed in a-wave amplitude. However, this increase in b-wave amplitude was not accompanied by any slow down in photoreceptor degeneration or apoptotic cell death. Delivery of rAAV.RPE65 also resulted in a decrease in retinyl ester lipid droplets and an increase in short wavelength cone opsin-positive cells, suggesting that the recovery of RPE65 expression has long-term benefits for retinal health.This work demonstrated the potential benefits of using the mice to study the effects and mechanism of rAAV.RPE65-mediated gene delivery into the retina. Although the functional recovery in this model was not as robust as in the dog model, these experiments provided important clues about the long-term physiological benefits of restoration of RPE65 expression in the retina

Evidence of Müller cell gliosis in mature Akimba mice with a severe phenotype.

<p>In the mature wt retina GFAP expression was localised and restricted to the astrocytes that reside on the GCL (arrowheads, C-D). In Akimba mice, GFAP expression was observed as thick processes in the GCL and long radial processes in the INL/OPL, indicating Müller cell gliosis (G-H). Edn2 only co-localised with GFAP in Müller cell processes in the INL/OPL (arrows, H) but not in the astrocytes of the GCL. Sections were counterstained with DAPI (B, F). GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer; PIS/POS: photoreceptor inner and outer segments. Scale bar: 200μm</p

Cellular localisation of Edn2 in the mature retina.

<p>Following 20 weeks of chronic hyperglycaemia in the mature Akita retina there was increased Edn2 staining in the IPL compared to young Akita retinae (F). Particularly in the mature Akimba retina GS staining was much reduced compared to the young retina, suggesting not only loss of photoreceptors (L) but also loss of Müller cells (I, arrowheads). Müller cell loss was also evident in mature Kimba retinae (M, arrowheads; O, arrows) compared to young Kimba retinae, but the loss was less pronounced. Scale bar: 100μm</p