Piroska E. Rakoczy

Overexpression of Vascular Endothelial Growth Factor (VEGF) in the Retinal Pigment Epithelium Leads to the Development of Choroidal Neovascularization

Vascular endothelial growth factor (VEGF) has been strongly implicated in the development of choroidal neovascularization found in age-related macular degeneration. Normally expressed in low levels, this study investigates whether the overexpression of VEGF in the retinal pigment epithelium is sufficient to cause choroidal neovascularization in the rat retina. A recombinant adenovirus vector expressing the rat VEGF(164) cDNA (AdCMV.VEGF) was constructed and injected into the subretinal space. The development of neovascularization was followed by fluorescein angiography, which indicates microvascular hyperpermeability of existing and/or newly forming blood vessels, and histology. VEGF mRNA was found to be overexpressed by retinal pigment epithelial cells and resulted in leaky blood vessels at 10 days postinjection, which was maintained for up to 31 days postinjection. By 80 days postinjection, new blood vessels had originated from the choriocapillaris, grown through the Bruchs membrane to the subretinal space, and disrupted the retinal pigment epithelium. This ultimately led to the formation of choroidal neovascular membranes and the death of overlying photoreceptor cells. By controlling the amount of virus delivered to the subretinal space, we were able to influence the severity and extent of the resulting choroidal neovascularization. These results show that even temporary overexpression of VEGF in retinal pigment epithelial cells is sufficient to induce choroidal neovascularization in the rat eye.

Lipofuscin of the retinal pigment epithelium: A review

Accumulation of lipofuscin is one of the most characteristic features of ageing observed in retinal pigment epithelial (RPE) cells. The lipofuscin found in RPE cells differs from that of other body tissues due to the fact that it is mainly derived from the chemically modified residues of incompletely digested photoreceptor outer segments. It is a heterogeneous material composed of a mixture of lipids, proteins, and different fluorescent compounds, the main fluorophore of which has recently been identified as a derivative of vitamin A. Research interest has variously focussed on the roles of age, light damage, free radicals, antioxidants, visual pigments, retinal locus, lysosomal enzymes, and pigmentation on lipofuscin formation, as well as the effects of lipofuscin on RPE cell function and causation of retinal disease. This article reviews the recent advances in knowledge of the composition, origin, and possible deleterious effects of RPE cell lipofuscin.

The use of synthetic polymers for delivery of therapeutic antisense oligodeoxynucleotides.

Abstract Developed over the past two decades, the antisense strategy has become a technology of recognised therapeutic potential, and many of the problems raised earlier in its application have been solved to varying extents. However, the adequate delivery of antisense oligodeoxynucleotides to individual cells remains an important and inordinately difficult challenge. Synthetic polymers appeared on this scene in the middle 1980s, and there is a surprisingly large variety used or proposed so far as agents for delivery of oligodeoxynucleotides. After discussing the principles of antisense strategy, certain aspects of the ingestion of macromolecules by cells, and the present situation of delivery procedures, this article analyses in detail the attempts to use synthetic polymers as carrier matrices and/or cell membrane permeabilisation agents for delivery of antisense oligodeoxynucleotides. Structural aspects of various polymers, as well as the results, promises and limitations of their use are critically evaluated.

Progressive Age-Related Changes Similar to Age-Related Macular Degeneration in a Transgenic Mouse Model

Age-related macular degeneration (AMD) is the major cause of blindness in the developed world. Its pathomechanism is unknown and its late onset, complex genetics and strong environmental components have all hampered investigations. Here we demonstrate the development of an animal model for AMD that reproduces features associated with geographic atrophy; a transgenic mouse line (mcd/mcd) expressing a mutated form of cathepsin D that is enzymatically inactive thus impairing processing of phagocytosed photoreceptor outer segments in the retinal pigment epithelial (RPE) cells. Pigmentary changes indicating RPE cell atrophy and a decreased response to flash electroretinograms were observed in 11- to 12-month-old mcd/mcd mice. Histological studies showed RPE cell proliferation, photoreceptor degeneration, shortening of photoreceptor outer segments, and accumulation of immunoreactive photoreceptor breakdown products in the RPE cells. An accelerated photoreceptor cell death was detected in 12-month-old mcd/mcd mice. Transmission electron microscopy demonstrated presence of basal laminar and linear deposits that are considered to be the hallmarks of AMD. Small hard drusen associated with human age-related maculopathy were absent in the mcd/mcd mouse model at the ages analyzed. In summary, this model presents several features of AMD, thus providing a valuable tool for investigating the underlying biological processes and pathomechanism of AMD.

Inhibition of angiogenesis by adenovirus-mediated sFlt-1 expression in a rat model of corneal neovascularization

Pathological angiogenesis, or the production of new capillary vessels from preexisting vasculature, within the eye is a serious event that often leads to blindness. Upregulation of vascular endothelial growth factor (VEGF) has been linked to neovascularization in the eye, suggesting that it could be a suitable target to inhibit angiogenic changes. This work investigated whether the presence of a proven antiangiogenic factor, the soluble variant of the VEGF receptor, sFlt-1, in the anterior chamber is sufficient to inhibit new vessel formation in the cornea in an animal model of corneal neovascularization. A recombinant adenovirus vector that can mediate efficient in vivo gene transfer and expression in ocular cells was selected as a delivery agent. We have shown that after the injection of Ad.betagal into the anterior chamber of normal and cauterized rat eyes, corneal endothelial cells and cells of the trabecular meshwork were efficiently transduced and that beta-galactosidase (beta-Gal) expression was maintained up to 10 days postinjection. Cauterization significantly increased the amount of immunoreactive VEGF in vehicle- or Ad.null-injected animals (t test, p < 0.001 and p < 0.001, respectively). However, when cauterization was combined with Ad.sflt injection there was no statistically significant increase in the amount of immunoreactive VEGF (p = 0.12). The injection of Ad.sflt into the anterior chamber slowed or inhibited VEGF-induced angiogenic changes. After cauterization, 100% of uninjected and vehicle-injected and 82% of Ad.null-injected animals developed moderate to severe corneal angiogenesis in contrast to 18% of Ad.sflt-injected animals. These in vivo results suggest that the transient presence of antiangiogenic agents in the anterior chamber can be successfully used to inhibit the development of corneal angiogenesis.

EVALUATION OF ADENO-ASSOCIATED VIRUS-MEDIATED GENE TRANSFER INTO THE RAT RETINA BY CLINICAL FLUORESCENCE PHOTOGRAPHY

The purpose of this study was to evaluate recombinant adeno-associated virus (AAV) as an in vivo gene transfer vector for the retina and to explore the possibility of monitoring the expression of green fluorescent protein (GFP) using a noninvasive method. Rats were injected subretinally with rAAV-gfp or rAAV-lacZ. Strong expression of the reporter gene in a circular area surrounding the injection site was observed in retinal whole mounts and tissue sections. Higher magnification revealed that cells demonstrating high levels of green fluorescence were hexagonal in shape, indicating they were retinal pigment epithelium (RPE) cells. Histological observation of retinal sections demonstrated that recombinant AAV specifically transduced RPE cells. Ten animals were injected with rAAV-gfp for longitudinal studies and the fluorescence was monitored by retinal fluorescence photography. The GFP signal was detected in 100% of the animals as early as 2 weeks postinjection and remained present throughout the experimental period of 4 months. After 2 weeks, a gradual increase in the number of transduced cells occurred before reaching maximal levels of GFP expression at 8 weeks. This was followed by a small decrease over 4 weeks before reaching stable expression at 16 weeks. Our results demonstrated that rAAV efficiently transduces rat RPE cells and that retinal fluorescence photography is suitable for monitoring GFP expression. By using this noninvasive technique, we demonstrated that repetitive measurements of GFP expression in vivo in the rAAV-gfp-transduced retina are possible. This study demonstrated that retinal fluorescence photography is a potent tool for studying AAV-mediated gene delivery in the retina.

In vivo use of oligonucleotides to inhibit choroidal neovascularisation in the eye

We have previously demonstrated the in vivo uptake of oligonucleotides in the rat eye and have continued with experiments to look at the effectiveness of targeted oligonucleotide sequences. Vascular endothelial growth factor (VEGF) is correlated with new blood vessel formation and has been implicated in numerous eye diseases characterised by abnormal blood vessel proliferation. An oligonucleotide targeted to the VEGF sequence was examined for its effect on VEGF production in vitro and the development of choroidal neovascularisation in vivo in the eye.

Changes in retinal pigment epithelial cell autofluorescence and protein expression associated with phagocytosis of rod outer segments in vitro.

Summary— The accumulation of autofluorescent lipofuscin was quantified in cultured human retinal pigment epithelial (RPE) cells phagocytosing bovine rod outer segments (BROS) and the expression of proteins in these cells was investigated. Results showed a steady increase in autofluorescence of RPE cells over a 4‐week period as measured by fluorophotometric flow cytometry. A significantly greater increase in autofluorescence was found in the cultured RPE cells from a 7‐year‐old donor compared with those from a 47‐year‐old donor. Within both groups the BROs‐challenged cells had significantly higher fluorescence readings than the control cells which were not challenged. Autoradiography of 35S‐labelled proteins separated by polyacrylamide gel electrophoresis (PAGE) revealed a small distinct band at 102 kDa in BROS‐challenged RPE cells of both bovine and human origin that did not appear in control or microsphere‐phagocytosing RPE cells. The intensity of the signal was unrelated to the duration of the challenge period.

A simple flow cytometric technique to quantify rod outer segment phagocytosis in cultured retinal pigment epithelial cells

PURPOSE The primary aim of this study was to develop and characterize a simple flow cytometric method of quantifying rod outer segment (ROS) phagocytosis in cultured retinal pigment epithelial (RPE) cells. A secondary aim was to compare the kinetics of ROS phagocytosis in an immortal human RPE cell line with untransformed human RPE cells. METHODS Flow cytometry was performed on RPE cells that had been challenged with fluorescein isothiocyanate-labeled ROS (FITC-ROS) and phagocytosis was calculated by subtracting background cellular autofluorescence. RESULTS Non-specific uptake of fluorescent label was negligible and RPE cells phagocytosed FITC-ROS and unlabeled ROS with equal efficacy. The kinetics of FITC-ROS phagocytosis in the D407 RPE cell line differed from early passage untransformed human RPE cultures. FITC-ROS phagocytosis proceeded at a fairly linear rate for the first 12 h in the 3 human cell cultures studied, but was rapid for the first 3 h before slowing in the D407 cells. Within all cell populations, there was a heterogeneity of phagocytic activity which varied with time. CONCLUSIONS This automated technique for measuring phagocytosis is rapid, simple, highly accurate, avoids radiation hazards, and permits study of heterogeneity within cell populations. The biochemistry, physiology and pathophysiology of the interactions between retinal pigment epithelial (RPE) cells and photoreceptors continue to be areas of considerable research interest (1, 2, 3). Vital to such work is the ability to accurately quantify rod outer segment (ROS) phagocytosis by RPE cells. Current in vitro techniques of measuring ROS phagocytosis use either automated or manual methods to count phagosomes. While manual counting techniques offer the advantage of visual quality control, they are highly labor intensive, there is a practical limitation to the number of phagosomes that can be counted, and measurements suffer from relatively large standard errors (3). Automated methods include scintillation counting and flow cytometry. Problems with radiolabels include radiation hazards, nonspecific radiolabel uptake, and limited visual control (3). Flow cytometry, on the other hand, circumvents nearly all of these problems and may prove to be the optimal phagocytosis assay.

Inhibition of diclofenac formulated in hyaluronan on angiogenesis in vitro and its intraocular tolerance in the rabbit eye.

Abstract Purpose: To investigate the effect of diclofenac, a potent nonsteroidal anti-inflammatory drug, formulated in hyaluronan (diclofenac/HA) on angiogenesis in vitro and its intraocular toxicity in vivo. Methods: The effect of diclofenac/HA on angiogenesis was determined by choriocapillary endothelial cells on Matrigel stimulated by vascular endothelial growth factor (VEGF). The tube areas were quantified by image digital analysis. For toxicity study, diclofenac/HA was injected intravitreally with a dose range from 100 to 1080 µg in 26 rabbits following gas compression vitrectomy. Potential toxicity was assessed by indirect ophthalmoscopy and by histological studies (light and electron microscopy). Retinal function was monitored by electroretinography (ERG) in six rabbits that received 400 µg of diclofenac/HA. Results: Diclofenac/HA, 180, 90 µg/ml, inhibited tube formation to 24% and 55% of the standard group (Media Ham’s F12 plus 5% fetal calf serum and 50 ng/ml VEGF) respectively (P<0.01). Intravitreal injection of 540 µg or higher doses of diclofenac/HA resulted in ocular toxicity in the rabbit, demonstrated as cataract, vitreous haze and retinal damage observed by indirect ophthalmoscopy and light- and electron- microscopic examinations. No toxicity was observed in the eyes that received 400 µg or less diclofenac/HA, which was further supported by the normal ERG examined at 4 and 25 days post injection. Conclusions: Diclofenac/HA inhibits tube formation in vitro and is nontoxic to the rabbit retina at concentrations that are inhibitory to tube formation. Our results suggest diclofenac/HA may be an effective candidate to inhibit ocular neovascularisation related to granulomatous reaction in the eye.