An improved method using densitometry for evaluating severity of laser photocoagulation induced CNV.

Laser photocoagulation induced choroidal neovascularization currently is the most effective model available for the study of this disease in terms of efficacy of new drugs and therapies. Previously, evaluating the extent of choroidal neovascularization using this model was time-consuming and required the use of experienced personnel. We describe a new method for simple and rapid evaluation of laser induced choroidal neovascularization using densitometry. Fluorescein angiograms of a laser photocoagulated rat eye were scanned into a computer. Densitometry software subsequently was used to calculate the severity of the laser lesions. The densitometry method proved effective for calculating the extent of laser induced choroidal neovascularization. In addition, this method was more rapid than visual evaluations and less likely to produce errors.

Long-term global retinal microvascular changes in a transgenic vascular endothelial growth factor mouse model.

AIMS/HYPOTHESIS: Vascular endothelial growth factor (VEGF) plays a pivotal role in the pathogenesis of diabetic retinopathy. We investigated whether transgenic mice with moderate VEGF expression in photoreceptors (trVEGF029) developed changes similar to diabetic retinopathy and whether retinopathy progressed with time. MATERIALS AND METHODS: Human VEGF(165) (hVEGF(165)) expression was analysed using ELISA and quantitative RT-PCR; serum glucose levels were also measured. Fundus fluorescein angiography (FA) was used to screen the degree of retinopathy from 6 weeks. Dynamic changes in the density of retinal microvasculature, as well as other changes similar to diabetic retinopathy, including retinal leucostasis, capillary endothelial cell and pericyte loss, and numbers of acellular capillaries, were quantified. RESULTS: trVEGF029 mice were normoglycaemic and showed a moderate, short-term hVEGF(165) upregulation for up to 3 weeks. Changes in the retinal microvasculature not only mimicked those seen in diabetic retinopathy, but also showed similar pathological progression with time. FA at 6 weeks identified two phenotypes, mild and moderate, which were distinguished by the extent of vascular leakage. Quantitative analysis of diabetic retinopathy-like changes revealed that these parameters were tightly correlated with the initial degree of vascular leakage; low levels reflected slow and limited retinal microvascular changes in mild cases and high levels reflected more rapid and extensive changes in moderate cases. CONCLUSIONS/INTERPRETATION: The data suggest that even an early short-term elevation in hVEGF(165) expression might set a train of events that lead to progressive retinopathy. Induction of many features characteristic of diabetic retinopathy in trVEGF029 enables mechanisms leading to the disease state to be examined, and provides a relevant animal model for testing novel therapeutics.

Eph/ephrin expression in the adult rat visual system following localized retinal lesions: localized and transneuronal up-regulation in the retina and superior colliculus.

Following unilateral optic nerve section in adult PVG hooded rat, the axon guidance cue ephrin-A2 is up-regulated in caudal but not rostral superior colliculus (SC) and the EphA5 receptor is down-regulated in axotomised retinal ganglion cells (RGCs). Changes occur bilaterally despite the retino-collicular projection being mostly crossed. Here we investigate the dynamics of Eph/ephrin expression using in situ hybridization and semi-quantitative immunohistochemistry after localized retinal lesions. Unilateral krypton laser lesions to dorso-nasal retina ablated contralaterally projecting RGCs (DN group); ventro-temporal lesions ablated contralaterally and ipsilaterally projecting RGCs (VT group). Lesions of the entire retina served as controls (Total group). Results are compared to normal animals in which tectal ephrin-A2 and retinal EphA5 are expressed, respectively, as shallow ascending rostro-caudal and naso-temporal gradients. In both SCs of DN and Total groups, tectal ephrin-A2 was up-regulated caudally; in the VT group, expression remained normal bilaterally. Unilateral collicular ablation indicated that bilateral changes in ephrin-A2 expression are mediated via intercollicular pathways. EphA5 expression in the VT group was elevated in the intact nasal region of experimental retinae. For each experimental group, EphA5 expression was also elevated in nasal retina of the opposite eye, resulting in uniform expression across the naso-temporal axis. Up-regulation of ephrin-A2 in caudal, but not rostral, SC suggests the enhancement of developmental positional information as a result of injury. Bilateral increases in retinal EphA5 expression demonstrate that signals for up-regulation operate interocularly. The study demonstrates that signals regulating guidance cue expression are both localized and relayed transneuronally.

Dendrimer delivery of an anti-VEGF oligonucleotide into the eye: a long-term study into inhibition of laser-induced CNV, distribution, uptake and toxicity.

We have performed a long-term study into the use of a lipophilic amino-acid dendrimer to deliver an anti-vascular endothelial growth factor (VEGF) oligonucleotide (ODN-1) into the eyes of rats and inhibit laser-induced choroidal neovascularization (CNV). In addition, the uptake, distribution and retinal tolerance of the dendrimer plus oligonucleotide conjugates were examined. Analysis of fluorescein angiograms of laser photocoagulated eyes revealed that dendrimer plus ODN-1 significantly inhibited (P<0.05) the development of CNV for 4-6 months by up to 95% in the initial stages. Eyes similarly injected with ODN-1 alone showed no significant difference (P>0.05) in mean severity score at 2 months (2.86+/-0.09), 4 months (2.15+/-0.17) or 6 months (2.7+/-0.12) compared to the vehicle-injected controls. Furthermore, we showed that intravitreally injected ODN-1 tagged with 6-fam was absorbed by a wide area of the retina and penetrated all of the retinal cell layers to the retinal pigment epithelium. Ophthalmological examinations indicated that the dendrimers plus ODN-1 conjugates were well tolerated in vivo, which was later confirmed using immunohistochemistry, which showed no observable increase in antigens associated with inflammation. We conclude that the use of such dendrimers may provide a viable mechanism for the delivery of therapeutic oligonucleotides for the treatment of angiogenic eye diseases.

Entrainment of circadian rhythm to a photoperiod reversal shows retinal dystrophy in RPE65(-/-) mice.

Light entrainment of circadian rhythms is mediated by classical "visual" photoreceptors (rods and cones) as well as "nonvisual" photoreceptive elements (light-detecting cells that do not contribute to classical "vision"). This paper aimed to assess whether light entrainment of locomotor circadian rhythms in mice with impaired rods and cones differs from normal controls and whether this technique, alongside existing techniques, could be used to assess visual function. The study was primarily interested in differences between the entrainment of circadian rhythms of normal-sighted C57Bl/6J mouse and the C57Bl/RPE65 knockout mouse (RPE65(-/-)), although C3H/HeJ (rd/rd) mice were included as a preexisting model of retinal degeneration. Circadian rhythms of motor activity before and after a 12-h light reversal were assessed in custom-built cages that continuously monitored movement. The controls showed a significantly higher mesor and amplitude when compared to the RPE65(-/-) and rd/rd mice. Despite the loss of rods and cones, the RPE65(-/-) and rd/rd maintained a 24-h circadian rhythm entrained to light similar to controls and were capable of circadian reentrainment to a 12-h light reversal. Importantly, this light reentrainment of the circadian phase occurred at a significantly slower rate in the retinal degenerate models than in the controls. The RPE65(-/-) model demonstrates a retinal degenerate reentrainment phenotype when compared to the rd/rd model. It is suggested that these retinal degenerate mice retain the ability to detect light for the purposes of circadian rhythm entrainment. However, alterations of specific parameters of the circadian rhythm with loss of rods and cones may provide measures of loss of visual function (sight).

Potential long-term inhibition of ocular neovascularisation by recombinant adeno-associated virus-mediated secretion gene therapy.

Neovascularisation (NV) within the eye often results in visual loss. Vascular endothelial growth factor (VEGF) has been implicated in the development of ocular NV. Previous studies have shown that VEGF antagonists successfully suppressed retinal and choroidal NV in animal models. However, the systemic approach and transient nature of the delivery systems used in these studies hinder therapeutic application. To achieve stable and localised ocular anti-angiogenic therapy, we explored the use of recombinant adeno-associated virus (rAAV)-mediated secretion gene therapy (SGT). In this study, we generated a rAAV vector encoding soluble VEGF receptor 1, sFlt-1 (AAV-CMV.sflt) and determined its ability to inhibit cautery-induced corneal NV and laser-induced choroidal NV. Delivery of AAV-CMV.sflt into the anterior chamber resulted in transgene expression in the iris pigment epithelium and corneal endothelium, which reduced the development of corneal NV in the stroma of cauterised rats by 36% compared with cauterised control groups (P = 0.009). Subretinal delivery of AAV-CMV.sflt near the equator of the eye also suppressed choroidal NV at the laser lesions around the optic nerve by 19% (P = 0.002), indicating that there was diffusion of the secreted anti-angiogenic protein across the retina. Both results suggest that the long-term suppression of ocular NV is possible through the use of stable rAAV-mediated SGT.

Uptake dynamics and retinal tolerance of phosphorothioate oligonucleotide and its direct delivery into the site of choroidal neovascularization through subretinal administration in the rat.

This study aimed to investigate uptake dynamics and retinal tolerance of phosphorothioate oligonucleotides (PS-oligos) following subretinal injection. A fluorescent-labeled PS-oligo (FL-oligo) with random sequence was administered into the subretinal space of rat by transsclera-choroid-retinal pigment epithelium (RPE) injection at doses of 0.129, 1.29, and 12.9 microg in 2.0 microl solution. The uptake dynamics were evaluated by fundus fluorescent photography in real time and by fluorescence microscopy using flat mounts and cryosections. Immunophenotyping for CD4+, CD8+ cytotoxic lymphocytes, and CD68+ macrophages was performed to assess cellular infiltration in the retina. In addition, the FL-oligo was injected subretinally in a rat model of choroidal neovascularization (CNV) for direct delivery into the site of CNV. Subretinal administration of FL-oligo resulted in both dose-dependent and time-dependent distribution in the retina, where it accessed the RPE and all layers of the neuroretina. CD4+, CD8+ cytotoxic lymphocytes, and CD68+ macrophages were observed at the site of needle penetration. However, in areas far from the injection site where the FL-oligo appeared strongly, cellular infiltration was absent, and the retinal morphology was preserved very well. The FL-oligo was successfully delivered into the site of intense laser photocoagulation. It was predominantly localized to the RPE, macrophages, and some choroid cells and remained detectable for at least 56 days after injection. Our results demonstrate for the first time that subretinal injection efficiently introduced PS-oligo into the RPE and neuroretina with an acceptable level of safety. Subretinal administration of antiangiogenic oligonucleotides may hold great potential for the treatment of CNV.

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

BACKGROUND: 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. METHODS: A series of sequences were assessed in an in vitro screening system using retinal pigment epithelial (RPE) cells to demonstrate a reduction in VEGF. A targeted sequence was further investigated using an animal model of choroidal neovascularisation where a krypton laser was used to produce a wound healing response in the choroid and retina. The oligonucleotide was injected into the vitreous and the development of choroidal neovascularisation assessed using fluorescein angiography. RESULTS: The targeted sequence was shown in vitro to downregulate the VEGF produced by RPE cells grown under hypoxic conditions and when injected into laser treated eyes was shown to be preferentially taken up in the laser lesion. Fluorescein angiography demonstrated that the test oligonucleotide was successful in reducing laser-mediated choroidal neovascularisation. CONCLUSIONS: A sequence corresponding to the 5'UTR of the VEGF gene has provided encouraging results for the treatment of neovascularisation.

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.

Combined effect of cyclosporine and sirolimus on improving the longevity of recombinant adenovirus-mediated transgene expression in the retina.

OBJECTIVES: To reevaluate the longevity and intraocular safety of recombinant adenovirus (rAd)-mediated gene delivery after subretinal injection, and to prolong transgene expression through the combination of 2 synergistic immunosuppressants. METHODS: An rAd vector carrying green fluorescent protein (GFP) gene was delivered subretinally in the rat eye. The GFP expression was monitored in real time by fundus fluorescent photography. Intraocular safety was examined by observation of changes of retinal pigmentation, cell infiltration in virus-contacted area, immunophenotyping for CD4(+) and CD8(+) cytotoxic T lymphocytes, and CD68(+) macrophages, histologic findings, and dark-adapted electroretinography. Two synergistic immunosuppressants, cyclosporine and sirolimus, were used alone or in combination to prolong transgene expression by temporary immunosuppression. RESULTS: The GFP expression peaked on day 4, dramatically decreased on day 10, and was not detectable on day 14. The decreased GFP expression was coincident with cell infiltration in virus-contacted area. Immunostaining showed that the infiltrating cells were CD4(+) and CD8(+) cytotoxic T lymphocytes and CD68(+) macrophages. Clumped retinal pigmentation and decreased b wave of dark-adapted electroretinogram were observed at 3 to 4 weeks after injection. Histologic examination confirmed rAd-induced retinal degeneration. Transient immunosuppression by cyclosporine and sirolimus, either alone or in combination, improved transgene expression, with the combination being the most efficient. The combined immunosuppression attenuated but did not retard the rAd-induced retinal damage. CONCLUSIONS: Transgene expression mediated by rAd after subretinal delivery is short-term and toxic to the retina. Combination of cyclosporine and sirolimus may act as an immunosuppressive adjunct to prolong rAd-mediated gene transfer. CLINICAL RELEVANCE: The intraocular safety of rAd should be carefully considered before clinical trials are performed.

Kinetics of efficient recombinant adeno-associated virus transduction in retinal pigment epithelial cells.

The aim of this study was to investigate the premise that retinal pigment epithelial (RPE) cells are more permissive to recombinant adeno-associated virus (rAAV) transduction than other cells. We investigated the kinetics and mechanisms of rAAV transduction in RPE cells and found that the transduction efficiencies of cultured RPE cells HRPE51 and ARPE19 were significantly higher than those of 293 (P < 0.008) and HeLa (P < 0.025) cells. In addition, RPE cells reached maximum transduction efficiency at a much lower m.o.i. (m.o.i. 10) than 293 cells (m.o.i. 25). Competition experiments using 1 microg/ml heparin inhibited the high level of transduction in RPE cells by 30%, but additional heparin failed to reduce rAAV transduction further. Southern hybridization of low-molecular-weight DNA from transduced RPE cells indicated that 42% of single-stranded rAAV DNA was translocated into the nucleus by 2 h postinfection. By 6 h postinfection, double-stranded rAAV DNA was observed, which coincided with the onset of transgene expression. Southern and fluorescence in situ hybridization of total genomic DNA indicated that long-term transgene expression in RPE cells was maintained by the integration of rAAV into the cellular chromosome. Together, these results suggest that the high permissiveness of RPE cells is not related to the presence of heparan sulfate receptors or nuclear trafficking but may be due to an enhanced rate of second-strand synthesis and that integration in RPE cells is responsible for long-term transgene expression.

Synthetic hydrogels as carriers in antisense therapy: preliminary evaluation of an oligodeoxynucleotide covalent conjugate with a copolymer of 1-vinyl-2-pyrrolidinone and 2-hydroxyethyl methacrylate.

A major challenge of the antisense therapeutic strategies is the development of improved systems for the delivery of antisense oligodeoxynucleotides (AS ODNs) in order to enhance the cellular uptake, to assure a better efficiency in reaching the target tissue, and to provide sustained delivery over longer periods of time. Because the current methods for delivery (liposomes and cationic polymers) present some disadvantages, the attention was directed toward the use of neutral polymers as carriers for the AS ODNs. Based on our previous work on synthetic hydrogels for vitreous substitution, we developed a poly[1-vinyl-2-pyrrolidinone-co-(2-hydroxyethyl methacrylate)] hydrogel as a potential carrier for AS ODNs. We have previously demonstrated that such hydrogels are not cytotoxic, and they may have growth-promoting effects on cultured fibroblasts. This copolymer also has the advantage of being injectable. In this study, a specific AS ODN was synthesized and then covalently bound to the copolymer via carbodiimide coupling method. The resulting conjugate was subjected to in vitro release experiments over 46 days in the presence of bovine vitreous humor. Compared with the control (no enzyme present), a significant amount of covalently bound ODN was released from the ODN-hydrogel conjugate, suggesting the possibility of using such systems for the sustained delivery of AS ODNs.

Addition of a c-myc epitope tag within the VEGF protein does not affect in vitro biological activity.

The overexpression of vascular endothelial growth factor (VEGF) has been strongly implicated in diseases involving neovascularization. VEGF exists in as many as six different isoforms, each showing a unique pattern of tissue distribution and activity. To investigate the effect of individual VEGF isoform overexpression in neovascular disease models, we inserted c-myc epitope tags into the three VEGF isoforms expressed in retinal pigment epithelial cells, VEGF121, VEGF165, and VEGF189. We found that the 12-amino acid insertion between the receptor binding and heparin binding domains did not affect VEGF transcription, translation, or secretion. In addition, VEGF isoforms containing the c-myc epitope tag were able to stimulate endothelial cell proliferation as efficiently as non-tagged VEGF isoforms and they could be individually identified by Western blotting and immunocytochemistry using the c-myc epitope specific monoclonal antibody 9E10.

Antisense DNA technology.

Antisense DNA technology is a method to inhibit or downregulate the production of a target protein by using antisense DNA or RNA molecules. An antisense sequence is a DNA or RNA that is perfectly complementary to the target nucleotide sequence present in the cell. There are two possible mechanisms for an antisense effect. The method that relies on targeting of the mRNA is called the antisense strategy. When the double-stranded DNA or genes situated in the nucleus are targeted, the approach is called the antigene strategy. Whereas the antisense strategy is well established with several examples of in vitro and in vivo applications (1), the antigene approach is still in its infancy and our understanding of the mechanism involved is limited. The antisense strategy utilizes the ability of a 100% complementary DNA or RNA sequence to interlock or hybridize with the target mRNA thus inhibiting the translation of the target protein. This inhibition can be achieved either by blocking the binding sites for the 40S ribosomal subunit and for other translation initiation signals. Alternatively, the formation of a double-stranded DNA/RNA complex can render the RNA susceptible to RNase H digestion (2). The antigene approach is based on the binding of an antisense or sense DNA to the complimentary DNA sequence in the nucleus thus forming a triplex structure. This triplex prevents the transcription of the DNA coding sequence into mRNA (2).

Long-term real-time monitoring of adeno-associated virus-mediated gene expression in the rat retina.

PURPOSE: Previous studies have demonstrated that adeno-associated virus (AAV) efficiently transduced retinal pigmented epithelial (RPE) cells. The goal of this study was to further evaluate and characterize transgene expression within the RPE cells over time in vivo. METHODS: Adeno-associated virus-mediated gene transfer was monitored and quantified by retinal photography following subretinal injection of a recombinant AAV encoding the green fluorescent protein gene (rAAVCMV-gfp) into rat eyes. Retinal function of transduced rat eyes was measured by electroretinography. RESULTS: The maximum level of transgene expression was reached at 8 weeks postinjection followed by a gradual decrease throughout the experimental period. Interestingly, it was observed that while gfp expression was stable in some RPE cells, gfp fluorescence completely disappeared in other cells over the duration of the experiment. The expression of AAV-mediated gfp in RPE cells did not alter the retinal function for over 1 year CONCLUSIONS: These results confirm the importance of this direct visualization system to study vector transgene expression in vivo and support the use of AAV for diseases treatable by targeting RPE cells.

Controlled production of active cathepsin D in retinal pigment epithelial cells following adenovirus-mediated gene delivery.

The transduction of a low cathepsin D-producing retinal pigment epithelial cell line with a recombinant adenovirus, Ad.proCatD, carrying a viral promoter and the precursor form of the lysosomal enzyme cathepsin D, procathepsin D, led to the upregulation of proCatD expression. However, the resultant aspartic protease activity did not exceed that observed in normal primary human retinal pigment epithelial cells. Following the injection of Ad. proCatD into rat eyes, immunohistochemistry and Western blot analysis localized the expression of procathepsin D to the retinal pigment epithelial cell layer and to the sclera/choroid/retinal epithelial cell layers, respectively. This upregulation of procathepsin D expression was accompanied by a limited increase in aspartic protease activity. The injected eyes did not demonstrate any of the retinal changes that have been associated with the overproduction and secretion of active cathepsin D. Immunoelectronmicroscopy of Ad.proCatD-transduced retinal pigment epithelial cells demonstrated the presence of cathepsin D not only in cytoplasmic vesicles and lysosomes but also in the nucleoli and, less strongly, elsewhere in euchromatic regions of some 10% of cells. In spite of the upregulated expression of procathepsin D, the production of active cathepsin D in Ad.proCatD-transduced retinal pigment epithelial cells was strictly controlled. It is proposed that active cathepsin D production is controlled at the point of posttranslational modification by an intranuclear feedback mechanism initiated by the relative excess of procathepsin D in Ad. proCatD-transduced retinal pigment epithelial cells.

A novel immunoassay for the evaluation of rod outer segment digestion in cultured retinal pigment epithelial cells.

In this work a novel enzyme-linked immunoabsorbent assay quantifying residual rod outer segments in the medium of rod outer segment-challenged retinal pigment epithelial cells is described. A retinal pigment epithelial cell line (D407) that produces low level of cathepsin D, and a primary human retinal pigment epithelial cell culture (HRPE51) that has normal cathepsin D levels, were challenged with bovine rod outer segments. At 3 days post-challenge, the amount of undigested or residual bovine rod outer segments left in the culture medium was quantified by an enzyme-linked immunoabsorbent assay. An antibody raised against bovine rod outer segments, which had been purified and labelled with nitroiodophenyl haptens, was used in the assay. The sensitivity of the immunoassay was less than 10(2) bovine rod outer segments per mL and the signal followed a linear curve, saturating around 10(6) bovine rod outer segments per mL. HRPE51 cells had no residual bovine rod outer segments present in the medium following a challenge with 10(4) bovine rod outer segments per mL. In the medium of D407 cells, residual bovine rod outer segment levels were higher at all bovine rod outer segment concentrations when compared to the residual bovine rod outer segment levels in HRPE51 cells, suggesting that D407 cells have a lower digestive capacity. These results demonstrated that the immunoassay for detecting bovine rod outer segments is a sensitive and reliable technique that can be used to quantify the amount of residual bovine rod outer segments, following bovine rod outer segment challenge of retinal pigment epithelial cells.

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 Bruch's 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.

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

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 microg 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 microg of diclofenac/HA. RESULTS: Diclofenac/HA, 180, 90 microg/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 microg 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 microg 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 non-toxic 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.