Long-term efficacy of ciliary muscle gene transfer of three sFlt-1 variants in a rat model of laser-induced choroidal neovascularization.

Inhibition of vascular endothelial growth factor (VEGF) has become the standard of care for patients presenting with wet age-related macular degeneration. However, monthly intravitreal injections are required for optimal efficacy. We have previously shown that electroporation enabled ciliary muscle gene transfer results in sustained protein secretion into the vitreous for up to 9 months. Here, we evaluated the long-term efficacy of ciliary muscle gene transfer of three soluble VEGF receptor-1 (sFlt-1) variants in a rat model of laser-induced choroidal neovascularization (CNV). All three sFlt-1 variants significantly diminished vascular leakage and neovascularization as measured by fluorescein angiography (FA) and flatmount choroid at 3 weeks. FA and infracyanine angiography demonstrated that inhibition of CNV was maintained for up to 6 months after gene transfer of the two shortest sFlt-1 variants. Throughout, clinical efficacy was correlated with sustained VEGF neutralization in the ocular media. Interestingly, treatment with sFlt-1 induced a 50% downregulation of VEGF messenger RNA levels in the retinal pigment epithelium and the choroid. We demonstrate for the first time that non-viral gene transfer can achieve a long-term reduction of VEGF levels and efficacy in the treatment of CNV.

Non-viral gene therapy for GDNF production in RCS rat: the crucial role of the plasmid dose.

Glial cell line-derived neurotrophic factor (GDNF) is one of the candidate molecules among neurotrophic factors proposed for a potential treatment of retinitis pigmentosa (RP). It must be administered repeatedly or through sustained releasing systems to exert prolonged neuroprotective effects. In the dystrophic Royal College of Surgeon's (RCS) rat model of RP, we found that endogenous GDNF levels dropped during retinal degeneration time course, opening a therapeutic window for GDNF supplementation. We showed that after a single electrotransfer of 30 µg of GDNF-encoding plasmid in the rat ciliary muscle, GDNF was produced for at least 7 months. Morphometric, electroretinographic and optokinetic analyses highlighted that this continuous release of GDNF delayed photoreceptors (PRs) as well as retinal functions loss until at least 70 days of age in RCS rats. Unexpectedly, increasing the GDNF secretion level accelerated PR degeneration and the loss of electrophysiological responses. This is the first report: (i) demonstrating the efficacy of GDNF delivery through non-viral gene therapy in RP; (ii) establishing the efficacy of intravitreal administration of GDNF in RP associated with a mutation in the retinal pigment epithelium; and (iii) warning against potential toxic effects of GDNF within the eye/retina.

Toxoplasma gondii: flat-mounting of retina as a new tool for the observation of ocular infection in mice.

Ocular toxoplasmosis is the principal cause of posterior uveitis and a leading cause of blindness. Animal models are required to improve our understanding of the pathogenesis of this disease. The method currently used for the detection of retinal cysts in animals involves the observation, under a microscope, of all the sections from infected eyes. However, this method is time-consuming and lacks sensitivity. We have developed a rapid, sensitive method for observing retinal cysts in mice infected with Toxoplasma gondii. This method involves combining the flat-mounting of retina - a compromise between macroscopic observation and global analysis of this tissue - and the use of an avirulent recombinant strain of T. gondii expressing the Escherichia coli beta-galactosidase gene, visually detectable at the submacroscopic level. Single cyst unilateral infection was found in six out of 17 mice killed within 28 days of infection, whereas a bilateral infection was found in only one mouse. There was no correlation between brain cysts number and ocular infection.

Influence of age on retinochoroidal healing processes after argon photocoagulation in C57bl/6j mice.

PURPOSE: To analyze the influence of age on retinochoroidal wound healing processes and on glial growth factor and cytokine mRNA expression profiles observed after argon laser photocoagulation. METHODS: A cellular and morphometric study was performed that used 44 C57Bl/6J mice: 4-week-old mice (group I, n=8), 6-week-old mice (group II, n=8), 10-12-week-old mice (group III, n=14), and 1-year-old mice (group IV, n=14). All mice in these groups underwent a standard argon laser photocoagulation (50 microm, 400 mW, 0.05 s). Two separated lesions were created in each retina using a slit lamp delivery system. At 1, 3, 7, 14, 60 days, and 4 months after photocoagulation, mice from each of the four groups were sacrificed by carbon dioxide inhalation. Groups III and IV were also studied at 6, 7, and 8 months after photocoagulation. At each time point the enucleated eyes were either mounted in Tissue Tek (OCT), snap frozen and processed for immunohistochemistry or either flat mounted (left eyes of groups III and IV). To determine, by RT-PCR, the time course of glial fibrillary acidic protein (GFAP), vascular endothelial growth factor (VEGF), and monocyte chemotactic protein-1 (MCP-1) gene expression, we delivered ten laser burns (50 microm, 400 mW, 0.05 s) to each retina in 10-12-week-old mice (group III', n=10) and 1-year-old mice (group IV', n=10). Animals from Groups III' and IV' had the same age than those from Groups III and IV, but they received ten laser impacts in each eye and served for the molecular analysis. Mice from Groups III and IV received only two laser impacts per eye and served for the cellular and morphologic study. Retinal and choroidal tissues from these treated mice were collected at 16 h, and 1, 2, 3, and 7 days after photocoagulation. Two mice of each group did not receive photocoagulation and were used as controls. RESULTS: In the cellular and morphologic study, the resultant retinal pigment epithelium interruption expanse was significantly different between the four groups. It was more concise and smaller in the oldest group IV (112.1 microm+/-11.4 versus 219.1 microm+/-12.2 in group III) p<0.0001 between groups III and IV. By contrast, while choroidal neovascularization (CNV) was mild and not readily identifiable in group I, at all time points studied, CNV was more prominent in the (1-year-old mice) Group IV than in the other groups. For instance, up to 14 days after photocoagulation, CNV reaction was statistically larger in group IV than in group III ((p=0.0049 between groups III and IV on slide sections and p<0.0001 between the same groups on flat mounts). Moreover, four months after photocoagulation, the CNV area (on slide sections) was 1,282 microm(2)+/-90 for group III and 2,999 microm(2)+/-115 for group IV (p<0.0001 between groups III and IV). Accordingly, GFAP, VEGF, and MCP-1 mRNA expression profiles, determined by RT-PCR at 16 h, 1, 2, 3, and 7 days postphotocoagulation, were modified with aging. In 1-year-old mice (group IV), GFAP mRNA expression was already significantly higher than in the younger (10-12 week) group III before photocoagulation. After laser burns, GFAP mRNA expression peaked at 16-24 h and on day 7, decreasing thereafter. VEGF mRNA expression was markedly increased after photocoagulation in old mice eyes, reaching 2.7 times its basal level at day 3, while it was only slightly increased in young mice (1.3 times its level in untreated young mice 3 days postphotocoagulation). At all time points after photocoagulation, MCP-1 mRNA expression was elevated in old mice, reaching high levels of expression at 16 h and day 3 respectively. CONCLUSIONS: Our results were based on the study of four different age groups and included not only data from morphological observations but also from a molecular analysis of the various alterations of cytokine signaling and expression. One-year-old mice demonstrated more extensive CNV formation and a slower pace of regression after laser photocoagulation than younger mice. These were accompanied by differences in growth factors and cytokine expression profiles indicate that aging is a factor that aggravates CNV. The above results may provide some insight into possible therapeutic strategies in the future.

PlGF-1 and VEGFR-1 pathway regulation of the external epithelial hemato-ocular barrier. A model for retinal edema.

VEGF is considered as an important factor in the pathogenesis of macular edema. VEGF induces the rupture of the blood retinal barrier and may also influence the retinal pigment epithelial (RPE) outer retinal barrier. The aim of this work was to analyze the influence of the VEGF receptor pathways in the modulation of the RPE barrier breakdown in vitro and in vivo. The ARPE19 human junctions in culture are modulated by VEGF through VEGFR-1 but not through VEGFR-2. PlGF-1, that is a pure agonist of VEGFR-1, is produced in ARPE-19 cells under hypoxic conditions and mimics VEGF effects on the external retinal barrier as measured by TER and inulin flux. In vivo, the intravitreous injection of PlGF-1 induces a rupture of the external retinal barrier together with a retinal edema. This effect is reversible within 4 days. VEGF-E, that is a pure agonist of VEGFR-2, does not induce any acute effect on the RPE barrier. These results demonstrate that PlGF-1 can reproduce alterations of the RPE barrier occurring during diabetic retinopathy.

[Distribution of transferrin and transferrin receptor of the type 1 in the process of formation of the rat eye retina in early postnatal ontogenesis].

By the method of indirect immunohistochemistry, distribution of transferrin and of transferrin receptor of the type (TFR1) was studied in the formed rat eye retina at the period of early postnatal ontogenesis (from birth to opening of eyelids). It has been established that the character of distribution of these proteins and intensity of specific staining change dependent on the retina formation stage. Retina of the newborn rat is characterized by diffuse transferrin distribution in nuclear retina layers (in the neuroblast layer--NBL) and in the ganglionic cell layer (GCL) as well as in the eye pigment epithelium (PE); relative immunoreactivity to transferrin is not high. At the 5th postnatal day, immunoreactivity to transferrin is maximal and is revealed both in nuclear and in plexiform layers of retina and in the eye PE, the greatest signal being characteristic of NBL. At the 10th postnatal day the transferrin signal intensity in retina decreases, specific staining is revealed in GCL, PE, and in the area of formed outer segments of photoreceptors. At the 15th postnatal day, transferrin is revealed in GCL, in outer and inner photoreceptor segments and in the eye PE. TFR1 is present in all retinal layers at all stages of the retina formation; the relative immunoreactivity to TFR1 sharply rises beginning from the 10th postnatal day; correlation between distribution of transferrin and TFR1 is detected in the entire retina of newborn rats as well as in the external retina area at subsequent stages of its development. A possible role of transferrin at various stages of formation of retinal is discussed.

Two distinct signalling pathways are involved in FGF2-stimulated proliferation of choriocapillary endothelial cells: a comparative study with VEGF.

In the retina, angiogenesis is an important component of normal physiological events such as embryonic vascular development. It is also involved in pathological processes including diabetic retinopathies and age-related macular degeneration, and tumour growth such as choroidal melanoma. Fibroblast growth factor (FGF) 2 and vascular endothelial cell growth factor (VEGF) are the two major angiogenic factors in the retina. We investigated the mechanism of proliferation and the regulation of the mitogenic properties of FGF2 and VEGF in cultures of chorocapillary endothelial cells (CEC). FGF2 is a strong mitogen for CEC and induced a 2.5-fold increase in cell proliferation after 4 days in culture in the absence of serum. In contrast, VEGF is a poor mitogen for CEC. FGF2, but not VEGF induces a large activation of MEK1, ERK1/2 and P90(RSK) during CEC proliferation. Pharmacological inhibition of Ras processing, and of MEK1 and ERK1/2 activation reduced only by 50% FGF2-induced cell proliferation, suggesting that there is another signalling pathway for CEC proliferation. Pharmacological inhibition of the PI 3-Kinase also inhibits by half FGF2-induced CEC proliferation. FGF2 stimulates the activation of the PI 3-K, P70(S6K) and Akt. Inhibition of both ERK1/2 and PI 3-K activities suppressed FGF2-induced CEC proliferation, demonstrating that CEC proliferation requires both ERKs and PI 3-K pathways. These data on the molecular mechanism and signalling may have important implications for providing more selective methods for anti-angiogenic and anti-tumoural therapy.

QRI, a retina-specific gene, encodes an extracellular matrix protein exclusively expressed during neural retina differentiation.

Neural retina development results from growth arrest of neuroectodermal precursors and differentiation of postmitotic cells. The QRI gene is specifically expressed in Müller retinal glial cells. Its expression coincides with the stage of withdrawal from the cell cycle and establishment of differentiation and is repressed upon induction of retinal cell proliferation by the v-src gene product. In this report, we show that the QR1 gene encodes several glycosylated proteins that are secreted and can either associate with the extracellular matrix or remain diffusible in the medium. By using pulse-chase experiments, the 100-103 kDa forms seem to appear first and are specifically incorporated into the extracellular matrix, whereas the 108 and 60 kDa polypeptides appear later and are detected as soluble forms in the culture medium. We also report that expression of the QR1 gene is developmentally regulated in the chicken. Its mRNA is first detectable at embryonic day 10, reaches a maximal level at embryonic day 15 and is no longer detected at embryonic day 18. Immunolocalization of the QR1 protein in chicken retina sections during development shows that expression of the protein parallels the differentiation pattern of post-miotic cells (in particular Müller cells and rods), corresponding to the two differentiation gradients in the retina: from the ganglion cell layer to the inner nuclear layer and outer nuclear layer, and from the optic nerve to the iris. At embryonic day 10, expression of the QR1 protein(s) is restricted to the optic nerve region and the inner nuclear layer, colocalizing with Müller cell bodies. As development proceeds, QR1 protein localization spreads towards the iris and towards the outer nuclear layer, following Müller cell elongations towards the photoreceptors. Between embryonic days 16 and 18, the QR1 protein is no longer detectable in the optic nerve region and is concentrated around the basal segment of the photoreceptors in the peripheral retina. Our results suggest a role for the QR1 gene product in the process of growth arrest and establishment of photoreceptor differentiation.

Localization of RIHB (retinoic acid-induced heparin-binding factor) transcript and protein during early chicken embryogenesis and in the developing wing.

Previously, we isolated an avian protein which we named retinoic acid induced heparin binding factor (RIHB). RIHB is a 121 amino acid secreted polypeptide, rich in basic and cysteine residues (Vigny et al., Eur. J. Biochem. 186: 733-740, 1989). Northern blot analysis indicates that the RIHB gene is transiently expressed during embryogenesis (Urios et al., Biochem. Biophys. Res. Com. 175:617-624, 1991). Here we present an investigation of RIHB expression during early chicken embryogenesis by in situ hybridization and immunofluorescence studies. In the 3-day embryo (stage 20-21), the RIHB transcript is observed throughout the embryo, with the notable exception of the neural tube. At this stage the protein can be visualized in almost all of the basement membranes and around many types of cells. The localization of the RIHB protein does not strictly parallel that of its messenger. Between days 3 and 11 we focused our attention on wing development. The level of both the mRNA and protein decreases during this period but the disappearance is not uniform. The level of both the mRNA and protein decreases during this period but the disappearance is not uniform. The transcript becomes progressively restricted to epithelia and regions surrounding the forming cartilage. In contrast to the transcript, the protein accumulates in the epithelial basement membrane and, interestingly, in the central part of the embryonic cartilage (diaphysis) but not in the distal parts (epiphysis). These data are discussed in relation to the putative role(s) of RIHB in development.

Developmental regulation of acidic fibroblast growth factor (aFGF) expression in bovine retina.

Acidic fibroblast growth factor (aFGF) is a signalling molecule implicated in a wide variety of biological processes such as cell growth, differentiation and survival. It has been purified from bovine retina. The present study was carried out to detect which cells in the bovine retina expressed aFGF at the different stages of embryonic and post-natal development. The specific aFGF mRNA and protein were detected by in situ hybridization employing riboprobes and immunocytochemistry using affinity purified polyclonal human recombinant aFGF antibodies respectively. No signal was detected by either technique until 4-5 months and then there was progressive expression of aFGF with terminal morphogenesis of the retina. By 8-9 months of embryonic development, nuclei of the 3 neuronal layers (ganglion cell layer, inner and outer nuclear layers) were all uniformly and intensely labeled. A slight labeling of the pigmented epithelium of the retina was also visible throughout development and maturation. These results showed a good correlation between message and protein expression in these cell types. In contrast, glial cells in the nerve fiber layer and vascular endothelial cells displayed a nuclear immunostaining for the protein in the absence of message. These data suggest that aFGF plays a role in the late steps of retinal differentiation by autocrine and paracrine mechanisms.

Analysis of the rat lens epithelial cell nucleus during ageing by scanning cytophotometry.

Nuclei of rat lens epithelial cells were stained with eosin and hematoxylin in order to investigate by cytophotometric analysis the effects of cellular ageing on their morphologic parameters and chromatin compaction. Our results demonstrate a decrease in the size of the nucleus without modification in their shape as a function of age. Older nuclei also have more condensed and heterogeneously distributed chromatin than young nuclei. The homogeneous population of nuclei in the young epithelium gave rise to a highly heterogeneous population in the old animal for both morphometric and densitometric parameters. In very old rats a small percentage of the cell nuclei retained young characteristics. These data support an earlier observation that growth of the lens epithelial cells declines with age.

Analysis of the changes in the morphology and staining density characteristics of cartilagenous cell nuclei from Triturus cristatus during aging and regeneration.

The modifications in the morphology and staining intensity of cell nuclei reflect their physiologic and functional state. In order to appreciate the variations in the metabolic activity of hind limb chondrocytes from Triturus cristatus during aging and regeneration, we have studied the nuclear size and shape, the DNA concentration, and the distribution and degree of condensation of the chromatin. This study was carried out using scanning cytophotometry on semithin 1 micrometer sections, Feulgen-stained for DNA, which is the major nuclear component. During aging, the nuclei decrease in size, becoming irregular in shape. The concentration of DNA increases and the chromatin distribution becomes more heterogeneous with an increase in condensed chromatin and a subsequent decrease in the diffuse chromatin fraction. During regeneration, however, these modifications are reversed whatever the age of the animal, but even more noticeably when the animal is old. The changes that occur in the chromatin fractions defined according to their state of condensation are discussed from a functional point of view.

Iron, ferritin, transferrin, and transferrin receptor in the adult rat retina.

PURPOSE: The retina and other tissues need iron to survive. However, the normal iron metabolism in rodent retinas had not been characterized. This study was intended to investigate iron and iron homeostasis protein (ferritin, transferrin [Tf] and transferrin receptor [Tf-R]) distribution in 20- to 55-day-old rat retinas. METHODS: Iron was revealed on retinal sections directly by proton-induced x-ray emission (PIXE) and indirectly by electron microscopy (EM). Ferritin, Tf, and Tf-R proteins were localized by immunohistochemistry. Transferrin expression was localized by in situ hybridization (ISH). Transferrin and ferritin proteins and mRNA were analyzed by Western blot analysis and reverse transcription-polymerase chain reaction (RT-PCR), respectively. RESULTS: Iron is widely and unevenly distributed throughout the adult rat retina. The highest concentration was observed by PIXE in the choroid and the retinal pigmented epithelial cell (RPE) layer, and in inner segments of photoreceptors (IS). Outer segments of photoreceptors (OS) also contain iron. EM studies suggested the presence of iron inclusions inside the photoreceptor discs. Choroid, RPE, and IS showed a strong immunoreactivity for ferritin. Transferrin accumulated mainly in the IS and OS areas and in RPE cells but can also be detected slightly in retinal capillaries. Western blot analysis for Tf and ferritin confirmed their presence in the adult neural retina. By RT-PCR, H- and L-chains of ferritin and Tf mRNAs were expressed in neural retina, but the main sites of Tf synthesis observed by ISH were the RPE and choroid cell layers. Tf-R immunoreactivity was detected in the ganglion cell layer, inner nuclear layer, outer plexiform layer, IS, RPE, and choroid. These results were similar for all stages studied. CONCLUSIONS: For the first time, the present study characterized both iron and iron homeostasis proteins in rodent retinas. In the outer retina, iron and ferritin shared the same distribution patterns. In contrast, Tf, mainly synthesized by RPE cells and detected in OS and IS areas, probably helps to transport iron to photoreceptors through their Tf-R. This is a likely pathway for filling iron needs in the outer retina.

Binding of basic fibroblast growth factor to normal and neovascularized rabbit cornea.

The labeling pattern of frozen sections of rabbit cornea incubated with radioiodinated basic fibroblast growth factor (bFGF) was investigated in normal corneas and prostaglandin-induced neovascularized corneas by autoradiography followed by image analysis. 125I-bFGF binds to Bowman's, Descemet's, and vascular basement membranes in a dose-dependent manner. The specificity of the binding of bFGF to basement membrane was demonstrated by the following experiments: 1) an excess of unlabeled growth factor displaced the labeling; 2) histones did not modify the labeling; and 3) 2 M NaCl washing and enzymatic treatment with heparitinase prevented binding of labeled growth factor without apparent destruction of the overall structure of the basement membrane. Our results suggest that bFGF binds to the heparan sulfate proteoglycan of basement membranes. Both normal limbal vessels and the newly formed corneal vessels exhibited the same type of labeling but with different intensities, according to the degree of maturation of the new vessels. bFGF binding also is located clearly on the endothelial cells in both types of vessels. This second binding site could correspond to the high affinity receptors on the cell surface and suggests a direct interaction of bFGF with endothelial cells during new vessel formation.

Long term light-induced retinal degeneration in the miniature pig.

In developing a model of slow light-induced retinal degeneration, ten miniature pigs were submitted to constant lighting for a period ranging from one to three months. Post-lighting survival time ranged from zero to two months. Control and illuminated animals were examined for pupillary reflex, underwent fundus examination and an electroretinogram. After euthanasia, retinas were processed for histology with measure of outer nuclear layer thickness. All animals illuminated one or more months had pupillar reflex alteration. Mean outer nuclear thickness was 24.12 microns in the control and ranged from 18.36 to 21.45 microns in illuminated animals (mean reduction 20%). Despite the pigmentation of miniature pigs, consistent results were obtained in the absence of pharmacologic pupil dilation.

Protection against light-induced retinal degeneration by an inhibitor of NO synthase.

The existence of nitric oxide synthase (NOS) in retinal rod outer segments and pigmented epithelial cells suggests that NO in excess could impair the interaction between these cells, resulting in photoreceptor degeneration. To test this hypothesis, NG-nitro-L-arginine methyl ester (L-NAME), an NOS inhibitor, was intraperitoneally injected daily into rats subjected to constant illumination for 7 days in order to destroy their photoreceptors. By measuring photoreceptor nuclear layer thicknesses, we found that L-NAME partially protects (by up to 35%) against the degeneration of photoreceptors and acts to maintain their organization. Thus NO may be involved in the process by which photoreceptor degeneration results from constant illumination of the retina.

Immunohistochemical analysis of fibroblast growth factor receptor in bovine retina.

The distribution of fibroblast growth factor (FGF) receptor in bovine retina was established using a polyclonal antibody against the extracellular domain of this receptor. Different conditions of tissue fixation and development of the secondary antibody were tested. The ability of the antiserum to map precisely the receptor was obtained on fresh-frozen sections which had been treated with paraformaldehyde prior to incubation with this antiserum. Positive staining was confined mainly to the synaptic and ganglion axon layers. These results suggest that the FGF receptor might act in the transmitter stability, and the plasticity of synapses and ganglion cell axons in adult retina.

Localization of acidic fibroblast growth factor (aFGF) mRNA in mouse and bovine retina by in situ hybridization.

Acidic fibroblast growth factor (aFGF) mRNA has been detected in adult mouse or bovine retina by in situ hybridization with bovine aFGF cDNA clones. It is localized on ganglion cell layer, inner nuclear layer, photoreceptors and slightly on pigmented epithelium. This synthesis of aFGF in highly specialized retinal cell types is discussed in the framework on current views about the role of FGF in retinal cell biology.

Acidic fibroblast growth factor (aFGF)-like immunoreactivity in the optic nerve.

Acidic fibroblast growth factor (aFGF)-like immunoreactivity was examined in the optic nerves of 1- to 25-month-old Wistar rats, 0.5- to 7-year-old bovine animals and normal human adults (24 and 35 years old), using cryostat sections incubated with a rabbit polyclonal antibody specific for aFGF. The immunoreactivity was associated with glial cells, and was localized predominantly in the nucleus. The presence of endogenous aFGF in the optic nerve of adult subjects and 'old' rats suggests that aFGF could play a role in the survival of retinal ganglion cells and their axons during aging.

Colocalization of fibroblast growth factor binding sites with extracellular matrix components in normal and keratoconus corneas.

Basic and acidic fibroblast growth factor (bFGF, aFGF) binding sites were determined in frozen sections of normal and keratoconus corneas. After incubation with I-125 radiolabelled growth factors, corneal binding sites were revealed by autoradiography. The growth factors were localized mainly to Descemet's membrane and to the epithelial basement membrane. FGF binding sites were generally similar in normal and keratoconus corneas. The binding specificity was demonstrated by competitive inhibition experiments with an excess of unlabelled growth factors. The binding sites were sensitive to pretreatment of the corneal sections with heparitinase. We have attributed FGF's basement membrane affinity to one of its constituents, proteoheparan sulfate. Proteoheparan sulfate, laminin, collagen type IV, and fibronectin were all revealed by immunofluorescent techniques. While keratoconus cornea stroma had less laminin but more fibronectin than normal corneas the main difference lied in type IV collagen which was overexpressed in keratoconus epithelium.