Upregulation of vascular endothelial growth factor (VEGF) in the retinas of transgenic mice overexpressing interleukin-1beta (IL-1beta) in the lens and mice undergoing retinal degeneration.

IL-1beta is a pro-inflammatory agent associated with angiogenesis and increased vascular permeability. To determine whether IL-1beta elicits these responses through an upregulation of VEGF, transgenic mice that overexpress IL-1beta in the lens were evaluated at various time points for the localization of VEGF, the location and extent of blood-retinal barrier (BRB) breakdown, and the origin and extent of neovascularization (NV). In homozygous and heterozygous transgenic mice, but not controls, intense VEGF immunoreactivity was scattered throughout the retina at postnatal days 5-7 (P5-7), just after the onset of inflammatory cell infiltration. VEGF staining in the retina remained widespread, but weak from P9-15. Beginning at P15, the intensity of VEGF immunoreactivity achieved a second peak, which it maintained through adulthood. This peak coincided with significant retinal destruction due to massive inflammation. The onset of BRB breakdown coincided with the upregulation of VEGF (P5-7) and widespread BRB breakdown was demonstrated from about P9. From P9-12, aggregates of cells positive for Griffonia simplicifolia isolectin-B4, a marker for vascular endothelial cells, formed on the retinal surface. These cells migrated into the retina at P12-15 with the more superficial cells forming a network of vessels and the deeper cells remaining in small clusters, thus demonstrating that NV occurs much later than BRB breakdown. Non-transgenic FVB/N mice, which undergo retinal degeneration beginning at about P9, also demonstrate the latter peak of VEGF upregulation and the accompanying BRB breakdown, but not the early upregulation. VEGF immunostaining of transgenic and non-transgenic mouse retinas was eliminated by pre-incubation of the VEGF antibodies with VEGF peptide. The data suggest that the early peak of VEGF upregulation (P5-7) and its accompanying BRB breakdown is due to IL-1beta expression and is likely to be dependent on inflammatory cell infiltration. The latter peak appears to be related to retinal destruction.

Vascular endothelial growth factor (VEGF), transforming growth factor-beta (TGFbeta), and interleukin-6 (IL-6) in experimental herpesvirus retinopathy: association with inflammation and viral infection.

Experimental herpesvirus retinopathy presents a unique model of a transient inflammatory response in the virus-injected eye and subsequent acute retinal necrosis and chronic inflammation in the contralateral eye. For 6 days after infection, VEGF, TGFbeta1, and TGFbeta2 were associated only with inflammatory cells in the injected eye. By 6 days (after viral antigens were no longer detected), VEGF and TGFbeta2 were upregulated in retinas of injected eyes until 8-10 days. In contralateral eyes, VEGF was first demonstrated in the retina at 6-7 days (prior to the appearance of viral antigens) and TGFbeta2 at 7-8 days. Staining for these factors was also evident around areas of necrosis. The VEGF receptor, flt-1, was associated with ganglion cells and the inner nuclear layer of normal and experimental mice and it was also demonstrated around areas of necrosis. Another VEGF receptor, flk-1, was localized to Muller cell processes and the outer plexiform layer in normal and experimental mice. Coincident with VEGF upregulation in the retinas of herpesvirus-1 injected mice, there was increased flk-1 in ganglion cells and the inner and outer nuclear layers. IL-6 was associated with Muller cell endfeet in normal mice. Following unilateral intraocular inoculation, IL-6 spread along the MUller cell processes and some astrocytes demonstrated IL-6 in both eyes at 6-8 days. The present study demonstrates that intraocular inoculation of herpesvirus is sufficient to induce VEGF, flk-1, TGFbeta2, and IL-6 in the retinas of injected and contralateral eyes. Further investigation of common signaling pathways for these factors during responses to viral infection and the development of acute retinal necrosis could provide information useful for therapeutic intervention in human herpesvirus retinopathy.

Blood-retinal barrier breakdown in experimental coronavirus retinopathy: association with viral antigen, inflammation, and VEGF in sensitive and resistant strains.

Intraocular coronavirus inoculation results in a biphasic retinal disease in susceptible mice (BALB/c) characterized by an acute inflammatory response, followed by retinal degeneration associated with autoimmune reactivity. Resistant mice (CD-1), when similarly inoculated, only develop the early phase of the disease. Blood-retinal barrier (BRB) breakdown occurs in the early phase in both strains, coincident with the onset of inflammation. As the inflammation subsides, the extent of retinal vascular leakage is decreased, indicating that BRB breakdown in experimental coronavirus retinopathy (ECOR) is primarily due to inflammation rather than to retinal cell destruction. Vascular endothelial growth factor (VEGF) is upregulated only in susceptible mice during the secondary (retinal degeneration) phase.

EMAP cytokine expression in developing retinas of normal and retinal degeneration (rd) mutant mice.

Endothelial-monocyte-activating polypeptide (EMAP) is a proinflammatory cytokine and a mediator of programmed endothelial cell death. To gain insight into its possible functions during retinal development and degeneration, the cellular distribution of EMAP protein was compared in control and retinal degeneration (rd) mice. EMAP immunoreactivity was confined to the ganglion cell layer (GCL) and the inner nuclear layer (INL). There were significant differences in the intensity of EMAP labeling in the GCL and the INL when comparing control and rd mouse retinas. Rd retinas contain much more EMAP immunoreactivity in the GCL and the INL than the control retinas at postnatal day 14, which is the time point immediately after the onset of the degeneration of the rd retina. Histopathologic examination showed no significant abnormalities in the GCL and INL in the rd mouse, despite a great degree of photoreceptor cell death from P12 to P18. Light and electron microscopic studies immunolocalize EMAP protein to the cytoplasm of retinal ganglion cells, amacrine cells, and horizontal cells. The data suggests that EMAP is synthesized and accumulated as an intracellular precursor protein that has a functional role in translation and protein synthesis as a cofactor for tRNA synthetase. The increased expression of EMAP precursor levels in rd mouse retina may reflect the enhanced rate of translation and protein synthesis in the production of endogenous factors that promote survival in the GCL and INL.

Photoreceptor-specific expression of platelet-derived growth factor-B results in traction retinal detachment.

Expression of platelet-derived growth factor (PDGF)-A and PDGF-B is increased in patients with proliferative retinopathies in which traction retinal detachments occur. Previous studies have demonstrated that increased expression of PDGF-A in the retina of transgenic mice results in retinal gliosis due to proliferation of astrocytes with different retinal phenotypes based on the time of onset and location of the PDGF-A production. In this study, we investigated the effects of PDGF-B in the retina using gain-of-function transgenic mice that express PDGF-B in photoreceptors. These mice show proliferation of astrocytes, pericytes, and, to a lesser extent, endothelial cells, resulting in ectopic cells on the surface and extending into the retina. The sheets of cells exert traction on the retina resulting in traction retinal detachments similar to those seen in humans with proliferative retinopathies. These studies suggest that PDGF-B has more dramatic effects in the retina than PDGF-A, because it acts on additional cell types, in particular on pericytes, which have a highly developed contractile apparatus. These studies in the retina suggest a means that could be used in other tissues throughout the body to achieve graded PDGF effects. They also provide a new model of traction retinal detachment that can be used to investigate new treatments for patients with proliferative retinopathies.

Sensitivity of different vascular beds in the eye to neovascularization and blood-retinal barrier breakdown in VEGF transgenic mice.

Neovascularization (NV) causes visual deficits in ocular disorders such as diabetic retinopathy, age-related macular degeneration, and retinopathy of prematurity. An understanding of the angiogenic factors promoting this abnormal vascular growth is necessary to devise a therapeutic approach to inhibit NV. One factor known to promote NV is vascular endothelial growth factor (VEGF), which can also induce a breakdown of the blood-retinal barrier (BRB) leading to macular edema, another major cause of visual loss in a variety of ocular disorders. To investigate the role of VEGF on ocular NV, transgenic mice have been produced that over-express VEGF in the photoreceptors under control of the rhodopsin promoter. Eyes from these mice and from littermates not expressing the transgene were examined using immunohistochemistry, griffonia simplicifolia isolectin-B4 (GSA) staining to clearly visualize vessels, and electron microscopy. Levels of transgene expression were determined by the polymerase chain reaction. In normal mice, retinal vessels are organized into a superficial and a deep capillary bed with some vessels forming a shunt between both beds. In a transgenic line of mice that over-expresses VEGF (V-6), NV originates from the deep capillary bed at about postnatal day 10 (P10) and extends through the photoreceptor layer to form vascular complexes in the subretinal space with BRB breakdown occurring only in the area of NV. The superficial capillary bed and the choroidal vasculature are unaffected. In another line of transgenic mice with a higher expression rate of VEGF (V-24), photoreceptor degeneration begins at P7-8, soon after the onset of transgene expression, without widespread NV, as was observed in V-6 mice. In conclusion, overexpression of VEGF in transgenic mice is sufficient to cause retinal NV, but only the deep capillary bed is responsive. Increasing the expression of VEGF does not necessarily increase the amount of NV. A better understanding of the specific factors and conditions that result in a particular pattern of ocular NV may provide clues regarding the pathogenesis of ocular neovascular disease.

Experimental models of growth factor-mediated angiogenesis and blood-retinal barrier breakdown.

Following chronic ischemia, vascular endothelial growth factor (VEGF) is induced primarily in the ganglion cell layer of the retina. This often results in neovascularization (NV) that originates from the vascular bed closest to the ganglion cell layer. To study the effects of VEGF, independent lines of transgenic mice that express VEGF in the lens and in the retina have been generated. Expression in the lens results in excessive proliferation and accumulation of angioblasts and endothelial cells in proximity to the lens. However, VEGF expression is not sufficient to direct blood vessel organization or maturation in the prenatal mouse. Abnormal vessels do form on the retinal surface, but not until the second postnatal week. In transgenic mice expressing VEGF in the photoreceptors, NV originates from the deep capillary bed--the vascular bed closest to the photoreceptors. NV is accompanied by localized blood-retinal barrier breakdown. NV is also induced in PDGF-B transgenic mice. PDGF-B expression in the lens occurs prenatally and, during this time, mainly affects the perilenticular vessels. Postnatally, transgenic mice expressing PDGF-B in the lens or photoreceptors show a similar phenotype. In both models, a highly vascularized cell mass containing endothelial cells, pericytes, and glia forms in the superficial retina, and the formation of the deep capillary bed is inhibited. The phenotype suggests that an additional factor is necessary for the maturation and penetration of vascular endothelial cells into the retina to form the deep capillary bed.

Cellular mechanisms of blood-retinal barrier dysfunction in macular edema.

PURPOSE: To determine the mechanism of blood-retinal barrier (BRB) dysfunction in human and experimental specimens using immunocytochemistry. METHODS: Extravascular albumin was localized in clinical specimens and retinas from transgenic mice that overexpress vascular endothelial growth factor (VEGF) in the photoreceptors. Transgenic mouse retinas were also labeled with Griffonia simplicifolia isolectin-B4 (GSA), a lectin that binds to endothelial cells. RESULTS: The BRB is established by the presence of tight junctions between the retinal vascular endothelial (RVE) cells and the RPE cells and by a paucity of intraendothelial cell vesicles. When BRB breakdown occurs in human ocular disorders such as diabetic retinopathy, retinitis pigmentosa, or cystoid macular edema, staining for extravascular albumin reveals leakage through the tight junctions, an upregulation of intraendothelial vesicles, and permeation of RVE or RPE cells that have undergone degenerative changes. VEGF, in addition to inducing neovascularization (NV), promotes vascular leakage. In VEGF transgenic mice, BRB failure is confined to the outer retina, the area where NV occurs. GSA binds to the luminal and abluminal surfaces of RVE cells in new and established vessels and to intraendothelial vesicles and interendothelial cell junctions in areas of vascular leakage. CONCLUSION: BRB dysfunction may be mediated by leakage through the tight junctions of RVE or RPE cells, by trans-endothelial vesicular transport, or by permeation of RVE or RPE cells that have undergone degenerative changes. GSA may be a useful marker to assist in recognizing open tight junctions and an increase in intraendothelial cell vesicles, which are indicative of BRB failure.

Targeted disruption of the FGF2 gene does not prevent choroidal neovascularization in a murine model.

Choroidal neovascularization (CNV) is the major cause of severe visual loss in patients with age-related macular degeneration. Laser treatment is helpful for a minority of patients with CNV, and development of new treatments is hampered by a poor understanding of the molecular signals involved. Several lines of evidence have suggested that basic fibroblast growth factor (FGF2) plays a role in stimulating CNV. In this study, we tested this hypothesis using mice with targeted disruption of the FGF2 gene in a newly developed murine model of laser-induced CNV. One week after krypton laser photocoagulation in C57BL/6J mice, 34 of 60 burns (57%) showed fluorescein leakage and 13 of 16 (81%) showed histopathological evidence of CNV. At 2 weeks, CNV was detected in 9 of 10 burns (90%) in which a bubble had been observed at the time of the laser treatment. Electron microscopy showed fenestrated vessels with large lumens within choroidal neovascular lesions. Two weeks after laser-induced rupture of Bruch's membrane, 27 of 36 burns (75%) contained CNV in FGF2-deficient mice compared with 26 of 30 (87%) in wild-type control mice, a difference that is not statistically significant. This study demonstrates that FGF2 is not required for the development of CNV after laser-induced rupture of Bruch's membrane and provides a new model to investigate molecular mechanisms and anti-angiogenic therapy in CNV.

Electron microscopic evidence for the mechanism of blood-retinal barrier breakdown in diabetic rabbits: comparison with magnetic resonance imaging.

Diabetes leads to a breakdown of the blood-retinal barrier (BRB), which can be demonstrated in experimental models by immunocytochemistry and magnetic resonance imaging (MRI). The present study utilizes these methods to investigate the mechanism of BRB breakdown in diabetic rabbits, a model ideally suited to both procedures. Rabbits were treated with alloxan and examined 2 months, 1 year, and 1.5 years after the development of diabetes to assess BRB breakdown using MRI and immunocytochemical staining for endogenous albumin. Using MRI, an increased incidence of retinal vascular leakage is first evident at 1 year of diabetes. Electron microscopic immunolocalization of albumin suggests that BRB compromise is principally mediated by transendothelial transport of serum proteins in endocytic vesicle-like structures of approximately 0.4-1 micron diameter. Some additional retinal vascular leakage is occasionally demonstrated through the interendothelial cell tight junctions, but only when adjacent vascular endothelial cells show degenerative changes. The similarity of these findings to those previously reported for diabetic humans and rats supports the use of the diabetic rabbit as a model for studying BRB dysfunction. MRI and electron microscopic (EM) immunocytochemistry are complementary methods for evaluating BRB dysfunction. MRI can provide an overall picture of the entire eye without sacrificing the animal. EM immunocytochemistry can provide a more detailed picture of a limited area of interest to gain insight into the mechanisms of extravasation. Together, both methods provide a more complete understanding of BRB breakdown in diabetic rabbits.

Increased vascular endothelial growth factor (VEGF) and transforming growth factor beta (TGFbeta) in experimental autoimmune uveoretinitis: upregulation of VEGF without neovascularization.

Experimental autoimmune uveoretinitis (EAU) was induced in Lewis rats and B10.A mice by immunization with S-antigen (S-Ag) to study the potential roles of vascular endothelial growth factor (VEGF) and the beta1 and beta2 isoforms of transforming growth factor (TGFbeta1 and TGFbeta2) during the progression of the disease. VEGF has been implicated as an angiogenic factor in ischemic retinopathies; however, Lewis rats developing EAU have high levels of VEGF in the retina, but no neovascularization. In the present study, immunohistochemical staining for VEGF, TGFbeta1 and TGFbeta2 was performed on the retinas of Lewis rats developing EAU or with oxygen-induced ischemic retinopathy. In rats immunized with S-antigen, a marked upregulation of VEGF was immunohistochemically visualized from the inner nuclear layer to the inner limiting membrane prior to blood-retinal barrier (BRB) failure and lymphocytic infiltration. VEGF is normally induced by hypoxia and its induction leads to neovascularization. Coincident with the increase in VEGF, there was increased immunoreactivity for TGFbeta1 and TGFbeta2 within the same layers of the retina. In contrast, rats with ischemic retinopathy and retinal neovascularization showed only a modest increase in VEGF immunoreactivity, which is largely confined to retinal ganglion cells and inner retinal vessels, and little or no increase in TGFbeta1 or TGFbeta2. In addition, in mice developing EAU, which does not have an abrupt onset as it does in rats and may involve neovascularization, a comparable upregulation of VEGF in the inner retina to that seen in rats developing EAU occurs with no increase in TGFbeta1 or TGFbeta2. Since TGFbeta can inhibit endothelial cell proliferation, it is likely that an increase in TGFbeta may prevent VEGF from exerting its endothelial growth activity in the rat EAU model, but VEGF may be operative in inducing BRB failure. These data suggest that there is a complex interaction among growth factors in the retina and that retinal neovascularization may require an imbalance between stimulatory and inhibitory factors.

Class III beta-tubulin isotype (beta III) in the adrenal medulla: I. Localization in the developing human adrenal medulla.

BACKGROUND: The class III beta-tubulin isotype (beta III) is present in neurons of the central and peripheral nervous systems at the earliest stages of morphological differentiation (Easter et al., J Neurosci 13:285-299, 1993; Katsetos et al., J Neuropathol Exp Neurol 52:655-666, 1993). The localization of this protein by immunohistochemistry in the different cell types of the developing human adrenal medulla is described. METHODS: A mouse monoclonal antibody, TuJ1, was used to localize beta III in formalin-fixed, paraffin-embedded sections from 18 human fetal and adult adrenal glands. Tissue sections were also studied with rabbit antisera recognizing either S-100 protein or glial fibrillary acidic protein (GFAP). RESULTS: In the developing human adrenal medulla, beta III immunoreactivity was maximal in migrating sympathoadrenal neuroblasts/immature neurons through the end of the second trimester. Clusters of beta III-positive migrating cells, focally forming Homer Wright rosettes, could be identified in a gradient of adrenocortical invasion, i.e., through the permanent cortex and within sinusoids of the fetal cortex en route to the medulla. Outside the adrenal gland, strong beta III staining was observed in peripheral nerve bundles, sympathetic ganglia, and paraganglia at various developmental stages. In adrenal glands from 23 weeks of gestation on, and throughout adult life, all ganglion cells were beta III immunoreactive. In contrast, not all chromaffin cells exhibited beta III staining, but when present, the staining was finely granular. Sustentacular and satellite cells, adrenocortical cells and other mesenchymal elements were betaIII-negative. In sections of fetal and adult adrenal glands, S-100 protein had a sustentacular localization. No GFAP staining was present in sustentacular cells from either fetal or adult adrenals. CONCLUSIONS: In the developing human adrenal medulla, there is a peak of beta III expression during the active wave of migration of sympathetic neuroblasts. In the mature medulla, beta III is invariably present in adrenergic neurons. However, not all chromaffin-like cells express beta III, suggesting that the presence or absence of this protein identifies two subpopulations of chromaffin cells.

Class III beta-tubulin isotype (beta III) in the adrenal medulla: II. Localization in primary human pheochromocytomas.

BACKGROUND: The Class III beta-tubulin isotype (beta III) is expressed specifically in central and peripheral nervous system neurons at various stages of neuronal differentiation. We have shown previously that beta III is expressed in a differentiation-dependent manner in human neuroblastomas arising in the adrenal medulla and sympathetic chains (Katsetos et al., Clin Neuropathol 13:241-255, 1994). The neuronal distribution of beta III in the developing and mature human adrenal medullae is detailed in the companion article (Katsetos et al., 1998A). METHODS: We have compared the localization of the neuronal beta III to S-100 protein, a sustentacular cell marker, in 14 formalin-fixed, paraffin-embedded primary human pheochromocytomas of the adrenal medulla and 14 adrenocortical tumors (adenomas and carcinomas). RESULTS: In pheochromocytomas, beta III staining was present in all tumors, but the number of stained cells varied in the two neural neoplastic phenotypes. Although the majority of chromaffin-like cells were beta III-positive, there was a lack of beta III in one-third of the tumor cells. Compared to chromaffin-like phenotypes, neuronal (ganglion-like cells) were invariably beta III-positive. Stromal sustentacular cells, stromal fibroblasts, and tumor blood vessels were beta III-negative. Sustentacular cells in pheochromocytomas were S-100 protein-positive, but beta III-negative. Primary adrenocortical tumors were beta III-negative with the exception of rare beta III-positive cells demonstrated in one case. CONCLUSIONS: The distribution of beta III in human pheochromocytomas of the adrenal gland is differentiation-dependent, closely recapitulating chromaffin cell and neuronal phenotypes of the normal adrenal medulla. Our findings indicate that beta III may be used as one of the adjuvant neural markers in the differential diagnosis of adrenal tumors, i.e., pheochromocytoma versus adrenocortical carcinoma. The occurrence of rare beta III-positive cells in cortical carcinomas is exceptional and probably represents the acquisition of a divergent neuroendocrine phenotype. The significance of the latter is unclear, although it may constitute a marker for malignancy.

Class III beta-tubulin isotype (beta III) in the adrenal medulla: III. Differential expression of neuronal and glial antigens identifies two distinct populations of neuronal and glial-like (sustentacular) cells in the PC12 rat pheochromocytoma cell line maintained in a Gelfoam matrix system.

BACKGROUND: The rat PC12 pheochromocytoma cell line provides an established system for the study of neuronal differentiation. To our knowledge, glial differentiation has not been reported in this cell line. METHODS: We have studied, by immunohistochemistry and immunoblotting, the presence of neuronal cytoskeletal antigens [class III beta-tubulin isotype (beta III), microtubule associated proteins MAP2, MAP1B and tau, and different neurofilament (NF) protein components], and synaptophysin in comparison with the glial fibrillary acidic protein (GFAP) and S-100 protein in the PC12 cell line. In three different experiments, PC12 cells were maintained in a three-dimensional gelatin foam (Gelfoam) matrix system for up to 34 days with and without treatment with 1 mM dibutyryl cyclic (dc)AMP. Immunohistochemistry was performed on explants ranging from 2 to 32 days-in vitro, which were fixed in either Bouin's solution, 70% ethanol, or 10% neutral-buffered formalin and embedded in paraffin. Immunoblotting was performed on Gelfoam explants with a panel of antibodies against all aforementioned neuronal and glial markers. Additional immunoblot experiments using anti-GFAP and anti-beta III monoclonal antibodies in cell suspensions and homogenates from PC12 monolayer cultures were carried out to compare growth conditions in relation to the expression of these proteins. RESULTS: Beta III and MAP2 were demonstrated by immunohistochemistry and immunoblotting of PC12 explants maintained for up to 32 days in Gelfoam matrices with and without treatment with dcAMP. Intense filamentous and granular beta III staining of PC12 cells was observed in dcAMP-treated cultures concomitant with neuronal morphologic alterations (neuritogenesis and ganglionic phenotype). In untreated cultures, beta III staining was present in less differentiated cells, as well in cells undergoing neuritic development. The neuronal phenotype of PC12 cells was confirmed by staining for MAP2, tau, and NF proteins, as well as for synaptophysin. The presence of beta III, MAP2, MAP1B, tau, and NF proteins was confirmed by immunoblotting. Clusters of GFAP-positive and S-100 protein-positive spindle cells, phenotypically distinct from the chromaffin-like or neuronal cells, were demonstrated in Gelfoam explants at 5-30 days in vitro. In 30-day-old cultures treated with dcAMP, there was strong filamentous GFAP and diffuse S-100 protein staining in an increased number of sustentacular-like PC12 cells. GFAP staining was corroborated by immunoblotting of explants maintained under identical conditions in vitro. In contrast, immunoblots performed on homogenates from PC12 suspension and monolayer cultures were GFAP-negative. CONCLUSIONS: Neuronal and glial-like, presumed sustentacular, phenotypes were demonstrated in PC12 cells grown in Gelfoam matrices with and without treatment with dcAMP for up to 34 days. To our knowledge, the occurrence of glial differentiation in the PC12 line is a hitherto unreported finding. Adult rat medullary sustentacular cells are known to express S-100 and GFA proteins (Suzuki and Kachi, Kaibogaku Zasshi-Anat 70(2): 130-139, 1995), and the organ culture system employed in our study may well have favored this direction of differentiation.

Evolution of neovascularization in mice with overexpression of vascular endothelial growth factor in photoreceptors.

PURPOSE: To determine the earliest changes that occur in the retina after the onset of ectopic expression of vascular endothelial growth factor (VEGF) by photoreceptors in transgenic mice, to characterize the development of neovascularization (NV), and to determine the feasibility of using these mice to test the efficacy of antiangiogenic agents. METHODS: The time course of expression of VEGF transgene mRNA was determined by reverse transcription-polymerase chain reaction (RT-PCR). Histopathologic changes in the retina were investigated by light and electron microscopy and immunocytochemistry. Standard and confocal fluorescence microscopy and image analysis were used to evaluate NV in retinal whole mounts. RESULTS: VEGF transgene mRNA was first detected in the retina by RT-PCR on postnatal day 6 (P6) and increased over the next several days to reach a constant steady-state level between P14 and P21. Abnormal cells were seen in the outer nuclear layer on P10 and among photoreceptors on P14; by P18 there were cell aggregates in the subretinal space with evidence of lumen formation. The invading cells were demonstrated to be endothelial cells by staining with an endothelial cell-specific lectin. Whole mounts of retinas perfused with fluorescein-labeled dextran showed a similar sequence of events, with sprouts from retinal vessels in the deep capillary bed seen on P14 and vessels reaching the subretinal space by P18. Confocal and standard fluorescence microscopy and changes in the number and area of neovascular lesions in the subretinal space over time measured by image analysis suggest gradual enlargement and coalescence of vascular complexes. The subretinal NV was progressively engulfed by the retinal pigmented epithelium. Invasion of blood vessels from the choroid was not identified in any specimen. CONCLUSIONS: These data support the feasibility of using rhodopsin-VEGF transgenic mice to study tissue-specific aspects of NV in the retina and to test antiangiogenic agents for inhibition of intraretinal and subretinal NV.

Blood-retinal barrier (BRB) breakdown in experimental autoimmune uveoretinitis: comparison with vascular endothelial growth factor, tumor necrosis factor alpha, and interleukin-1beta-mediated breakdown.

Experimental autoimmune uveoretinitis (EAU) induced in Lewis rats by immunization with S-antigen is a model of human uveitis. By using immunocytochemical staining for albumin, relatively minor blood-retinal barrier (BRB) breakdown was initially shown in the peripheral retina 8 days after immunization and in the posterior retina by 10 days. Albumin extravasation appeared to occur by opening of the retinal vascular endothelial (RVE) and the retinal pigmented epithelial (RPE) tight junctions, by transendothelial vesicular transport, and by permeating damaged RVE cells. Each of three anti-inflammatory agents reduced or delayed autoimmune-mediated cell destruction but did not eliminate any particular route of extravasation. Vascular endothelial growth factor (VEGF), tumor necrosis factor alpha (TNF alpha), and interleukin-1beta (IL-1beta) are intimately associated with the development of EAU and are capable of causing BRB dysfunction. A high percentage of RVE tight junctions appeared open ultrastructurally after intravitreal injection of VEGF (26.7%), TNF alpha (35.6%), or IL-1beta (22.1%) compared with saline-injected control (11.4%) or normal, untreated rabbits (4.1%). Heat treatment abolished the effect of IL-1beta on the BRB but only partially reduced the effect of VEGF. By 24 hr after injection, the effect of TNF alpha had reversed, but that of IL-1beta had not; VEGF-mediated BRB dysfunction was partially reversible. In addition, albumin-filled vesicle-like structures were seen in the RVE cytoplasm following treatment with each mediator. This study shows that VEGF, TNF alpha, and IL-1beta each cause BRB breakdown by opening tight junctions between RVE cells and possibly by increasing transendothelial vesicular transport. Each of these agents may contribute to BRB breakdown in EAU and in patients with uveitis.

Intravitreal sustained release of VEGF causes retinal neovascularization in rabbits and breakdown of the blood-retinal barrier in rabbits and primates.

Vascular endothelial growth factor (VEGF) has been identified as a possible mediator of retinal neovascularisation (NV), but it is not certain if VEGF alone is sufficient to cause retinal NV. We sought to investigate this issue by implanting ethylene-vinyl acetate copolymer pellets that slowly release VEGF into the vitreous cavity of rabbits and primates. Eyes were examined by indirect ophthalmoscopy, fundus photography, and fluorescein angiography and then animals were killed at various time points and immunocytochemical and ultrastructural evaluations were carried out. Seven days after implantation of a pellet containing 30 micrograms of human recombinant VEGF into the vitreous cavity of rabbits, retinal blood vessels became dilated and tortuous, and between days 14 and 21, retinal NV was noted in all eyes. Fluorescein angiography showed profuse leakage of dye from the anomalous vessels. Immunohistochemical staining for proliferating cell nuclear antigen (PCNA) showed positively staining nuclei in many of the endothelial cells of new blood vessels on the surface of the retina. Six eyes implanted with control pellets containing vehicle and two eyes implanted with pellets containing 30 micrograms of human serum albumin alone showed no retinal vascular abnormalities. Implantation of pellets containing 100 micrograms of VEGF into the vitreous cavity of primates resulted in iris NV and retinal vascular dilation and tortuosity very much like that seen in humans with ischemic retinopathies. Immunohistochemical staining for serum albumin showed widespread severe breakdown of the blood-retinal barrier (BRB). Histology showed dilated thin-walled retinal vessels, but unequivocal retinal NV could not be identified and staining for PCNA was negative. These findings indicate that sustained intravitreal release of VEGF causes widespread retinal vascular dilation and breakdown of the BRB. Retinal NV seems to require persistent high levels of VEGF at the retinal surface and can be achieved in rabbits providing a potentially useful model of retinal NV, but is difficult to achieve in primates. The extensive VEGF-induced disruption of the blood-retinal barrier suggests that VEGF antagonists may provide a new therapy for patients with ischemic retinopathies and macular edema.

Upregulation of vascular endothelial growth factor in ischemic and non-ischemic human and experimental retinal disease.

Vascular endothelial growth factor (VEGF) is induced by hypoxia and it has been implicated in the development of iris and retinal neovascularization (NV) in ischemic retinopathies in which it has been suggested that Muller cells are responsible for increased VEGF production. VEGF, however, is also known to be a potent mediator of vascular permeability in other tissues and may perform this function in retina. Immunohistochemical staining for VEGF was performed on a variety of human and experimental ischemic and non-ischemic ocular disorders in which blood retinal barrier (BRB) breakdown is known to occur to determine if there is an upregulation of VEGF in these conditions. We found increased VEGF immunoreactivity in ganglion cells of rats with oxygen-induced ischemic retinopathy and in ganglion cells, the inner plexiform layer, and some cells in the inner nuclear layer of rats with experimental autoimmune uveoretinitis (EAU), in which there was no identifiable ischemia or NV. In rats with EAU, VEGF staining intensity increased from 8 to 11 days after immunization, coincident with BRB failure. These results were confirmed using two distinct anti-VEGF antibodies and by immunoblot and the immunohistochemical staining was eliminated by pre-incubating the antibodies with VEGF peptide. VEGF staining was also increased in the retina and iris of patients with ischemic retinopathies, such as diabetic retinopathy and retinal vascular occlusive disease, and in patients with disorders in which retinal ischemia does not play a major role, such as aphakic/ pseudophakic cystoid macular edema, retinoblastoma, ocular inflammatory disease or infection, and choroidal melanoma. VEGF was primarily localized within retinal neurons and retinal pigmented epithelial cells in these cases. In addition or in association with its role of inducing NV, VEGF may contribute to BRB breakdown in a variety of ocular disorders and blockage of VEGF signaling may help to reduce some types of macular edema.

Feasibility of laser-targeted photoocclusion of the choriocapillary layer in rats.

PURPOSE: A new method, laser-targeted photoocclusion, was developed to occlude choroidal neovascularization while minimizing damage to the overlying retina. The ability to occlude normal choriocapillary layer in rats was evaluated as a first test of the feasibility of treating choroidal neovascularization with this method. METHOD: A photosensitive agent, aluminum phthalocyanine tetrasulfonate, encapsulated in heat-sensitive liposomes, was administered intravenously along with carboxyfluorescein liposomes. A low-power argon laser (retinal power density of 5.7 W/cm2) locally released a photosensitizer bolus, monitored by the simultaneous release of carboxyfluorescein. A diode laser (operating at 675 nm with a retinal power density of 0.27 W/cm2) activated the photosensitizer with its release. RESULTS: Vessels in the choriocapillary layer were occluded at day 3 after laser treatment and remained unchanged during the 30-day follow-up. Larger choroidal vessels and retinal capillaries remained perfused. Control experiments excluded possible effects of heat or activation of free photosensitizer. Pilot histologic studies showed no damage to the retinal pigment epithelium. CONCLUSIONS: Laser-targeted photoocclusion caused selective occlusion of normal choriocapillaries while sparing overlying retinal pigment epithelium and retinal vessels. The method has potential as a treatment of choroidal neovascularization that may minimize iatrogenic loss of vision.