Anti-angiogenic effect of the basement membrane protein nidogen-1 in a mouse model of choroidal neovascularization.

In patients with age-related macular degeneration disruption of the integrity of the retinal pigment epithelium (RPE) and Bruch's membrane (BrM), precedes choroidal neovascularization (CNV). We investigated the role of the basement membrane (BM) proteins nidogen-1 and nidogen-2 for the development of experimental CNV. Laser-induced CNV was studied in Nid1(-/-) and Nid2(-/-) mice and wild type (WT) controls by fluorescein angiography, by immune histochemistry of flat-mounts or paraffin sections to analyze expression pattern of nidogen-1 and -2 and nidogen binding BM proteins, and by western blotting. The influence of VEGF and bFGF on the mRNA expression of nidogen-1 was studied in vitro. Nidogen-1 protein is present in the BM of the inner limiting membrane (ILM), the retinal capillaries, and the choroid/sclera and CNV. Nidogen-2 protein is also found in these BMs but with a weaker expression in the ILM. In the retina the absence of nidogen-1 does not influence the expression of nidogen-2 and vice versa and does not influence the expression of the BM components collagen IV, laminin γ1, and perlecan. In Nid1(-/-) mice, CNV lesions showed increased vessel leakage during angiography and the CNV area was larger than in WT or nidogen-2 deficient mice. Laser treatment led to up-regulation of nidogen-1 protein expression in the sclera/choroid of nidogen-2 deficient or WT mice. The treatment of HUVECs with VEGF leads to a reduced expression of nidogen-1 mRNA whereas its expression remained unchanged in RPE cells. In conclusion, nidogen-1 produced by the endothelial cells acts as a factor to help stabilizing the BM, thus preventing the sprouting of new vessels or the infiltration of endothelial cells. In this sense nidogen-1 is essential to provide an anti-angiogenic environment of differentiated vessels.

Multifocal ERG recordings under visual control of the stimulated fundus in mice.

PURPOSE: Therapeutic approaches to retinal disease require a continuous monitoring of functional improvement over lesion areas that sometimes cannot be shown in full-field ERG. The aim of this study was to assess the usefulness of multifocal electroretinograms (mfERGs) under visual control using scanning laser ophthalmoscopy (SLO) for evaluation of local retinopathy in mice. METHODS: mfERGs were optimized for recordings in C57BL/6 mice by varying dark steps between each stimuli, background intensity, and the numbers of hexagons. Local retinopathy was induced by argon laser photocoagulation with different spot sizes and retinal irradiances. mfERG recordings were performed before, and 10 days and 4 weeks after laser treatment. In each recording, the central hexagon was positioned on the optic nerve head visualized by SLO images. The amplitudes of the P1 response components were analyzed as a function of retinal location. RESULTS: The mfERG amplitudes depended on stimulus condition. The P1 amplitudes increased with increasing number of dark frames in the m-sequence and with decreasing number of hexagons. A stimulus with 19 hexagons and four dark frames was chosen because substantial response amplitudes could be achieved while preserving sufficient spatial resolution. In the untreated eyes, the response to the central hexagon, stimulating the optic nerve head, was smaller than those to the surrounding hexagons. The responses to hexagons stimulating photocoagulated areas were reduced compared with the responses of surrounding areas. The amplitude reduction was more pronounced when the coagulated areas were larger and when higher energies were used. CONCLUSIONS: Areas with decreased sensitivities to light stimulation (either the optic nerve head or damaged retinal areas) can be detected and correlated with the retinal images and in the mfERG responses. We demonstrate that the mfERG technique is able to reproducibly detect the functional consequences of a local treatment.

Recruitment of blood-derived inflammatory cells mediated via tumor necrosis factor-α receptor 1b exacerbates choroidal neovascularization.

Purpose. Tumor necrosis factor (TNF)-α contributes to inflammation-associated angiogenesis, and TNF-α receptor 1b is selectively expressed on immuno-competent and endothelial cells. This study investigated the role of TNF-α receptor 1b in the recruitment of circulating inflammatory cells and the development of choroidal neovascularization (CNV). Methods. Lethally irradiated Tnfrsf1b(-/-) mice and their wild-type (WT) controls were transplanted with whole adult bone marrow (BM) cells, competent for both TNF-α receptors 1a and 1b (gfp(+) labeled), as well as with BM cells deficient for TNF-α receptor 1b. One month after transplantation CNV was induced by laser damage of Bruch's membrane. Pathologic angiogenesis was estimated qualitatively and quantitatively by histology on choroidal flatmounts and paraffin cross sections. Macrophage invasion was investigated by immunochemistry. Results. One month after transplantation the reconstitution rate measured by FACS analysis was >80% in gfp(+)-chimeric mice. Two weeks after laser injury reduced gfp(+)-cell invasion to the laser scars and decreased pathologic angiogenesis were observed in Tnfrsf1b(-/-) versus WT recipients. Approximately 70% of the invaded gfp(+) cells were labeled with macrophage marker F4/80. Transplantation of TNF-α receptor 1b-deficient BM cells in WT recipients reduced the CNV lesion compared with WT and Tnfrsf1b(-/-) recipients that received TNF-α receptor-competent BM cells. Transplantation of receptor 1b-deficient cells to Tnfrsf1b(-/-) recipients further reduced the degree of CNV formation. Conclusions. Signals through TNF-α receptor 1b expressed on BM -derived inflammatory cells mediate an increased inflammatory cell invasion and enhanced angiogenic response after laser-induced rupture of Bruch's membrane.

Conditions of retinal glial and inflammatory cell activation after irradiation in a GFP-chimeric mouse model.

PURPOSE: Microglia cells have been associated with immunologic defense and repair. The course of retinal disease after lethal irradiation for bone marrow depletion and substitution was evaluated with respect to macrophage and microglial involvement. METHODS: Lethal irradiation in C57BL/6 mice was conducted with a low-voltage radiation unit. The animals were randomized to shielded or unshielded radiation and subsequently received transplants of GFP+ bone marrow cells (beta-actin promoter). The GFP transformation rate was analyzed by flow cytometry. GFP+ cells in the retina were examined for co-localization with macrophage and dendritic cell markers at various time points between 1 and 7 months after irradiation. Clodronate liposomes were used to investigate the fate of migrated and residential microglia cells. Pathologic angiogenesis was investigated in laser-induced choroidal neovascularization (CNV) after unshielded and shielded irradiation. RESULTS: Flow cytometry revealed average transformation rates of 78.2% in unshielded and 64.1% in shielded group. Four weeks after transplantation, perfused flat mounts were virtually free of extravasal GFP+ cells in both groups, whereas 4 months after irradiation, cluster cell infiltrations, preferentially in the peripheral retina, became apparent exclusively in the unshielded group. Cell morphology ranged from oval, to a few extensions, to dendritiform with long-branched extensions. Clodronate treatment resulted in a reduction of GFP+ cells in the retinal tissue when applied 3 months after unshielded irradiation. Although GFP+ cells accumulated in the choroidal scar after laser treatment, in both the shielded and unshielded groups, GFP+ cells in the overlying retina were restricted to the unshielded group. CONCLUSIONS: Approximately 3 months after lethal full-body irradiation including the eye, bone marrow-derived leukocytes exhibit a wound-healing reaction, and unlike physiological turnover, infiltrate the retina and form microglial cells.

Differential role of tumor necrosis factor (TNF)-alpha receptors in the development of choroidal neovascularization.

PURPOSE: Tumor necrosis factor alpha (TNF)-alpha contributes to inflammation-associated angiogenesis. This study investigates the role of TNF-alpha receptors 1a and 1b in the development of choroidal neovascularization (CNV). METHODS: CNV was induced in Tnfrsf1a(-/-) and Tnfrsf1b(-/-) mice with C57Bl6/J background and their wild-type (WT) (C57Bl/6J) controls by laser damage to the Bruch's membrane. TNF-alpha expression in RPE/choroid was determined by Western blot analysis. Pathologic angiogenesis was estimated qualitatively and quantitatively by fluorescein angiography and histology on choroidal flat mounts and paraffin cross-sections. Inflammatory cell invasion was investigated by clodronic acid depletion of circulating macrophages and immunochemistry, and the apoptotic activity was investigated by TUNEL assay and by caspase-3 and caspase-8 expression. Receptor 1b-specific Bmx/Etk kinase was detected by immunochemistry. RESULTS: TNF-alpha levels were elevated after laser treatment. Severe CNV lesions and increased macrophage invasion were observed in Tnfrsf1a(-/-) compared with WT and Tnfrsf1b(-/-) mice. Increased immunoreactivity for Bmx/Etk kinase corresponded to the severity of CNV formation. Reduced pathologic angiogenesis and macrophage invasion in Tnfrsf1b(-/-) mice (vs. WT and Tnfrsf1a(-/-)) was accompanied by enhanced endothelial cell apoptosis and by caspase-3 and caspase-8 activation. CONCLUSIONS: Receptor 1b promotes the recruitment of inflammatory cells to the site of injury and exacerbated pathologic angiogenesis probably by way of the Bmx/Etk-kinase-dependent pathway in the absence of receptor 1a. On the other hand, receptor 1a-dependent apoptosis in the absence of receptor 1b leads to reduced inflammatory response and CNV lesions after laser treatment. This demonstrates the potential for specific targeting of TNF-alpha receptors for future therapies of inflammation-associated choroidal neovascularization.

Deposition of nidogens and other basement membrane proteins in the young and aging mouse retina.

AIM: To determine the distribution of major basement membrane constituents, particularly nidogen 1 and 2, in young and aging mouse retinae. METHODS: The specificity of antibodies against basement membrane proteins was ascertained by immunoblotting with proteins extracted from mouse retinae. The same antibodies were used in indirect immunofluorescence microscopy to localize basement membrane proteins in paraffin sections of retinae from 1-, 12- and 18-month-old C57BL/6 mice. RESULTS: At a young age, laminin, perlecan and collagen IV were most abundant in Bruch's membrane. Later, the proteins were clearly detected in capillary basement membranes and the inner limiting membrane. In both of these basement membranes, a massive increase in protein amount was seen upon aging, whereas in Bruch's membrane the staining intensity was less drastically changed. Both nidogen 1 and 2 were present in vascular basement membranes and Bruch's membrane throughout the age periods studied. In the inner limiting membrane, the nidogens were more strongly expressed at higher ages, with an earlier and more extensive deposition of nidogen 1. CONCLUSIONS: All major basement membrane constituents are present in the mouse retina, but the onset of deposition differs among the different proteins and between the various retinal basement membranes. In general, basement membrane protein deposition increases with age.

Retinal localization of the glutamate receptor GluR2 and GluR2-regulating proteins in diabetic rats.

Impaired glutamatergic activity and synaptic dysfunction contributing to excitotoxicity and neuronal degeneration has been observed in the diabetic retina. Here we analyzed the expression changes and trafficking abnormalities of the AMPA glutamate receptor 2 subunit (GluR2) and its regulators protein kinase Calpha (PKCalpha) and PKC-interacting protein 1 (PICK1) in the rat retina during the early phases of streptozotocin-(STZ-) induced diabetes. Diabetes was induced in Long Evans rats by injection of STZ. Two and six weeks after induction of diabetes, immunohistochemistry and in situ hybridization were performed on retinal paraffin sections to investigate the expression and localization of GluR2 and its regulators PKCalpha and PICK1. The cellular distribution and trafficking of these proteins in retinae were also investigated by subcellular fractionation and western blotting. While no significant changes were observed for GluR2 transcripts, we observed a strong increase in GluR2 immunoreactivity, predominantly in the ganglion cell layer (GCL) and the inner plexiform layer (IPL), as early as two weeks of diabetes. GluR2/3 immunoreactivity was further increased from the GCL to OPL after 6 weeks of diabetes. Increased expression of a phosphorylated non-synaptic population of GluR2 was detected in the GCL, the IPL and in distinct photoreceptor cells within the outer nuclear layer (ONL) of diabetic animals. Further, the PICK1 retinal distribution was unchanged two and six weeks after onset of diabetes and in both control and diabetic rat retinae the PKCalpha immunoreactivity remained the same. However, phosphorylated PKCalpha immunoreactivity was increased in diabetic retina as compared to control and peaked after 6 weeks of diabetes. Activated PKCalpha was almost completely lost in all membrane fractions and primarily recovered in the cytosolic fraction. These results are consistent with PKCalpha being re-localized in the diabetic retina. The observations indicate a diabetes-dependent increase in the activation of PKCalpha and a disturbed GluR2 regulation by altered internalization and recycling.

TNF-alpha mediated apoptosis plays an important role in the development of early diabetic retinopathy and long-term histopathological alterations.

PURPOSE: The pathophysiology of diabetic retinopathy involves leukocyte adhesion to retinal vasculature, early blood-retinal barrier breakdown, capillary nonperfusion, and endothelial cell death. We investigated the involvement of tumor necrosis factor alpha (TNF-alpha) in diabetes-related histopathological changes in two relevant rodent models. METHODS: In short-term studies, Long-Evans rats with streptozotocin-induced diabetes were treated with or without the TNF-alpha inhibitor, etanercept. For long-term studies, tumor necrosis factor receptor I (TNF-RI)-deficient mice and TNF-RII-deficient mice, as well as C57/Bl6 wild-type mice, were fed 30% galactose for up to 20 months. The retinal histopathological alterations of hypergalactosemia were analyzed in trypsin digest preparations. Endothelial cell injury and apoptosis in rat retinas were evaluated by propidium iodide, TUNEL, CytoDeath staining, and DNA fragmentation ELISA. Caspase 3 and 8 activity was evaluated by immunoblotting and quantitative enzymatic activity assay. RESULTS: Etanercept suppressed caspase activation, retinal cell injury, and apoptosis in short-term diabetic rats. Pericyte and endothelial cell loss were also reduced in long-term hypergalactosemic mice. Long-term studies demonstrated that pericyte loss and endothelial cell loss were reduced in comparison to wild-type diabetic controls. CONCLUSIONS: Our study identifies an important role for TNF-alpha in the pathogenesis of signature diabetic retinopathy pathologies and demonstrates that etanercept can inhibit retinal cell death and long-term complication of diabetes. Taken together, our results suggest that etanercept could prove beneficial in preventing both early and late vascular diabetic complications.

ICAM-1 depletion does not alter retinal vascular development in a model of oxygen-mediated neovascularization.

ICAM-1 has been identified as a mediator of inflammatory and VEGF-dependent corneal neovascularization. Furthermore, ICAM-1 has been demonstrated to be involved in leukocyte-mediated endothelial injury in diabetic retinopathy. Here we investigated the role of ICAM-1 in retinal vaso-obliteration and vascularization. ICAM-1 deficient mice as well as their respective wild-type controls were exposed to 75% oxygen from postnatal day 7 to day 12. Retinal vascularization was investigated after lectin labeling of endothelial cells on day 14, 17, and 20 in flat mount preparations. Retinal mRNA expression of VEGF, Angiopoietin 1 and 2 as well as PDGFbeta was examined at day 14 and 20 by Real Time RT-PCR. ICAM-1(-/-) mice and their respective wild-type controls demonstrated similar retinal development and vascularization under normoxic conditions. Similarly, after oxygen challenge, the vascular area, the avascularized area as well as the area of neovascular tufts did not differ between ICAM-1(-/-) and the respective wild-type mice although the mRNA expression of VEGF, ang-1, ang-2, and PDGFbeta differed clearly. This study demonstrates that lack of ICAM-1 leads to an altered expression of angiogenic factors that in combination may neutralize each other and do not alter retinal development and angiogenesis in oxygen-induced retinopathy.

[Effect of anti-TNF-alpha on laser-induced choroidal neovascularization].

OBJECTIVE: To investigate the role of TNF-alpha in the development of laser-induced choroidal neovascularization (CNV) in the mouse. METHODS: Laser photocoagulation was used to induce CNV in wild-type C57BL/6J mice by making four separate choroidal bums in each eye. Animals were treated 3 days before or after laser injury with recombinant TNF receptor P75 (etanercept, 5 microg/h, group 1, n = 12), chimeric monoclonal antibody (infliximab, 5 microg/h, group 2, n = 12) for 7 days by intraperitoneally implanted osmotic pumps. PBS was used as control (group 3, n = 12). The left eyes were removed for histopathologic examination and the right eyes were removed for flatmounts immunohistochemistry immediately after fluorescein angiography. In mice treated with medications 3 days before laser injury, left eyes were collected at 1 or 2 weeks after laser injury. In mice treated with medications 3 days after laser injury, left eyes were collected at 10 days after laser injury. CNV responses were compared by flatmount analysis of CNV-related fluorescence area and by determination of fluorescein angiographic leakage. The level of protein expression of TNF-alpha was semiquantitatively evaluated by Western blot analysis of the choroidal and RPE layer from mice with or without laser treatment. RESULTS: Western blotting demonstrated that TNF-alpha was highly expressed in choroidal and RPE cells of wild type mice 1 week after laser treatment as compared to the control mice without laser treatment. Etanercept and infliximab administrated 3 days before laser-damage significantly reduced CNV size and pathological fluorescein leakage in comparison to the control group one and two weeks after laser injury. Only etanercept administered 3 days after laser injury still significantly reduced the development of CNV lesions. Histopathological examination confirmed that CNV lesions in treated mice had smaller diameter and thinner center as compared to the control animals. CONCLUSIONS: Anti-TNF-alpha treatment reduces the size and leakage of laser-induced CNV. These results suggest the involvement of TNF-alpha in the development of laser-induced CNV and its potential use as a therapeutic agent in the age related macular degeneration.

Inhibition of TNF-alpha reduces laser-induced choroidal neovascularization.

To investigate the role of the TNF-alpha in the development of laser-induced choroidal neovascularization (CNV) in a mouse model. Four separate laser burns were applied to induce ruptures of Bruch's membrane and subsequent choroidal neovascularization in C57BL/6J mice. TNF-alpha protein expression was semiquantitatively assessed by Western blot analysis of the choroidal and RPE layer from mice with or without laser treatment. To investigate the effect of TNF-alpha inhibition on CNV formation, animals were treated for 7 days via intraperitonealy implanted osmotic pumps either 3 days before or after laser injury with recombinant TNF receptor P75 (etanercept), a chimeric monoclonal antibody (infliximab), or a purified rat anti-mouse/rat TNF monoclonal antibody (TNF-mAb), respectively. Fluorescein angiography, flat-mount preparations, and histopathology were performed at day 7, 10, or 14 after laser treatment. Western blotting demonstrated that TNF-alpha expression was 4.57-fold higher in the choroid and RPE one week after laser injury compared to control mice without laser. When evaluated one and two weeks after laser injury, etanercept and infliximab given from the 3rd day before laser-damage significantly reduced CNV size and pathological fluorescein leakage compared to the control group after laser treatment only. The inhibitory effect of the monoclonal TNF-alpha antibody on CNV formation was evident two weeks after photocoagulation but not after one week. Only etanercept administered 3 days after laser injury still reduced significantly the development of CNV lesions. Histopathology confirmed that CNV lesions in treated mice were smaller in size compared to the control animals without TNF inhibitor treatment. In conclusion, anti-TNF-alpha treatment with different inhibitors reduces both the size and the leakage of laser-induced CNV. These results suggest the involvement of TNF-alpha in the development of laser-induced CNV and its potential use as a therapeutic agent in the age-related macular degeneration.

Overexpression of FasL in retinal pigment epithelial cells reduces choroidal neovascularization.

Choroidal neovascularization (CNV) is responsible for the severe visual loss in age-related macular degeneration. CNV formation is considered to be due to an imbalance between pro- and antiangiogenic factors that lead to neovascular growth from the choriocapillaris into the subretinal space. To define whether FasL overexpression in retinal pigment epithelial cells (RPE) can inhibit choroidal neovascularization through Fas-FasL-mediated apoptosis, we examined the role of this pathway in a mouse model of laser-induced choroidal neovascularization. FasL was expressed in the retinal pigment epithelium of transgenic mice. Polymerase chain reaction (PCR), immunoblot, and immunohistochemistry confirmed that the transgene FasL was specifically expressed in RPE. The established laser model was used to induce choroidal neovascularization (CNV) in wild-type (WT) and transgenic mice. CNV formation was compared with respect to fluorescein angiographic leakage (at days 0 and 14 after laser injury) and histological appearance. The lesions were assessed on RPE-choroidal flatmounts after CD31-labeling and with confocal microscopy after perfusion with rhodamine-labeled concanavalin A (Con A). Apoptosis was quantified by TUNEL positivity and caspase activation. FasL mRNA and protein were highly expressed in the RPE of the transgenic mice before and after laser photocoagulation. In contrast, FasL was only weakly expressed in the RPE layer of WT C57BL/6J mice. While ruptures of Bruch's membrane and CNV formation were observed histologically two weeks after laser photocoagulation in transgenic as well as control eyes, the shape and size of CNV lesions were reduced in the transgenic mice. The area of leakage was decreased by 70% in FasL transgenic mice compared with WT mice (P<0.005). The number of TUNEL-positive cells was greater in FasL-overexpressing mice and correlated with the expression of activated caspases. Th expression of other antiangiogenic factors such as PEDF remained unchanged. The specific overexpression of FasL in RPE layer reduced CNV formation in our laser model. Our results strongly point to the FasL-Fas pathway as a potential therapeutic target in controlling pathological choroidal neovascularization.

Neurally selected embryonic stem cells induce tumor formation after long-term survival following engraftment into the subretinal space.

PURPOSE: To determine whether transplantation of embryonic stem (ES) cells into the subretinal space of rhodopsin-knockout mice has a tumorigenic effect. METHODS: Mouse ES-cell-derived neural precursor cells carrying the sequence for the green fluorescent protein (GFP) gene were grafted subretinally into the eyes of rhodopsin(-/-) mice, whereas control animals underwent sham surgery. Eyes were retrieved after 2, 4, and 8 weeks after cell injection or sham surgery for histologic analysis. RESULTS: Gross morphologic, histologic, and immunohistochemical analysis of eyes at 2 and 4 weeks after engraftment exhibited no morphologic alterations, whereas neoplasia formation was detected in 50% of the eyes evaluated at 8 weeks after engraftment. Because the neoplasias expressed differentiation characteristics of the different germ layers, they were considered to be teratomas. The resultant tumor formation affected almost all layers of the eye, including the retina, the vitreous, and the choroid. CONCLUSIONS: Although ES cells may provide treatment for degenerative disease in the future, their unlimited self-renewal and high differentiation potential poses the risk of tumor induction after engraftment. Thus, more care must be taken before using ES cell transplantation as a therapeutic option for patients with degenerative disease.

Investigation of laser-induced choroidal neovascularization in the rat.

PURPOSE: Choroidal neovascularization plays an important role in pathogenesis of age-related macular degeneration. Induction of neovascularization by laser photocoagulation in the rat fundus is an established animal model in which the effects of new therapeutic approaches are assessed. The purpose of this study was to compare different detection methods of laser-induced neovascularization in the rat. METHODS: Laser spots were applied to the fundus of Long-Evans rats. Ten days after, four different methods were used to detect laser-induced neovascularization: (1) high-resolution angiography with fluorescein isothiocyanate-dextran, (2) immunohistochemical visualization of platelet endothelial cell adhesion molecule (PECAM)-1, (3) visualization of intravascular lumens by peroxidase perfusion in the living rat with subsequent histologic analysis, and (4) histochemical representation of alkaline phosphatase in endothelial cells. RESULTS: At the rim of the laser scars vessel-forming endothelial cells with intravasal dextran and peroxidase were present. Cross-sections demonstrated that these vessels originated from the retina. The center of the scars contained homogenous endothelial cells of choroidal origin, which was confirmed by immunohistochemistry and electron microscopy. In laser-treated eyes without FITC-dextran perfusion, scars showed unspecific fluorescence, making differentiation from specific FITC-dextran-associated fluorescence difficult. CONCLUSIONS: In the rat model of laser-induced neovascularization, newly developed endothelial cells originate from the retina and the choroid. Whereas ring-like surrounding vessels come from the retina, flat endothelial cells in deeper layers are of choroidal origin or may originate from circulating endothelial precursor cells. Dextran angiography has to be regarded critically for visualizing the choriocapillaris and CNV in laser scars. PECAM-1 immunohistochemistry is best for detection and quantification of neovascularization in laser scars.

Autologous transplantation of genetically modified iris pigment epithelial cells: a promising concept for the treatment of age-related macular degeneration and other disorders of the eye.

Age-related macular degeneration (ARMD) is the leading cause for visual impairment and blindness in the elder population. Laser photocoagulation, photodynamic therapy and excision of neovascular membranes have met with limited success. Submacular transplantation of autologous iris pigment epithelial (IPE) cells has been proposed to replace the damaged retinal pigment epithelium following surgical removal of the membranes. We tested our hypothesis that the subretinal transplantation of genetically modified autologous IPE cells expressing biological therapeutics might be a promising strategy for the treatment of ARMD and other retinal disorders. Pigment epithelium-derived factor (PEDF) has strong antiangiogenic and neuroprotective activities in the eye. Subretinal transplantation of PEDF expressing IPE cells inhibited pathological choroidal neovascularization in rat models of laser-induced rupture of Bruch's membrane and of oxygen induced ischemic retinopathy. PEDF expressing IPE transplants also increased the survival and preserved rhodopsin expression of photoreceptor cells in the RCS rat, a model of retinal degeneration. These findings suggest a promising concept for the treatment of ARMD and other retinal disorders.

Iris pigment epithelial cells transplanted into the vitreous accumulate at the optic nerve head.

BACKGROUND: Iris pigment epithelial (IPE) cells have mainly been investigated in the past for their proposed potential to rescue or even replace degenerated retinal pigment epithelial (RPE) cells after subretinal transplantation in patients with age-related macular degeneration (AMD). More recent reports have characterised the IPE cell as a potent source of trophic factors and cytokines. In our study we investigated the spatial distribution of IPE cells that were injected into the vitreous instead of being injected subretinally. METHODS: IPE cells from Long Evans rats were isolated and injected into the vitreous cavity of Wistar rats without preculturing. Free melanin granules were injected into the vitreous in the same manner. After a period of 2 months, eyes were prepared for histological analysis. Localisation of the injected IPE cells was defined by topographical mapping of the analysed sections. RESULTS: PVR was not observed in any eye. In 8 of 10 injected eyes, IPE cells had accumulated in the prepapillary region. In 2 of 10 eyes, no IPE cells could be detected. The injected melanin granules also accumulated at the optic nerve head, indicating that this is most likely a passive process. In sections of the papillary region containing retinal vessels, the IPE cells seemed to have migrated into the superficial tissue of the optic nerve head. CONCLUSION: Our results demonstrate a way to access the optic nerve head easily and securely without the danger of damaging its fragile structure. This could have important implications for new therapeutic strategies in ocular neurodegenerative diseases like glaucoma. New prospects in gene therapy will require further characterisation of the potential of the IPE cell to produce neuroprotective trophic factors at the optic nerve head.

Long-term transgene expression in the RPE after gene transfer with a high-capacity adenoviral vector.

PURPOSE: To analyze duration of gene expression in the retinal pigment epithelium (RPE) in immunocompetent animals after gene transfer with a high-capacity adenoviral (HC-Ad) vector. METHODS: An HC-Ad vector was constructed to express the enhanced green fluorescence protein (EGFP) from the human CMV promoter. This vector (HC-AdFK7) was used to transduce rat RPE cells in cell culture and after subretinal injection in vivo in adult immunocompetent Wistar rats. In cell culture, expression of EGFP was analyzed by fluorescence microscopy. In vivo expression was monitored by scanning laser ophthalmoscopy and stereo fluorescence microscopy. After enucleation of the eyes, immunohistochemical and morphologic analyses by fluorescence light microscopy and electron microscopy were performed. RESULTS: In vitro, RPE cells were efficiently transduced with HC-AdFK7. Expression persisted for the observation time of 8 weeks. In vivo, the RPE was efficiently transduced with low doses of HC-AdFK7. EGFP synthesis was confirmed by antibody staining and found to be stable for the complete study period of 6 months. The neuroretina was well preserved over areas of subretinal vector administration, and significant morphologic changes were not detected. There was no accumulation of inflammatory T cells or macrophages. CONCLUSIONS: In contrast to previous results with earlier generation adenoviral vectors, subretinal injection of an HC-Ad vector in immunocompetent rats resulted in long-term transgene expression without evidence of adverse immune reactions or significant toxicity, probably because of the absence of expression of the viral gene from this vector. Thus, HC-Ad vectors are suitable for the treatment of eye disorders that require durable gene expression in the RPE.