Reduction of laser-induced choroidal neovascularization by intravitreal vasohibin-1 in monkey eyes.

PURPOSE: To determine whether intravitreal vasohibin-1 will reduce the grade of the choroidal neovascularization in monkey eyes. METHODS: Choroidal neovascularizations were induced in 12 monkey eyes by laser photocoagulation. Three monkeys were evaluated for the safety of the vasohibin-1 injections, 6 monkeys for the effects of a single injection, and 3 monkeys for repeated injections of vasohibin-1. Ophthalmoscopy, fluorescein angiography, focal electroretinograms, and optical coherence tomography were used for the evaluations. The level of vascular endothelial growth factor in the aqueous was determined by enzyme-linked immunosorbent assay. Immunohistochemistry was performed. RESULTS: An intravitreal injection of 10 µg of vasohibin-1 induced mild intraocular inflammation. Eyes with an intravitreal injection of 0.1 µg and 1.0 µg of vasohibin-1 had significant less fluorescein leakage from the choroidal neovascularizations and larger amplitude focal electroretinograms than that of vehicle-injected eyes. Similar results were obtained by repeated injections of 0.1 µg of vasohibin-1. Immunohistochemistry showed that vasohibin-1 was expressed mainly in the endothelial cells within the choroidal neovascularizations. The vascular endothelial growth factor level was not significantly altered by intravitreal vasohibin-1. CONCLUSION: The reduction of the laser-induced choroidal neovascularizations and preservation of macular function in monkey by intravitreal vasohibin-1 suggest that it should be considered for suppressing choroidal neovascularizations in humans.

Suppression of choroidal neovascularization by vasohibin-1, a vascular endothelium-derived angiogenic inhibitor.

PURPOSE. To determine the expression of vasohibin-1 during the development of experimentally induced choroidal neovascularization (CNV) and to investigate the effect of vasohibin-1 on the generation of CNV. METHODS. CNV lesions were induced in the eyes of wild-type (WT) and vasohibin-1 knockout (KO) mice by laser photocoagulation. The expression of vasohibin-1, vascular endothelial growth factor (VEGF), VEGF receptor-1 (VEGFR1), VEGFR2, and pigment epithelial-derived factor (PEDF) was determined by semiquantitative reverse transcription-polymerase chain reaction. The expression of vasohibin-1 was also examined by immunohistochemistry with anti-CD68, anti-alpha smooth muscle actin (αSMA), anti-cytokeratin, and anti-CD31. Vasohibin-1 was injected into the vitreous and the activity and size of the CNV were determined by fluorescein angiography and in choroidal flat mounts. RESULTS. Vasohibin-1 was detected not only in CD31-positive endothelial cells but also in CD68-positive macrophages and αSMA-positive retinal pigment epithelial cells. Strong vasohibin-1 expression was observed at day 28, when the CNV lesions had regressed by histologic examination. The vasohibin-1 level was significantly decreased at day 14 and increased at day 28 after laser application. Significantly less VEGFR2 expression was observed on day 4 after vasohibin-1. The expression of PEDF was not significantly changed by vasohibin-1 injection. Vasohibin-1 injection significantly suppressed the CNV, with no adverse side effects. The CNV lesions in the vasohibin-1-KO mice were significantly larger than those in the WT mice. CONCLUSIONS. The endogenous expression of vasohibin-1 is associated with the natural course of the development of CNV. Intravitreal injections of vasohibin-1 may be a method for inhibiting CNV.

Polymorphisms in ARMS2 (LOC387715) and LOXL1 genes in the Japanese with age-related macular degeneration.

PURPOSE: To determine whether polymorphisms in the ARMS2 (LOC387715) gene and the lysyl oxidase-like 1 (LOXL1) gene are associated with age-related macular degeneration (AMD) in Japanese patients. DESIGN: Clinically relevant laboratory investigation. METHODS: Forty-one unrelated Japanese subjects with dry AMD, 50 subjects with exudative (wet) AMD, and 60 subjects with polypoidal choroidal vasculopathy (PCV) were studied. The single nucleotide polymorphisms (SNPs), p.Ala69Ser of the ARMS2 gene and p.Arg141Leu of the LOXL1 gene, were amplified by polymerase chain reaction, directly sequenced, and genotyped. RESULTS: For the ARMS2 gene, the genotype frequency of the p.Ala69Ser single nucleotide polymorphism in eyes with dry AMD was not significantly different from that in the controls (P = .04), but the frequency was significantly higher in the exudative AMD group (P = 3.1 × 10(-8)) and PCV group (P = 6.9 × 10(-3)). For the LOXL1 gene, the genotype frequency of the p.Arg141Leu single nucleotide polymorphism was not statistically higher in the dry AMD and PCV groups than in the control group (dry AMD, P = .05; PCV, P = .16), but was statistically higher in the exudative AMD group (P = 6.8 × 10(-3)). Regression analyses showed significant associations between the ARMS2 gene and LOXL1 gene in patients with exudative AMD. CONCLUSIONS: The p.Ala69Ser polymorphism of the ARMS2 gene is strongly associated with exudative AMD and PCV and is associated marginally with dry AMD. The polymorphisms in the LOXL1 gene did not predispose the individual to dry AMD and PCV. These findings suggest that there is a significant association between the ARMS2 gene and LOXL1 gene in exudative AMD.

Evaluation of retinal degeneration in P27KIP1 null mouse.

PURPOSE: p27kip1 is well-known as a cell cycle inhibitor and also plays an important role for cell differentiation. We hypothesized that if we caused retinal degeneration in a p27(-/-) mouse, then the appropriate method of restoration may be different from that of wild mice and therefore suggest a therapeutic methodology for retinal regeneration. METHODS: Histological and electrophysiological (ERG) examination was performed on p27(-/-) mice retina. We injected N-methy-N-nitrosourea (MNU) to induce retinal degeneration. BrdU was used to identify the dividing cells in the retina. RESULTS: Thicker retina were observed in the p27(-/-) mice when compared to those of the p27(-/+) mice or wild type mice. Almost all retinal layers were thick and optic nerves were also enlarged. A statistically significant decrease of a and b waves amplitudes of ERG was observed in p27(-/-) mice when compared to those of the other mice. BrdU and nestin positive cells were present at the outer nuclear layer with no difference between p27(-/-) and wild type mice after MNU injection. CONCLUSION: p27(-/-) mice showed thicker retina and less retinal function than those of other mice. The MNU-induced retinal degeneration in p27(-/-) mice closely resembled the reaction of the other mice with no retinal regeneration observed in our experimental condition.

TrkB-T1 receptors on Muller cells play critical role in brain-derived neurotrophic factor-mediated photoreceptor protection against phototoxicity.

PURPOSE: To determine how brain-derived neurotrophic factor (BDNF) protects photoreceptors against phototoxicity. METHODS: Iris pigment epithelial cells (IPE) that were transduced with different concentrations of adeno-associated virus (AAV) mediated BDNF (AAV-BDNF-IPE) were transplanted into the subretinal space of rats. We also injected small interfering RNAs (siRNAs) for TrkB, a BDNF receptor. The rats were exposed to continuous light to induce phototoxicity. We examined the expression of TrkB in the retina by Western blot and immunohistochemistry. RESULTS: Significant photoreceptor protection was detected when more than 1 x 10(7) capsids/ml AAV-BDNF was transplanted. An intravitreal injection of siRNAs showed that the photoreceptor protection by AAV-BDNF-IPE was reduced by injecting the siRNA of TrkB-T1, one of the TrkB isoforms. TrkB-T1 was slightly upregulated by Western blot, and one of the cells that upregulated TrkB-T1 was Muller cells by immunohistochemistry. CONCLUSION: We conclude that Muller cells are one of the cells responsible for the expression of TrkBs, and TrkB-T1 may play a role in the protection of photoreceptors against phototoxicity.

Topical doxycycline can induce expression of BDNF in transduced retinal pigment epithelial cells transplanted into the subretinal space.

PURPOSE: To determine whether topical doxycycline (DOX) induces the expression of brain-derived neurotrophic factor (BDNF) by BDNF-transduced retinal pigment epithelial (RPE) cells transplanted into the subretinal space of rats. METHODS: A rat RPE cell line that can express BDNF by exposure to DOX was created (Tet-BDNF-RPE). The expression of BDNF was examined by ELISA, Western blot analysis, and real-time PCR. The expression of BDNF was controlled by exposure to DOX in vitro. Tet-BDNF-RPE cells were transplanted into the subretinal space of rats, and the rats were exposed to constant light 1 day or 1 month after the transplantation. The rats were followed with or without topical DOX and examined electrophysiologically and histologically. RESULTS: The expression of BDNF was upregulated by exposure of Tet-BDNF-RPE cells to DOX in vitro. The optimal concentration for inducing BDNF expression was 0.5 to 1.0 microg/mL DOX. BDNF expression was also increased in vivo by topical DOX after subretinal transplantation of Tet-BDNF-RPE cells. Statistically significant protection of the electroretinogram amplitudes were found 3 days or 1 month after transplantation, and the outer nuclear layer was better preserved 7 days or 1 month after transplantation in the rats treated by 5 or 10 mg/mL/d topical DOX than rats treated by other conditions or sham-operation rats. CONCLUSIONS: The expression of BDNF can be significantly increased by topical DOX after Tet-BDNF-RPE subretinal transplantation. Better photoreceptor protection against phototoxicity was achieved by DOX eye drops after the cell transplantation.

Expression of vasohibin, an antiangiogenic factor, in human choroidal neovascular membranes.

PURPOSE: To determine whether vasohibin, an antiangiogenic factor produced by vascular endothelial cells, is expressed in the choroidal neovascular (CNV) membranes obtained from human eyes with age-related macular degeneration (AMD) or polypoidal choroidal vasculopathy (PCV). DESIGN: Retrospective, interventional case series. METHODS: The medical charts of 21 eyes of 21 patients with AMD or PCV who underwent surgical removal of the CNV membrane were reviewed. The removed tissues were immunostained for von Willebrand Factor (vWF), vascular endothelial growth factor (VEGF), and vasohibin. The levels of the messenger ribonucleic acid of VEGF, VEGFR2, and vasohibin were determined by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) from the CNV membranes excised from nine AMD and nine PCV patients. RESULTS: The patients were divided into three groups; four patients were placed in the most active group (Group H), 13 in the less active group (Group E), and four in the nonactive group (Group S). Immunohistochemistry showed that vasohibin, vWF, and VEGF were expressed in the vascular endothelial cells in the CNV membranes and in the polypoidal vessels. RT-PCR showed that there was a strong correlation between the level of expression of VEGFR2 and vasohibin (P = .0002). Eyes with a lower vasohibin-to-VEGF ratio tended to have larger subretinal hemorrhages or vitreous hemorrhages, whereas eyes with higher vasohibin-to-VEGF ratio had subretinal fibrosislike lesions. Statistical analysis of the vasohibin-to-VEGF ratio among the three groups was significant (P = .0209). CONCLUSIONS: Vasohibin is expressed in human CNV membranes. Our results indicate that the vasohibin-to-VEGF ratio may be related with the activity of the CNV.

Iris pigment epithelial cell transplantation for degenerative retinal diseases.

The transplantation of different types of cells into the eye to treat retinal diseases has advanced in the past 20 years. One of the types of cells used for transplantation is the iris pigment epithelial (IPE) cell, because autologous IPE cells are easily obtained and their properties are similar to those of retinal pigment epithelial (RPE) cells and retinal cells. IPE cells are transplanted as; freshly isolated or cultured cells to replace defective or diseased RPE cells, genetically modified IPE cells for delivering target molecules to the retina or RPE, and retinal progenitor cells. IPE cells have also been transplanted for non-retinal disorders. The survival of the transplanted cells in the host is an important factor for the success of transplantation. Autologous IPE cells have been found in the transplanted subretinal space and were able to phagocytose rod outer segments even 6 months after transplantation. Allogeneic and xenogenic cells will not remain in the region longer than autologous cells. Allogenic cells transplanted into the subretinal space are rejected in humans. Thus, we have transplanted cultured autologous IPE cells in 56 patients with age-related macular degeneration. The long-term results (more than 2 years with a maximum of 8 years) showed that the visual acuity (VA) was significantly improved over the pre-transplantation VA, although a slight decrease of VA was observed 2 weeks after the transplantation. One patient showed a vasculitis-like lesion. IPE cells that were transduced with neurotrophic factors by plasmid or viral vectors have also been transplanted in animals. We have transduced several neurotrophic factor genes into IPE cells with a plasmid vector, adeno-associated virus, or adenovirus. Transplantation of these transduced IPE cells into the subretinal space rescued photoreceptor cells from several types of photoreceptor toxicities. In addition, transduction of a gene into the IPE cells suppressed the systemic dissemination of the viral genome. The neuroprotective effects of the IPE cells were different for the different types of neurotrophic factor, and some of the neurotrophic factors may enhance systemic immune reaction after transplantation. IPE cells have also been used as retinal progenital cells because they originate from the same cell lines that give rise to the neural retina and RPE cells. The transduction of the photoreceptor-related homeobox gene was reported to induce photoreceptor phenotypes in IPE cells. Furthermore, transplantations of IPE cells have been performed to treat central nervous system disorders. In this review, we summarize recent progress on IPE transplantation.

Polymorphisms in Complement Factor H and Hemicentin-1 genes in a Japanese population with dry-type age-related macular degeneration.

PURPOSE: To determine whether polymorphisms in the Complement Factor H (CFH) gene and the Hemicentin-1 gene at the ARMD1 locus are associated with dry age-related macular degeneration (AMD) in Japanese patients. DESIGN: Clinically relevant laboratory investigation. METHODS: Eighty unrelated Japanese patients with dry AMD and 196 Japanese control patients were studied. Two exons of the CFH gene and four exons of the Hemicentin-1 gene were amplified by polymerase chain reaction and sequenced directly. RESULTS: For the CFH gene, the frequency of the previously reported Tyr402His variant was not significantly higher in the AMD group than in the control group (P = .31). In the Hemicentin-1 gene, three sequence alterations (Asp5088Val, IVS99-13C/T, and His5245Gln) were detected, and the originally reported Gln5346Arg was not detected. CONCLUSION: The CFH gene and Hemicentin-1 genes do not appear to be involved in a statistically significant fraction of dry AMD cases in the Japanese population.

Hypothermia of 8 degrees C protects cultured retinal pigment epithelial cells and retinal ganglion cells against trypan blue toxicity.

PURPOSE: To determine whether hypothermia of 8 degrees C can protect cultured human retinal pigment epithelial (ARPE-19) cells and rat retinal ganglion cells (RGC-5) against trypan blue (TB) toxicity. DESIGN: Laboratory investigation. METHODS: ARPE-19 cells and RGC-5 were exposed to balanced salt solution as controls, and 0.05% and 0.5% TB at 37 degrees C, and at 8 degrees C for one minute. The percentage of surviving cells was determined by the resazurin test. RESULTS: TB induced a statistically significant decrease in the percentage of ARPE-19 cells surviving at 0.5% TB at 37 degrees C (P < .01). Conversely, TB induced a statistically significant decrease in the percentage of RGC-5 surviving at all conditions except for 0.05% TB at 8 degrees C (0.05% 37 degrees C; P < .05, 0.5% 37 degrees C and 8 degrees C; P < .01). CONCLUSIONS: These results indicate that reducing the temperature to 8 degrees C has a protective effect against the TB toxicity for ARPE-19 cells and RGC-5 in culture.

Recombinant AAV-transduced iris pigment epithelial cell transplantation may transfer vector to native RPE but suppress systemic dissemination.

PURPOSE: To determine whether adenoassociated virus (AAV) vectors transduced into iris pigment epithelial (IPE) cells and transplanted into the subretinal space of rats will transfer the AAV genome to the host cells and whether the vectors are disseminated systemically. METHODS: Recombinant (r)AAV was transduced into rat IPE cells and transplanted into the subretinal space of rats. For the control, rAAVs alone were injected subretinally. The transplanted IPE cells were detected by LacZ staining. Immunohistochemistry, electron microscopy, electroretinography, and fluorescein-dextran angiography were performed. DNA was extracted from various organs and blood and examined for the AAV genome by polymerase chain reaction. RESULTS: No toxicity from rAAV transduction was observed in vitro. LacZ was expressed in the transplanted cells 1 and 2 weeks after transplantation. At 4 and 12 weeks, fewer transplanted cells were detected than at 1 week, and LacZ expression was occasionally detected at the level of host retinal pigment epithelial (RPE) cells. Expression was also detected in ciliary body epithelial cells. The electroretinograms and fluorescein-dextran angiography were only mildly altered. Significantly lower levels of AAV genome were detected in the organs and blood of rats receiving rAAV-IPE cell transplants than with direct intravenous injection of AAV vectors. CONCLUSIONS: AAV-mediated LacZ was expressed in the transplanted cells after subretinal transplantation, and the transplanted IPE cells may transfer the rAAV to host tissues, such as RPE cells, long after the transplantation. This method of gene delivery did not lead to systemic dissemination of the vectors.

Introduction of the Low Vision Evaluator (LoVE) for children.

PURPOSE: To determine whether the Low Vision Evaluator (LoVE) can grade the visual acuity of young children with light perception and hand movement acuity into finer acuity steps and at what age reliable measurements can be obtained. METHODS: Two hundred twenty children were tested with the LoVE. Each eye was tested separately, and each stimulus magnitude (intensity x duration) was presented three times. Three catch trials per eye also were presented. RESULTS: Scores ranged from -8 to -1 on variable-duration tests and from 22.5 to 37.5 dB on fixed-duration tests for four children with hand movement vision. Scores ranged from -12 to 0 on variable-duration tests and from 12.5 to 40 dB on fixed-duration tests for five children with light perception vision. Reliable measurements were obtained at different times on different days. Mean scores for children with counting finger vision or better were significantly better than scores for eyes with light perception and hand movement (P < .001 and P < .01, respectively). Reliability was less for children younger than age 4 years. CONCLUSIONS: The LoVE is capable of grading the visual function of children with light perception and hand movement vision into finer steps. Reliable measurements can be obtained for children age 4 years and older.

Protection of photoreceptor cells from phototoxicity by transplanted retinal pigment epithelial cells expressing different neurotrophic factors.

Transplantation of cells or tissues and the intravitreal injection of neurotrophic factors are two methods that have been used to treat retinal diseases. The purpose of this study was to examine the effects of combining both methods: the transplantation of retinal pigment epithelial (RPE) cells expressing different neurotrophic factors. The neutrophic factors were Axokine, brain derived-neurotrophic factor (BDNF), and basic fibroblast growth factor (bFGF). The enhanced green fluorescence protein (eGFP) gene was used as a reporter gene. These genes were transduced into RPE cells by lipofection, selected by antibiotics, and transplanted into the subretinal space of 108 rats. The rats were examined at 1 week and 3 months after the transplantation to determine whether the transduced cells were present, were expressing the protein, and were able to protect photoreceptors against phototoxicity. The survival of the transplanted cells was monitored by the presence of eGFP. The degree of protection was determined by the thickness of the outer nuclear layer. Our results showed that the degree of photoreceptor protection was different for the different types of neurotrophic factors at 1 week. After 3 months, the number of surviving transplanted cell was markedly reduced, and protection was observed only with the BDNF-transduced RPE cells. A significant degree of rescue was also observed by BDNF-transduced RPE cells in the nontransplanted area of the retina at both the early and late times. Lymphocytic infiltration was not detected in the vitreous, retina, and choroid at any time. We conclude that the transplantation of BDNF-transduced RPE cells can reduce the photoreceptor damage induced by phototoxicity in the transplanted area and weakly in the nontransplanted area.

Photoreceptor protection by iris pigment epithelial transplantation transduced with AAV-mediated brain-derived neurotrophic factor gene.

PURPOSE: To determine whether subretinal transplantation of iris pigment epithelial (IPE) cells transduced with the adeno-associated virus (AAV2)-mediated brain-derived neurotrophic factor (BDNF) gene can protect photoreceptors against phototoxicity. METHODS: The BDNF gene was inserted into AAV2 (AAV2-BDNF), and the recombinant AAV2 was transduced into rat IPE (AAV2-BDNF-IPE) cells at various multiplicities of infection (MOI). The concentrations of AAV capsids and BDNF were determined by enzyme-linked immunosorbent assay (ELISA). The AAV2-BDNF-IPE cells were transplanted into the subretinal space of rats, and the rats were placed under constant light on days 1 and 90 after the transplantation. The thickness of the outer nuclear layer was measured in histologic sections and compared to that of control sections. The expression of beta-galactosidase (LacZ) in the subretinal space was confirmed by LacZ staining after AAV2-LacZ-IPE transplantation. BDNF gene expression after transplantation was confirmed by real-time polymerase chain reaction (PCR). RESULTS: Transduction efficiency increased with successive days in culture and increased with higher MOI in vitro. The expression of the BDNF gene in the subretinal space was higher in AAV-BDNF-IPE than with AAV2-LacZ-IPE or with IPE-only transplantation. LacZ expression was observed in the subretinal space 7 and 90 days after transplantation. A statistically significant photoreceptor protection was observed on days 1 and 90 in eyes receiving the AAV2-BDNF-IPE transplant, in both the superior transplant site and the inferior hemispheres which did not receive the transplant. CONCLUSIONS: Transplantation of AAV2-BDNF-IPE cells may be an alternative method of delivering neurotrophic factors to the lesion.