Ryosuke Wakusawa

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.

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.

Polymorphisms in ARMS2 (LOC387715) and LOXL1 Genes in the Japanese With Age-Related Macular Degeneration

a t e a g The point of this is to say that we do not believe that evaluation of the posterior curvature, Descemet membrane stripping, and careful peripheral scraping is the ONLY way to make the tissue attach, but we do believe that these steps decrease the rate of detachment to less than 3%, not only in this setting, but also for our published series of hundreds of routine cases. We also have observed that thinner donor tissue has a greater propensity to conform to the variable curvature of the recipient bed. It may be that Mifflin and associates were using tissue thinner than prior reports of endothelial keratoplasty under PK and, because of this, had better conformation of their tissue to the posterior protuberances of the PK edge recipient beds, enhancing their adherence rate in this unique setting. In our series, by fitting the tissue between protuberances, we could use donor tissues of any thickness, without worrying about conforming to the recipient bed edges. We recommend that Mifflin and associates perform donor tissue thickness analysis when they submit their longer manuscript for peer review of their study. Finally, although adherence of the donor tissue is enhanced by the removal of interface fluid, we caution Mifflin and associates about the routine use of venting incisions to accomplish this. There is now a plethora of publications demonstrating the short-term and the longterm liabilities, such as infections, melting, and epithelial downgrowth, that can occur because of these incisions. We have used surface sweeping without venting incisions to evacuate interface fluid for essentially all of our 1300 cases over the past 11 years and have avoided these liabilities, yet retained the lowest dislocation rates in the world. Once again, we thank Mifflin and associates for their interest in our article, and we look forward to reading their expanded manuscript in the future.

Vitreous levels of vasohibin-1 and vascular endothelial growth factor in patients with proliferative diabetic retinopathy.

Abbreviations PDR proliferative diabetic retinopathy PEDF pigment epithelium-derived factor VEGF vascular endothelial growth factor To the Editor: Intraocular neovascularisation develops in many ischaemic retinal diseases, e.g. diabetic retinopathy, ischaemic retinal vein occlusion and retinopathy of prematurity. The new vessels are fragile and often rupture, leading to vitreous haemorrhage, tractional retinal detachment, neovascular glaucoma and subsequent vision decrease. The formation of new vessels is dependent on a local balance of stimulators and inhibitors of angiogenesis [1]. Among the stimulators, vascular endothelial growth factor (VEGF) has been shown to play a major role in mediating active neovascularisation in patients with diabetic retinopathy [2]. In addition, several studies have shown that the concentration of VEGF in the intraocular fluids was significantly elevated in eyes with proliferative diabetic retinopathy (PDR) [3–5]. On the other hand, pigment epithelium-derived factor (PEDF) is a potent inhibitor of angiogenesis, and lower levels of PEDF have been found in the vitreous of eyes with active diabetic retinopathy [4]. It has also been shown that the vitreous level of endostatin, another inhibitor of angiogenesis, is correlated with the level of VEGF and that endostatin is produced in the fibrovascular membrane of eyes with PDR [5]. Vasohibin-1, a novel angiogenesis inhibitor, is mainly produced in endothelial cells and is induced by stimulation with VEGF or fibroblast growth factor 2. Vasohibin-1 selectively affects endothelial cells and inhibits angiogenesis [6]. We therefore hypothesised that vasohibin-1 is present in the vitreous of eyes with PDR and is associated with the vitreous level of VEGF. To examine this hypothesis, we measured the vitreous levels of vasohibin-1 and VEGF in 49 samples from 46 patients. This study was conducted in accordance with the tenets of the Declaration of Helsinki as revised in 2000 and was carried out with the approval of the Institutional Review Board of Tohoku University. Informed consent was obtained from all patients Diabetologia (2009) 52:359–361 DOI 10.1007/s00125-008-1229-z

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.

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 × 107 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.

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.

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.

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 &mgr;g of vasohibin-1 induced mild intraocular inflammation. Eyes with an intravitreal injection of 0.1 &mgr;g and 1.0 &mgr;g 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 &mgr;g 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.

Vasohibin-1 and Retinal Pigment Epithelium

Vascular endothelial growth factor (VEGF) is one of the main factors for inducing choroidal neovascularization in patients with age-related macular degeneration (AMD). Retinal pigment epithelium (RPE) is one of the main sources for expressing VEGF. Vasohibin-1 is a VEGF-inducible gene in human cultured endothelial cells with antiangiogenic properties. We examined the effects of vasohibin-1 against RPE. We used rat RPE cell line, RPE-J. Cobalt chloride and low glucose and oxygen supply were used for hypoxic stress. Western blot analysis and real-time PCR were performed to detect the expression of vasohibin-1 and VEGF in the RPE-J. Cobalt chloride or low oxygen and low glucose enhanced VEGF expression, whereas statistically significant less vasohibin-1 expression was observed. RPE cell dynamics was monitored using xCELLigence System for real-time cell analysis during culture. External VEGF (0.2–2 nM) enhanced Cell Index (CI), such as cell viability, number, and adhesion to the plates significantly at 2% oxygen and no glucose when compared to those of vehicle or other concentration of VEGF. Conversely, external vasohibin-1 (2 nM) showed lower CI at the indicated condition. Vasohibin-1 also reduced VEGF-induced CI index. These results were not observed under standard culture condition. When we performed MTS assay or cell count, external vasohibin-1 also showed comparable results. When we transduced full-length vasohibin-1 cDNA in RPE-J, the internal vasohibin-1 showed less VEGF expression than that of control vector-transduced RPE-J. In summary, vasohibin-1 showed opposite results as that of VEGF on the RPE-J, especially under hypoxic condition.