Leukocytes mediate retinal vascular remodeling during development and vaso-obliteration in disease.

Retinal ischemia can cause vision-threatening pathological neovascularization. The mechanisms of retinal ischemia are not fully understood, however. Here we have shown that leukocytes prune the retinal vasculature during normal development and obliterate it in disease. Beginning at postnatal day 5 (P5) in the normal rat, vascular pruning began centrally and extended peripherally, leaving behind a less dense, smaller-caliber vasculature. The pruning was correlated with retinal vascular expression of intercellular adhesion molecule-1 (ICAM-1) and coincided with an outward-moving wave of adherent leukocytes composed in part of cytotoxic T lymphocytes. The leukocytes adhered to the vasculature through CD18 and remodeled it through Fas ligand (FasL)-mediated endothelial cell apoptosis. In a model of oxygen-induced ischemic retinopathy, this process was exaggerated. Leukocytes used CD18 and FasL to obliterate the retinal vasculature, leaving behind large areas of ischemic retina. In vitro, T lymphocytes isolated from oxygen-exposed neonates induced a FasL-mediated apoptosis of hyperoxygenated endothelial cells. Targeting these pathways may prove useful in the treatment of retinal ischemia, a leading cause of vision loss and blindness.

[Pilot study to resolve problems of visual acuity assessment in grading vision of the physically handicapped--visual acuity and difficulties in daily life of age-related macular degeneration-].

PURPOSE: To resolve the problems of visual acuity assessment in grading the vision of the physically handicapped as proposed by the Subcommittee for Promoting the Realization of a Cohesive Society with the Visually Disabled, Science Council of Japan, a method suitable for assessing visual disturbances, and the relationship between the degree of visual disturbances and the degree of difficulty in activities of daily life are clarified. SUBJECTS AND METHODS: 151 persons with age-related macular degeneration were studied. Examination methods for measuring visual acuity and reading performance were studied, and interviews using the daily living task dependent on vision (DLTV) questionnaire were performed. The correlations between total DLTV score and each examination method were analyzed. The median total DLTV score for each grade of visual acuity of the better eye was calculated. RESULTS: Spearman's correlation coefficient between distance corrected visual acuity of the better eye and total DLTV score was 0.76. Median DLTV scores for visual acuities (better eye) of 0.2, 0.3, 0.4, 0.5 were 65, 73.5, 62, 79 respectively. CONCLUSION: Visual acuity can be assessed by measuring distant corrected visual acuity of the better eye and setting the upper limit of visual disturbance at either 0.3 or 0.4.

Recombinant angiopoietin-1 restores higher-order architecture of growing blood vessels in mice in the absence of mural cells.

Interactions between endothelial cells (ECs) and perivascular mural cells (MCs) via signaling molecules or physical contacts are implicated both in vascular remodeling and maintenance of vascular integrity. However, it remains unclear how MCs regulate the morphogenic activity of ECs to form an organized vascular architecture, comprising distinct artery, vein, and capillary, from a simple mesh-like network. A clear elucidation of this question requires an experimental model system in which ECs are separated from MCs and yet form vascular structures. Here we report that injection of an antagonistic mAb against PDGFR-beta into murine neonates provides such an experimental system in the retina by completely blocking MC recruitment to developing vessels. While a vascular network was formed even in the absence of MCs, it was poorly remodeled and leaky. Using this vascular system ideal for direct assessment of the activities of MC-derived molecules, we show that addition of recombinant modified angiopoietin-1 restored a hierarchical vasculature, and also rescued retinal edema and hemorrhage in the complete absence of MCs. These observations demonstrate the potential of Ang1 as a new therapeutic modality for MC dropout in diseases such as diabetic retinopathies.

Phosphatidylinositol 3-kinase/Akt regulates angiotensin II-induced inhibition of apoptosis in microvascular endothelial cells by governing survivin expression and suppression of caspase-3 activity.

Angiotensin II (Ang II) plays essential roles in vascular homeostasis, neointimal formation, and postinfarct remodeling. Although Ang II has been shown to regulate apoptosis in cardiomyocytes and vascular smooth muscle cells, its role in vascular endothelial cells (ECs) remains elusive. To address this issue, we first performed TUNEL and caspase-3 activity assays with porcine microvascular ECs challenged by serum deprivation. Ang II significantly reduced the ratio of apoptotic cells and caspase-3 activity. The Ang II type 1 receptor (AT1) was responsible for these effects. Among the signaling molecules downstream of AT1, we revealed that PI3-kinase/Akt pathway plays a predominant role in the antiapoptotic effect of Ang II. Interestingly, the expression of survivin, a central molecule of cell survival, increased after Ang II stimulation. Overexpression of a dominant-negative form of Akt abolished both Ang II-induced antiapoptosis and survivin protein expression. In a murine model of hyperoxygen-induced retinal vascular regression, AT1a knockout mice showed a significant increase in retinal avascular areas. Our data indicate that Ang II plays a critical antiapoptotic role in vascular ECs by a mechanism involving PI3-kinase/Akt activation, subsequent upregulation of survivin, and suppression of caspase-3 activity.

Recovery of rod-mediated a-wave during light-adaptation in mGluR6-deficient mice.

The purpose of this study was to compare the a-waves of mGluR6-deficient mice (KO) to that of wild-type mice (WT), and to determine whether the light-adapted electroretinogram of the KO mice originate exclusively from cones. Dark-adapted a-waves were recorded under the same conditions from both types of mice. With a 96-cd/m(2) background, the a-wave from both types of mice showed a rapid recovery over a 50-min period. The analysis of the a-waves in KO mice indicated that the recovery was determined mainly by the rod component. The light-adapted b-wave of WT mice showed no corresponding recovery. We conclude that rod contribution must be considered in the analyses of the light-adapted a-waves of KO mice.

Tlx, an orphan nuclear receptor, regulates cell numbers and astrocyte development in the developing retina.

Tlx belongs to a class of orphan nuclear receptors that underlies many aspects of neural development in the CNS. However, the fundamental roles played by Tlx in the control of eye developmental programs remain elusive. By using Tlx knock-out (KO) mice, we show here that Tlx is expressed by retinal progenitor cells in the neuroblastic layer during the period of retinal layer formation, and it is critical for controlling the generation of appropriate numbers of retinal progenies through the activities of cell cycle-related molecules, cyclin D1 and p27Kip1. Tlx expression is restricted to Müller cells in the mature retina and appears to control their proper development. Furthermore, we show that Tlx is expressed by immature astrocytes that migrate from the optic nerve onto the inner surface of the retina and is required for their generation and maturation, as assessed by honeycomb network formation and expression of R-cadherin, a critical component for vasculogenesis. The impaired astrocyte network formation on the inner retinal surface is accompanied by the loss of vasculogenesis in Tlx KO retinas. Our studies thus indicate that Tlx underlies a fundamental developmental program of retinal organization and controls the generation of the proper numbers of retinal progenies and development of glial cells during the protracted period of retinogenesis.

Iris-derived cells from adult rodents and primates adopt photoreceptor-specific phenotypes.

PURPOSE: The purpose of this study was to investigate the effects of various genes related to photoreceptor development on rodent and primate iris cells and the potential of iris cells as donor cells for retinal transplantation. METHODS: Adult rat and monkey iris tissue were cultured in serum-free medium containing basic fibroblast growth factor. Gene deliveries of Crx, Nrl, NeuroD and some combinations (Crx-Nrl, Crx-NeuroD) were performed with recombinant retrovirus. Immunocytochemistry, Western blot analysis, RT-PCR, and intracellular recording were used to examine the expression of photoreceptor-specific phenotypes in the iris-derived cells after gene transfer, . Coculture of the iris-derived cells with embryonic retinal explant was conducted, to investigate the potential integration of these cells in coculture conditions. RESULTS: Misexpression of Crx induced adult rat iris cells to express several photoreceptor-specific antigens and transcripts, such as rhodopsin, recoverin, cGMP-gated channel, arrestin, interphotoreceptor retinal-binding protein, rhodopsin kinase, and NeuroD. In primates, a combination of Crx and NeuroD was needed to induce monkey iris-derived cells to adopt photoreceptor-specific phenotypes. Furthermore, the photoreceptor-like cells derived from both rat- and primate-iris tissues showed rod photoreceptor-specific electrophysiological response to light stimuli after Crx and Crx-NeuroD gene transfer, respectively. The results further showed that iris-derived cells integrated in the developing host retina in coculture conditions. CONCLUSIONS: Adult iris-derived cultured cells of both rodents and primates expressed photoreceptor-specific phenotypes by inductions of transcription factors. These iris-derived photoreceptor-like cells have electrophysiological characteristics of rod photoreceptors. Furthermore, they can integrate in the developing retina under coculture conditions.

Drug delivery systems for vitreoretinal diseases.

The eye has an environment that is specific unto itself in terms of pharmacokinetics: the inner and outer blood-retinal barriers separate the retina and the vitreous from the systemic circulation and vitreous body, which physiologically has no cellular components, occupies the vitreous cavity, an inner space of the eye, and reduces practical convection of molecules. Considering this, development of a drug delivery system (DDS) is becoming increasingly important in the treatment of vitreoretinal diseases not only to facilitate drug efficacy but also to attenuate adverse effects. The DDS has three major goals: enhances drug permeation (e.g., iontophoresis and transscleral DDS), controls release of drugs (e.g., microspheres, liposomes, and intraocular implants), and targets drugs (e.g., prodrugs with high molecular weight and immunoconjugates). Comprehensive knowledge of these should lead to development of innovative treatment modalities.

Generation of knock-in mice carrying third cones with spectral sensitivity different from S and L cones.

Red-green color vision in primates is unique in the sense that it is mediated by two photoreceptor cells that are indistinguishable in all aspects except for their visual pigments. In order to generate an animal model for investigation of the interaction between red-green inputs at the molecular level, we applied knock-in technology and X-chromosome inactivation machinery to make a mouse model with cone cells possessing visual pigments with different spectral sensitivities. We introduced a S308A point mutation into the Green opsin gene allele on the X-chromosome. This manipulation generated a 24 nm red-shift of absorption maximum in the cone pigment with negligible functional differences in other molecular properties. Amplitudes of responses in ERG and ganglion cell recordings of homozygotes were similar to those of wild-types, although the spectral sensitivities differed. Heterozygotes showed variable spectral sensitivities of ganglion cell responses due to the different integration of the native and the S308A cone inputs on the dendritic fields. In situ hybridization experiments showed that cone cells with respective pigments formed patch-like clusters of specific L cone-types, approximately 30 mum in diameter, which were randomly distributed in the dorsal region of the retinas. Since the patch-like clustering was arranged by X-inactivation, such clustering could be present in the peripheral retinas of New World monkeys with polymorphic L pigments, indicating that our mice would be a suitable model to study evolution of the mammalian color vision system.

Argatroban attenuates leukocyte- and platelet-endothelial cell interactions after transient retinal ischemia.

BACKGROUND AND PURPOSE: Argatroban, a direct thrombin inhibitor, has been shown to reduce neural injury after transient cerebral ischemia. It has also been reported that this neuroprotective effect results from an anticoagulant function. This study was designed to evaluate quantitatively the inhibitory effects of argatroban on leukocyte- and platelet-endothelial cell interactions after transient retinal ischemia. METHODS: Retinal ischemia was induced for 60 minutes in male Long-Evans rats by temporary ligation of the optic sheath (n=342). Argatroban was administered just after induction of ischemia. Leukocyte and platelet behavior in the retinal microcirculation was then evaluated in vivo with scanning laser ophthalmoscopy. The expression of P-selectin and intracellular adhesion molecule-1 (ICAM-1) was evaluated by reverse transcription-polymerase chain reaction. After 10 days of reperfusion, ischemia-induced retinal damage was evaluated histologically. RESULTS: Treatment with argatroban suppressed leukocyte-endothelial cell interactions; the maximum numbers of rolling and accumulated leukocytes were reduced by 90.1% (P<0.05) and 58.7% (P<0.05), respectively, at 12 hours after reperfusion. Treatment with argatroban also suppressed platelet-endothelial cell interactions; the maximum numbers of rolling and adhering platelets were reduced by 91.8% (P<0.01) and 78.9% (P<0.01), respectively, at 12 hours after reperfusion. The expression of P-selectin and ICAM-1 mRNA was suppressed significantly in the argatroban-treated retinas (P<0.01). Histologic examination demonstrated the protective effect of argatroban on ischemia-induced retinal damage (P<0.01). CONCLUSIONS: Argatroban treatment suppressed leukocyte- and platelet-endothelial cell interactions after transient retinal ischemia. This inhibitory effect on postischemic blood cell-endothelial cell interactions might partially contribute to its neuroprotective effects.

Localization and activity of membrane dipeptidase in bovine ciliary epithelium.

PURPOSE: To investigate the localization and the activity of membrane dipeptidase (MDP) in the bovine eye. METHODS: A monoclonal antibody (mAb), 49C mAb, raised against bovine ciliary process was used to examine the localization of MDP. Conversion of leukotriene (LT)D4 to LTE4 was evaluated by enzyme-linked immunosorbent assay for LTE4. Hydrolytic activity (beta-lactamase activity) was evaluated with a fluorometric assay. To clarify the contribution of MDP to conversion of LTD4 and beta-lactamase activity, we separated MDP from other enzymes by 49C mAb-conjugated gel. RESULTS: The antigenic molecule of 49C mAb was shown to be MDP by amino acid sequencing. MDP was immunohistochemically detected in the ciliary pigmented and nonpigmented epithelial cells. Conversion of LTD4 to LTE4 in the ciliary process was much greater than that of the neural retina (NR). beta-Lactamase activity in the ciliary process was apparent, but that in the NR or the retinal pigment epithelium was negligible. Approximately 100% of beta-lactamase activity in the ciliary process was catalyzed by the 49C mAb-bound fraction. Conversion of LTD4 was catalyzed by the 49C mAb-bound fraction (55% of total activity) and by the unbound fraction (45% of total activity). CONCLUSIONS: This study produced the first evidence of the presence of MDP in ciliary epithelial cells. The ciliary epithelium converts LTD4 to LTE4 and shows beta-lactamase activity. Conversion of LTD4 is catalyzed by at least two enzymes, and a major part of the conversion is induced by MDP.

Potential role of the angiopoietin/tie2 system in ischemia-induced retinal neovascularization.

PURPOSE: Ischemia-induced neovascularization can cause catastrophic loss of vision in retinal disorders such as diabetic retinopathy. Recent studies have shown that the angiopoietin-Tie2 system is a major regulator of vascular integrity and is involved in pathologic angiogenesis. In the study described herein, the role of these molecules in ischemic retinal disorders was investigated. METHODS: Human epiretinal membranes were examined by immunohistochemistry, In situ hybridization, and reverse transcription-polymerase chain reaction (RT-PCR) analysis. Effects of angiopoietins on tube formation were studied in vitro in bovine retinal capillary endothelial cells (BRECs) and in a murine model of ischemia-induced retinal neovascularization. RESULTS: In human epiretinal membranes surgically obtained from eyes with ischemic retinal disorders, substantial upregulation of angiopoietin 2 (Ang2) and the receptor Tie2 was recorded than in those from eyes with nonischemic diseases, whereas expression of Ang1 was constant in all membranes. Both Ang1 and Ang2 promoted tube-forming activity and enhanced the effects of vascular endothelial growth factor (VEGF) in cultured BRECs. Soluble Tie2 fusion protein (sTie2-Fc), which precluded modulation of VEGF-dependent tube formation by the angiopoietins, suppressed both VEGF and hypoxia-conditioned, medium-induced tube-forming activity in BRECs. Intravitreal injection of sTie2-Fc, soluble Flt-1 fusion protein (sFlt-1-Fc), and both chimeric proteins suppressed retinal angiogenesis in a murine model of retinal ischemia in the order of sTie2-Fc < sFlt-1-Fc < sTie2-Fc+sFlt-1-Fc. CONCLUSIONS: These results reinforce the substantial role of the angiopoietins/Tie2 system in ischemia-induced angiogenesis as well as the VEGF system and suggest that combined inhibition of Tie2 and VEGF signaling may be more effective in halting or preventing pathologic angiogenesis in ischemic retinal disorders.

Induction of the differentiation of lentoids from primate embryonic stem cells.

PURPOSE: To produce lens cells from primate embryonic stem (ES) cells in a reproducible, controlled manner. METHODS: Cynomologus monkey ES cells were induced to differentiate by stromal cell-derived inducing activity (SDIA). The lentoids produced by this treatment were processed for immunohistochemical and immunoblotting analysis. The effect of varying the concentration of fibroblast growth factor (FGF)-2 and the density of the ES colonies plated during the differentiation process were also examined. RESULTS: After a 2- to 3-week induction period, lentoids were produced by a subpopulation of ES colonies. Western blot analysis and immunohistochemistry revealed that these lentoids expressed alphaA-crystallin and Pax6. The number of lentoids resulting from treatment increased with increasing FGF-2 concentration and plated colony density. CONCLUSIONS: The differentiation of primate ES cells into lentoids can be achieved by treatment with SIDA. ES cells can be used to facilitate a greater understanding of the mechanisms functioning in differentiation in vivo and in vitro.

Noninvasive technique for monitoring chorioretinal temperature during transpupillary thermotherapy, with a thermosensitive liposome.

PURPOSE: To develop a technique for noninvasive and real-time monitoring of chorioretinal temperature in transpupillary thermotherapy (TTT). METHOD: A modified slit lamp, which was equipped with two laser wavelengths (490 nm for illumination and fluorescein excitation and 810 nm for hyperthermia), was developed for TTT and temperature monitoring. Five types of liposomes were prepared, and their phase-transition temperatures were 40 degrees C, 46 degrees C, 47 degrees C, 48 degrees C, and 52 degrees C, respectively. Carboxyfluorescein was encapsulated in each liposome. After intravenous injection of each liposome, TTT with the modified slit lamp was performed on normal rat choroid or tissue with choroidal neovascularization (CNV). During TTT, chorioretinal temperature was monitored by observing release of fluorescein from circulating liposomes. RESULTS: Fluorescence from liposomes was initially observed around the heated lesion immediately after TTT began and disappeared rapidly when irradiation stopped. Choroidal and retinal temperatures were monitored separately. TTT for normal retina required higher power than that for normal choroid to observe fluorescence from a 40 degrees C, 46 degrees C, and 47 degrees C liposome. Retinal whitening was observed after TTT at a high-power setting. TTT for CNV required higher laser power than that for the normal choroid and retina. CONCLUSIONS: The results demonstrate the potential use of a noninvasive monitoring technique of chorioretinal temperature during TTT. The method should be useful to establish the TTT setting and achieve the optimal temperature increase in CNV.

Mitochondrial ATP-sensitive potassium channel: a novel site for neuroprotection.

PURPOSE: It has been shown that bradykinin (BK) protects retinal neurons against glutamate excitotoxicity, but it was not clear how BK inhibits glutamate excitotoxicity. The purpose of this study was to investigate the effect of opening the mitochondrial adenosine triphosphate (ATP)-sensitive potassium (Mit K (ATP)) channel on glutamate excitotoxicity and the protective effect of BK using cultured retinal neurons. METHODS: Primary cultures were obtained from the retina of fetal rats (gestation days 17-19). Glutamate neurotoxicity was assessed by 10-minute exposure to 1 mM glutamate followed by 1-hour incubation in glutamate-free medium, using the trypan blue exclusion method. BK, diazoxide (the opener of the Mit K (ATP) channel), 5HD, and glibenclamide (blockers of the Mit K (ATP) channel) were applied simultaneously with glutamate. Mitochondrial membrane potential was measured as the ratio of 590:527 nm fluorescence of JC-1. RESULTS: Cell viability was markedly reduced by 10-minute exposure to 1 mM glutamate followed by 1-hour incubation in glutamate-free medium, and glutamate induced mitochondrial depolarization of retinal neurons. BK and diazoxide protected retinal neurons against glutamate excitotoxicity and inhibited glutamate-induced mitochondrial depolarization. These actions of BK and diazoxide were inhibited by the coapplication of 5HD and glibenclamide. Furthermore, diazoxide inhibited the sodium nitroprusside (SNP, NO donor) toxicity, but did not inhibit the 3-morpholinosydnonimine (SIN-1, NO, and superoxide donor) toxicity. CONCLUSIONS: These results suggest that BK and diazoxide protect retinal neurons against glutamate excitotoxicity by opening the Mit K (ATP) channel. It is suggested that opening of the Mit K (ATP) channel inhibited glutamate-induced generation of superoxide.

In vivo analysis of choroidal circulation by continuous laser-targeted angiography in the rat.

PURPOSE: To obtain high-quality angiograms of the rat choriocapillaris with continuous laser-targeted angiography (LTA), for the purpose of assessing the choroidal circulation system in vivo by studying the patterns of the images. METHODS: A slit lamp was modified to incorporate two kinds of lasers (argon and diode). Carboxyfluorescein was encapsulated in heat-sensitive liposomes and injected intravenously. Encapsulated carboxyfluorescein was released locally by applying a continuous heat beam provided by diode laser (810 nm) with various powers. Video angiograms were generated with excitation illumination provided by argon laser (488 and 514 nm) to observe highly selective images of the choriocapillaris. RESULTS: Three distinct phases (filling, plateau, and draining) were observed in fluorescent images of choriocapillaris by applying the diode laser continuously. In the plateau phase, a lobe-shaped area of choriocapillaris peripheral to the laser site was illuminated, and this finite area did not change in size with continuous laser application to the same spot. When laser power was increased, a larger area of choriocapillaris was illuminated in the plateau phase. The filling and draining phases demonstrated the flow patterns in choriocapillaris lobules, which filled from a central spot and drained along a peripheral ring. CONCLUSIONS: This study showed that the rat choriocapillaris is divided into independent functional units and that the choroidal circulation is segmental under normal conditions. The results implied that in LTA, the diode laser warms up a choroidal artery and the released fluorescein flows downstream to an area of choriocapillaris fed by the same artery. LTA appeared to be a powerful method to analyze choroidal circulation in vivo.

Analysis of choriocapillaris flow patterns by continuous laser-targeted angiography in monkeys.

PURPOSE: To investigate choriocapillaris flow patterns and its segmental distribution in monkeys by continuous laser-targeted angiography (LTA). METHODS: A slit lamp was modified to incorporate two kinds of lasers (argon and diode). Carboxyfluorescein (CF) was encapsulated in heat-sensitive liposomes and injected intravenously. Encapsulated CF was released locally by applying a continuous heat beam provided by the diode laser (810 nm). Video angiograms were generated with excitation illumination provided by the argon laser (488 and 514 nm), to observe selective images of the choriocapillaris. RESULTS: Continuous application of the diode laser disclosed three distinct phases (filling, plateau, and draining) of fluorescent images of the choriocapillaris. In the plateau phase, a cluster of lobules fed by a common arteriole was uniformly illuminated. This defined area did not change in size while a continuous diode laser was applied to the same spot. Only in posterior regions did the angiograms demonstrate that during the filling and draining phases each lobule was filled from a central spot and drained along a peripheral ring, showing honeycomb flow patterns. In peripheral regions, large choroidal vessels as well as choriocapillaris were observed. CONCLUSIONS: Continuous LTA demonstrated clusters of lobules fed by a common arteriole, and each cluster was found to be functionally independent. There were regional differences in choriocapillaris flow patterns, which suggests that the choriocapillaris provides a more highly efficient system of outflow in posterior regions than in peripheral regions. This modified LTA method appears to be useful in analyzing choroidal circulation in vivo.

Spatiotemporal expression patterns of N-syndecan, a transmembrane heparan sulfate proteoglycan, in developing retina.

PURPOSE: N-syndecan is a transmembrane heparan sulfate proteoglycan, that is highly expressed in neural tissues. In the current study, changes in N-syndecan expression during retinal development were examined. METHODS: Localization of N-syndecan in developing rat retina was examined by immunohistochemistry and in situ hybridization. The amount of the core protein was evaluated by immunoblot analysis, using retinal homogenates at various developmental stages. In addition, mRNA expression was semiquantified by reverse transcription-polymerase chain reaction (RT-PCR). To understand better the localization of N-syndecan in retinal neuronal cells, we performed immunocytochemistry using retinal ganglion cells in culture. RESULTS: N-syndecan is highly expressed in nerve fiber-rich layers of the retina at early postnatal stages (between postnatal day [P]0 and P14). In contrast, immunoreactivity was faint during embryonic stages and late postnatal stages. In addition, in retinal flatmounted sections, N-syndecan immunoreactivity was observed on the axons of retinal ganglion cells. Intense signals were observed in the ganglion cell layer during in situ hybridization. Immunoblot analyses demonstrated that the amount of N-syndecan core protein reached a peak at approximately P14. The RT-PCR analyses using N-syndecan primers showed that an intense amplified band was observed in the cDNA derived from P14 retinas, whereas only faint bands were detected in the embryonic day (E)16 and P42 retinas. In retinal ganglion cells in culture, N-syndecan was located on the long, extended neurites. CONCLUSIONS: The data show that N-syndecan is transiently expressed, primarily in retinal neural fibers, during retinal development, indicating that it may be involved in formation of the retinal neural network.

Targeting of interferon to choroidal neovascularization by use of dextran and metal coordination.

PURPOSE: Bioactive proteins such as interferon (IFN) have been reported to be combined with water-soluble polymers, such as dextran, through metal coordination, without need for complicated procedures. In the current study, the targeting and inhibitory effects of IFN combined with dextran on experimental choroidal neovascularization (CNV) were studied in vivo. METHODS: Interferon (IFN)beta was conjugated to dextran, which has metal-chelating, diethylenetriaminepentaacetic acid (DTPA) residues. Based on metal coordination, conjugation of IFNbeta with DTPA-dextran resulted from simply mixing both substances in an aqueous solution containing Zn(2+). The effects of IFNbeta on the proliferation of human umbilical vein endothelial cells (HUVECs) and bovine retinal pigment epithelial cells (BRPECs) were evaluated. To evaluate the activity loss of IFNbeta by conjugation, the effect of the conjugate on HUVECs was compared with that of free IFNbeta. Experimental CNV was induced by subretinal injection of gelatin microspheres containing basic fibroblast growth factor in rabbits. The rabbits with CNV were intravenously treated twice weekly with 7.5 million international units (MIU)/kg per day free IFNbeta (for 4 weeks), with IFNbeta-DTPA-dextran conjugate containing 7.5 (for 2 weeks) or 0.75 (for 4 weeks) MIU/kg per day IFNbeta, or with saline. The effects of these substances were evaluated by fluorescein angiography and histology. To observe the accumulation of conjugate, the doses of IFNbeta in CNV tissues were measured by enzyme-linked immunosorbent assay. RESULTS: IFNbeta inhibited the growth of HUVECs and enhanced the proliferation of BRPECs. The conjugate seemed to preserve approximately 44% of IFNbeta activity. Although both doses of IFNbeta-DTPA-dextran inhibited progression of CNV in rabbits, longer term administration of a lower dose of IFNbeta-DTPA-dextran had a sustained inhibitory effect on progression of CNV (P < 0.05). Histologic studies revealed the inhibitory effect of IFNbeta-DTPA-dextran on progression of CNV. This conjugate prolonged the plasma half-life of IFNbeta and enabled IFNbeta to accumulate in the CNV in rabbits. CONCLUSIONS: In this study, human IFNbeta was successfully used to target CNV, an enhanced antiangiogenic effect was achieved by combining it with dextran, based on metal coordination. This targeted delivery of IFNbeta may have potential as a treatment modality for CNV.

New insight into the functional role of acetylcholine in developing embryonic rat retinal neurons.

PURPOSE: To examine the effects of acetylcholine (ACh) on glutamate-induced neurotoxicity in embryonic rat retinal neurons. METHODS: Primary cultures were obtained from rat retinas at embryonic days 17 to 19. Cultured cells were exposed to glutamate for 10 minutes, followed by incubation in glutamate-free medium for 1 hour. Drugs were added to the incubation medium for 1 to 24 hours until immediately before glutamate exposure and were removed from culture medium during glutamate exposure and the postincubation period. The neurotoxic effects on retinal cultures were quantitatively assessed by the trypan blue exclusion method. RESULTS: Cell viability was markedly reduced by 10-minute exposure to 500 microM glutamate followed by a 1-hour incubation in glutamate-free medium. Incubating the cultures with 1 microM ACh for 12 hours before glutamate exposure reduced glutamate neurotoxicity. A similar effect was induced by application of carbachol (1 microM). The protective effect of ACh against glutamate neurotoxicity was inhibited by a nicotinic acetylcholine receptor (nAChR) antagonist, mecamylamine (0.5 microM), whereas a muscarinic acetylcholine receptor (mAChR) antagonist, atropine (0.5 microM) did not affect ACh-induced protection. In addition, a similar protection was induced by application of nicotine (1 microM), but not by muscarine (1 microM). Pretreatment with nicotine induced a protective effect in a time-dependent manner, ranging from 1 to 12 hours. Pretreatment with nicotine at concentrations ranging from 0.001 to 1 microM induced dose-dependent protection against glutamate neurotoxicity. Furthermore, the protective action of nicotine was inhibited by simultaneous application of dopamine D1 receptor antagonist, SCH23390 (1 microM), with nicotine, whereas a dopamine D2 receptor antagonist, domperidone (1 microM), did not affect nicotine-induced protection. CONCLUSIONS: These results suggest that pretreatment of cultured rat retinal neurons with ACh or the nAChR agonists, nicotine and carbachol, has a protective action against glutamate neurotoxicity through nAChRs and that the dopamine release induced by nicotinic stimulation subsequently protects the retinal neurons by way of dopamine D1 receptors.