Activator of G-protein signaling 8 is involved in VEGF-induced choroidal neovascularization.

Choroidal neovascularization (CNV) is associated with age-related macular degeneration (AMD), a major cause of vision loss among elderly people. Vascular endothelial cell growth factor (VEGF) is essential for the development and progression of AMD, and VEGF signaling molecules are effective targets for the treatment of AMD. We recently reported that activator of G-protein signaling 8 (AGS8), a receptor-independent Gßγ regulator, is involved in VEGF-induced angiogenesis in cultured endothelial cells (EC); however, the role of AGS8 in CNV is not yet understood. This study aimed to explore the role of AGS8 in CNV in cultured cells, explanted choroid tissue, and laser-induced CNV in a mouse AMD model. AGS8 knockdown in cultured choroidal EC inhibited VEGF-induced VEGFR-2 phosphorylation, cell proliferation, and migration. AGS8 knockdown also downregulated cell sprouting from mouse choroidal tissue in ex vivo culture. A mouse model of laser-induced CNV, created to analyze the roles of AGS8 in vivo, demonstrated that AGS8 mRNA was significantly upregulated in choroidal lesions and AGS8 was specifically expressed in the neovasculature. Local AGS8 knockdown in intravitreal tissue significantly inhibited laser-induced AGS8 upregulation and suppressed CNV, suggesting that AGS8 knockdown in the choroid has therapeutic potential for AMD. Together, these results demonstrate that AGS8 plays critical roles in VEGF-induced CNV.

Effect of oral tranexamic acid on macular edema associated with retinal vein occlusion or diabetes.

PURPOSE: Tranexamic acid (TXA) is a widely used antifibrinolytic agent that can also cause a decrease in vascular permeability. We hypothesized that TXA could improve macular edema (ME) that is caused by an increase in retinal vascular permeability. The aim of this study is to evaluate the efficacy of oral TXA for ME associated with retinal vein occlusion (RVO) or diabetic ME (DME). PATIENTS AND METHODS: Oral TXA (1,500 mg daily for 2 weeks) was administered to patients with persistent ME secondary to RVO (7 eyes) and DME (7 eyes). After 2 weeks (ie, the final day of administration) and 6 weeks (ie, 4 weeks after the final administration), best-corrected visual acuity and central macular thickness (CMT) were measured and compared with baseline. Analyses were performed for RVO and DME cases. No other treatment was performed during the study period. RESULTS: In RVO cases, significant improvement in CMT was found between baseline (467.7±121.4 µm) and 2-week measurements after treatment (428.7±110.5 µm, p=0.024). No significant change was found in CMT between measurements taken at baseline and 6 weeks after treatment. In DME cases, no significant change was found in CMT between measurements taken at baseline and 2 or 6 weeks after treatment. In all analyses of best-corrected visual acuity, no significant change was observed. CONCLUSION: The results support the hypothesis that plasmin plays a role in the development of ME associated with RVO, and oral TXA administration may be useful as an adjuvant treatment when combined with other agents such as anti-vascular endothelial growth factor.

Decreased VEGF-A and sustained PEDF expression in a human retinal pigment epithelium cell line cultured under hypothermia.

BACKGROUND: Previous reports have described a decrease in retinal temperature and clinical improvement of wet age-related macular degeneration (AMD) after vitrectomy. We hypothesized that the retinal temperature decrease after vitrectomy plays a part in the suppression of wet AMD development. To test this hypothesis, we evaluated the temperature dependence of the expression of vascular endothelial growth factor-A (VEGF-A) and in vitro angiogenesis in retinal pigment epithelium (RPE). RESULTS: We cultured ARPE-19 cells at 37, 35, 33 and 31 °C and measured the expression of VEGF-A, VEGF-A splicing variants, and pigment epithelium-derived factor (PEDF). We performed an in vitro tube formation assay. The dehydrogenase activity was also evaluated at each temperature. Expression of VEGF-A significantly decreased with decreased temperature while PEDF expression did not. VEGF165 expression and in vitro angiogenesis also were temperature dependent. The dehydrogenase activity significantly decreased as the culture temperature decreased. CONCLUSIONS: RPE cultured under hypothermia that decreased cellular metabolism also had decreased VEGF-A and sustained PEDF expression, creating an anti-angiogenic environment. This mechanism may be associated with a beneficial effect after vitrectomy in patients with wet AMD.

Valsalva-like retinopathy spontaneously occurred after ocular massage.

PURPOSE: The aim of this study is to describe a case of Valsalva-like retinopathy that occurred after ocular massage. CASE PRESENTATION: A healthy 44-year-old Japanese female had massaged her eye with strong pressure several times. Subsequently, she noticed a loss in the left central vision. A left-eye fundus examination showed a dense preretinal hemorrhage located under the internal limiting membrane at the posterior pole and a mild vitreous hemorrhage. We performed a neodymium-doped yttrium-aluminium-garnet laser membranotomy to perforate the internal limiting membrane. Her best-corrected visual acuity improved from 0.01 to 1.0. No retinal vascular abnormalities in the macular area were found. CONCLUSION: Ocular massage can cause Valsalva-like retinopathy.

Decreased VEGF-A and sustained PEDF expression in a human retinal pigment epithelium cell line cultured under hypothermia

BACKGROUND: Previous reports have described a decrease in retinal temperature and clinical improvement of wet age-related macular degeneration (AMD) after vitrectomy. We hypothesized that the retinal temperature decrease after vitrectomy plays a part in the suppression of wet AMD development. To test this hypothesis, we evaluated the temperature dependence of the expression of vascular endothelial growth factor-A (VEGF-A) and in vitro angiogen-esis in retinal pigment epithelium (RPE). RESULTS: We cultured ARPE-19 cells at 37, 35, 33 and 31°C and measured the expression of VEGF-A, VEGF-A splicing variants, and pigment epithelium-derived factor (PEDF). We performed an in vitro tube formation assay. The dehydrogenase activity was also evaluated at each temperature. Expression of VEGF-A significantly decreased with decreased temperature while PEDF expression did not. VEGF165 expression and in vitro angiogenesis also were temperature dependent. The dehydrogenase activity significantly decreased as the culture temperature decreased. CONCLUSIONS: RPE cultured under hypothermia that decreased cellular metabolism also had decreased VEGF-A and sustained PEDF expression, creating an anti-angiogenic environment. This mechanism may be associated with a beneficial effect after vitrectomy in patients with wet AMD.

Recurrent vitreous hemorrhage associated with regressed retinopathy of prematurity in a 47-year-old patient: a case report.

INTRODUCTION: Vitreous hemorrhage associated with retinopathy of prematurity is often seen in childhood, but adult onset without retinal break is rare. We describe a case of recurrent vitreous hemorrhage associated with regressed retinopathy of prematurity in a 47-year-old patient. CASE PRESENTATION: A 47-year-old Japanese woman with a history of retinopathy of prematurity presented with a visual disturbance in her left eye due to vitreous hemorrhage. Because the vitreous hemorrhage was recurrent and refractory, we performed pars plana vitrectomy combined with lens extraction by phacoemulsification and intraocular lens implantation. No retinal break or retinal detachment was found. No vitreous hemorrhage or other complication occurred in the first six months after surgery. CONCLUSIONS: Vitrectomy, potentially in combination with lens extraction, should be considered in adult-onset recurrent vitreous hemorrhage associated with retinopathy of prematurity.

Macroaneurysm on the optic disc in a patient with aortic dissection.

PURPOSE: We report a case of macroaneurysm on the optic disc, a rare location, accompanied by vitreous hemorrhage in a patient with aortic dissection. METHODS: A 60-year-old female with a history of aortic dissection at the age of 51 presented with visual disturbance owing to vitreous hemorrhage in her right eye. During vitrectomy, we found a large macroaneurysm on the optic disc that was beating and oozing blood. However, the macroaneurysm was not treated. RESULTS: The macroaneurysm gradually shrunk, and the beating and oozing of blood disappeared accordingly. CONCLUSION: Up to now, there have been no reports of macroaneurysm on the optic disc in aortic dissection patients. The association between macroaneurysm on the optic disc and aortic dissection is unclear; therefore, additional case reports may be necessary. To the best of our knowledge, our case is the first one reported in the literature.

Intravitreal Injection of Bevacizumab to Treat a Macular Edema Caused by Leber's Miliary Aneurysm.

PURPOSE: Leber's miliary aneurysm is a variant of Coats' disease and similar to type 1 idiopathic macular telangiectasia. A recent report showed that an intravitreal injection of bevacizumab (IVB) was effective in an adult patient with type 1 idiopathic macular telangiectasia. We describe our experience with an adult patient with a macular edema caused by Leber's miliary aneurysm, which had not been resolved by prior retinal laser photocoagulation, who underwent IVB. METHODS: We investigated the ocular characteristics of the patient before and after a single administration of IVB. RESULTS: The macular edema had partially improved 1 month after IVB, but then it worsened. We performed laser photocoagulation to treat the aneurysms near the fovea. The macular edema improved, but there was no improvement in visual acuity. CONCLUSION: In this patient, the effect of IVB was limited.

Choroidal and optic disc metastases from breast cancer and their response to combination pharmacotherapy with tamoxifen, cyclophosphamide hydrate, letrozole, and bevacizumab.

PURPOSE: The purpose of this study was to report a patient with choroidal and optic disc metastases from breast cancer and the response to combination pharmacotherapy with tamoxifen, cyclophosphamide hydrate, letrozole, and bevacizumab. METHODS: We report a case of a 51-year-old woman with a medical history of breast cancer treated with mastectomy at 16 years earlier followed by 5'-deoxy-5-fluorouridine and tamoxifen for 12 years, who has been followed by annual check-up, with no additional findings. Two weeks prior to her first visit to our hospital, she noted blurred and abnormal color vision and field loss in her right eye. Her right eye showed choroidal metastasis from breast cancer, with retinal detachment involving the macula and inferior periphery. RESULTS: Multiple metastases involving bones, lymph nodes in the mediastinum, ovaries, and pelvic nodes were diagnosed after ocular metastases were established. Combination therapy with tamoxifen, cyclophosphamide hydrate, letrozole, and bevacizumab was administered to the patient. The retinal detachment and optic disc metastasis improved dramatically. CONCLUSIONS: The patient underwent combination pharmacotherapy with tamoxifen, cyclophosphamide hydrate, letrozole, and bevacizumab, which was effective in the treatment of ocular metastases from breast cancer.

Effect of infliximab on tumor necrosis factor-alpha-induced alterations in retinal microvascular endothelial cells and retinal pigment epithelial cells.

PURPOSE: Tumor necrosis factor-alpha (TNF-α) may disrupt the extracellular matrix components comprising the blood-retinal barrier (BRB) in patients with posterior uveitis, such as Behçet's disease. We investigated changes in the mRNA expression levels of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in human BRB cells in the presence of TNF-α in vitro and examined the effect of infliximab addition. METHODS: Cells were cultured in the presence or absence of TNF-α, and TNF-α-exposed cells were treated with or without infliximab. We measured the expression levels of MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1, and TIMP-2 mRNA in human retinal microvascular endothelial ACBRI181 cells and retinal pigment epithelial ARPE-19 cells by real-time polymerase chain reaction. The cell-derived proteins degraded by MMP were observed after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. RESULTS: Expression of MMP-3 increased and TIMP-1 decreased in the presence of 10 ng/mL TNF-α in ACBRI181 cells. Expression of MMP-1 increased and TIMP-2 decreased in the presence of 10 ng/mL TNF-α in ARPE-19 cells. These altered levels of expression were reversed by the addition of infliximab. The cell-derived proteins degraded by MMP-1 and -3 were detected in each set of cells. CONCLUSIONS: The presence of TNF-α altered expression of MMPs and TIMPs in cells comprising the BRB, and infliximab counteracted this alteration.

Increase in matrix metalloproteinase-2 level in the chicken retina after laser photocoagulation.

BACKGROUND AND OBJECTIVE: We investigated the levels of matrix metalloproteinase-2 (MMP-2), which has been implicated in various vitreoretinal diseases, in the retina after laser photocoagulation (LPC). MATERIALS AND METHODS: The time course of MMP-2 expression in 2-day-old chicken retinas before and 6 hours, 12 hours, 1 day, 2 days, 4 days, 8 days, 16 days, and 32 days after LPC was determined by real-time PCR and gelatin zymography. The basal level of MMP-2 in the retina and vitreous was also measured by gelatin zymography. MMP-2 localization in the retina was examined by immunohistochemistry. The localization of MMP-2 mRNA was determined by fluorescent in situ hybridization. The internal limiting membrane (ILM) was observed by scanning electron microscopy. RESULTS: MMP-2 mRNA expression in the retina peaked at day 4, but gelatin zymography showed that MMP-2 peaked 6 hours after LPC and the significant increase in the level of active MMP-2 lasted for more than 4 days. The concentration of MMP-2 in the vitreous was significantly higher than that in the retina. A distinct MMP-2 signal around the ILM was identified 6 hours after LPC, but MMP-2 mRNA was not detected there. Electron microscopy showed a damaged retinal surface after LPC. CONCLUSION AND OUTLOOK: The significant increase in retinal MMP-2 which lasted for more than 4 days after LPC may be induced by influx from the vitreous into the retina. This MMP-2 dynamics may contribute to pathological processes in the retina after LPC.

Distinct response to heparin by two chicken brain type creatine kinase subunits.

In the chicken, two creatine kinase-type B (B-CK) isoproteins, Ba- and Bb-CK, both of which are derived from a single copy gene by alternative splicing, dimerize in neural tissues. The two isoproteins contain distinct N-terminal portions, but their functional difference remains unknown. We investigated the binding affinities of Ba- and Bb-CK to heparin, hyaluronan and chondroitin sulfates, and examined the influence of these glycosaminoglycans on enzyme activity. Chicken retinal samples analyzed by Western blotting and amino acid sequence study after two-dimensional gel electrophoresis showed that heparin binds Bb-CK, but not Ba-CK, while hyaluronan and chondroitin sulfates showed no interaction with either isoprotein. Using fusion proteins covering the distinct N-terminal portions, we also showed that heparin did not react with the N-terminus of Ba-CK, but did react with that of Bb-CK. Site-directed mutagenesis of basic amino acids found in the N-terminal portion of Bb-CK identified three basic amino acids critical for this binding. Furthermore, heparin dose-dependently inhibited the enzymatic activities of Ba-CK; Bb-CK activities were less intensely inhibited. Hyaluronan and chondroitin sulfates had no effects on the activities of these enzymes. Thus, the N-terminal portion of B-CK is critical to mediate its affinity to heparin and control enzyme activity, which may be important for regulating energy metabolism in neural tissues such as brain and retina, unique organs abundant in heparan sulfates.

Competitive binding of heparin with hyaluronan to a specific motif in SPACR.

The critical hyaluronan binding motif (HABM) in sialoprotein associated with cones and rods (SPACR) has already been determined. As sialoproteoglycan associated with cones and rods, another interphotoreceptor matrix molecule, binds to chondroitin sulfate and heparin with or without the employment of HABMs, respectively, we evaluated and compared the binding of these glycosaminoglycans to SPACR. A western blotting study in combination with inhibition assays showed that heparin bound specifically to SPACR. A series of GST fusion proteins covering the whole SPACR molecule narrowed down the region responsible for the binding. Finally, a site-directed mutagenesis assay demonstrated that the critical HABM also acts as a specific binding site for heparin. These results were supported with mutual inhibitions by hyaluronan and heparin in analyses using GST fusion proteins and native SPACR derived from retina. Thus, these glycosaminoglycans bind to SPACR in a different manner than to sialoproteoglycan associated with cones and rods. The competitive binding between hyaluronan and heparin to SPACR, mediated through the identical HABM, may dominate the functions of SPACR, in turn involving physiological and pathological processes involved in retinal development, aging and other related disorders.

Characterization of a motif for specific binding to hyaluronan in chicken SPACR.

The chicken sialoprotein associated with cones and rods (SPACR) binds to hyaluronan (HA) in the interphotoreceptor matrix space, but the motif for HA binding is still unknown. This study was conducted to determine the critical site required for specific binding to HA. Western blotting study showed that SPACR binds biotinylated HA, and this interaction was specifically inhibited by unlabeled HA. A series of GST fusion proteins covering whole SPACR was prepared, and reactivity with HA was individually screened to narrow down the region for the binding. Further, putative HA-binding motif found near the carboxyl-terminus of SPACR was mutated by site-directed mutagenesis to identify the critical binding site. Finally, we showed that native SPACR derived from retina similarly binds to HA-affinity column under both reducing and non-reducing conditions. These results revealed that the specific putative HA-binding motif is located near the carboxyl-terminus of chicken SPACR, and suggested that a structural integrity such as folded structure is not largely involved in the HA binding.

Versican and fibrillin-1 form a major hyaluronan-binding complex in the ciliary body.

PURPOSE: In this study, biochemistry, molecular biology, immunohistochemistry, and electron microscopy techniques were used to examine whether versican, which is known to bind fibrillin-1, interacts with fibrillin-1 in the ciliary body and vitreous, and whether the versican in this complex binds to hyaluronan. METHODS: The new polyclonal antibodies against the amino and carboxyl termini of versican were raised and characterized. The mRNA expression levels of versican and fibrillin-1 were analyzed by RT-PCR and real-time PCR, and their protein levels were evaluated by Western blot analysis and immunohistochemistry. Isolation of versican bound to fibrillin-1-containing microfibrils from ciliary bodies was performed by extraction studies. Slot-blot analyses and rotary shadowing electron microscopy were applied to identify versican associated with fibrillin-1-containing microfibrils after gel filtration chromatography and density gradient centrifugation. RESULTS: The newly prepared polyclonal antibodies recognized amino and carboxyl termini of chicken versican. Versican, principally V0 and V1, was found to be securely bound to fibrillin-1-containing microfibrils, forming a major hyaluronan-binding structure in the ciliary nonpigmented epithelium. In addition, Western blot analysis revealed two cleaved complexes, the carboxyl-terminal end of versican bound to fibrillin microfibrils and the amino terminal end of versican bound to hyaluronan in the vitreous body. CONCLUSIONS: Fibrillin-1, versican, and hyaluronan form a unique complex in the ciliary nonpigmented epithelium, and two cleavage products of this complex were shown to exist in the vitreous body. This newly clarified fibrillin-versican-hyaluronan (FiVerHy) complex and its cleavage products may be indispensable for the physiological properties important to the ciliary body and vitreous.

Cooling prevents induction of corneal damage by argon laser peripheral iridotomy.

PURPOSE: There are many reports of corneal complications caused by argon laser peripheral iridotomy. In this study, we investigated whether cooling the anterior ocular segment during laser iridotomy prevented corneal damage. METHODS: A space for cooling the anterior ocular segment by perfusion with ice-cold water was made between the cornea and a contact lens. Dutch pigmented rabbits were excessively irradiated by an argon green laser, aiming at the peripheral iris. We used a contact lens without a cooling system as a control. Temperature in the anterior chamber and intraocular pressure were also monitored throughout the experiment. RESULTS: During laser treatment, the temperature without the cooling system rose to a maximum of 44.5 degrees C in the anterior chamber, whereas use of the cooling system consistently kept this temperature at 11.1 degrees -16.1 degrees C. Although most eyes treated without cooling showed corneal damage, damage was seen in only a few or in no eyes cooled during laser treatment. CONCLUSIONS: Argon laser treatment using contact lenses with a cooling system drastically reduced the corneal damage induced by argon laser peripheral iridotomy. This technique may be acceptable for clinical use, considering its technical simplicity and low incidence of treatment-related complications.