Characteristic distribution of microaneurysms and capillary dropouts in diabetic macular edema.

PURPOSE: To investigate the distribution pattern of microaneurysms (MAs) and capillary dropouts (CDOs) related to retinal thickness in patients with diabetic macular edema (DME). METHODS: We designed a cross-sectional observational study in which we manually merged fluorescein angiography and optical coherence tomography (OCT) map and located MAs and CDOs areas. The density of MAs, the width and the length of circumference of CDOs, and the number of MAs adjacent to CDOs were compared between highly thickened (white area (WA) in OCT map) and border areas (red area (RA)). RESULTS: We examined 115 eyes of 115 patients with DME. The density of MAs in RA (1.086 ± 0.616) was significantly higher than that in WA (0.8601 ± 1.086) (p = 0.002). The MA rates adjacent to CDOs in WA and RA were 79.1% and 80.7%, respectively. In the RA, the size of CDO adjacent to MAs was smaller (p = 0.013), but its circumference was longer (p = 0.018), and the number of MAs adjacent to CDOs was larger than those in WA (p = 0.002). The total length of circumference of CDOs was significantly correlated with the number of MAs adjacent to CDOs in WA (p = 0.011, R2 = 0.68) and RA (p = 0.008, R2 = 0.81). CONCLUSION: Smaller but more CDOs with longer circumference adjacent to MAs contribute to the higher density of MAs in the surrounding areas of DME.

Cytoprotective Effect of Astaxanthin in a Model of Normal Intraocular Pressure Glaucoma.

Glaucoma is characterized by axonal degeneration of retinal ganglion cells (RGCs) and apoptotic death of their cell bodies. Lowering intraocular pressure is currently the only way to treat glaucoma, but it is often insufficient to inhibit the progression of the disease. Glaucoma is a multifactorial disease, and the involvement of oxidative stress has recently received much attention. In the present study, we investigated the cytoprotective effect of astaxanthin (AST) on RGC degeneration using a normal-tension glaucoma (NTG) mouse model, which lacks the glutamate/aspartate transporter (Glast) and demonstrates spontaneous RGC and optic nerve degeneration without elevated intraocular pressure. Three-week-old Glast± mice were given intraperitoneal injections of AST at 10, 30, or 60 mg/kg/day or vehicle alone, and littermate control mice were given vehicle alone for 14 days, respectively. Five weeks after birth, the number of RGCs was counted in paraffin sections of retinal tissues stained with hematoxylin and eosin. We also used a retrograde labeling technique to quantify the number of RGCs. Additionally, the phosphorylated (p) IκB/total IκB ratio and the 4-hydroxynonenal (HNE) were measured in retinal tissues. The number of RGCs in Glast± mice was significantly decreased compared with that in control mice. RGC loss was suppressed by the administration of AST at 60 mg/kg/day, compared with vehicle alone. Following AST administration, the concentration of 4-HNE in the retina was also suppressed, but the pIκB/IκB ratio did not change. Our study revealed that the antioxidative stress effects of AST inhibit RGC degeneration in the retina and may be useful in the treatment of NTG.

Photodynamic therapy induces caspase-dependent apoptosis in rat CNV model.

PURPOSE: To investigate the mechanism of cell death in laser-induced choroidal neovascularization (CNV) after photodynamic therapy (PDT). METHODS: PDT was performed in Brown-Norway rats using laser light at a wavelength of 689 nm, irradiance of 600 mW/cm(2), and fluence of 25 J/cm(2) after intravenous injection of verteporfin at the doses of 3, 6, and 12 mg/m(2). Apoptotic cells in CNV were detected by TUNEL assay at 1, 3, 6, 15, 24, and 48 hours after PDT. Caspase activation at 1, 3, 6, 15, and 24 hours after PDT was determined by immunohistochemistry (IHC) with a cleaved caspase-3 or -9 antibody. Akt activity was determined by Western blot and IHC with a phosphorylated-Akt (pAkt) antibody. To investigate the roles of Akt in PDT-induced apoptosis, insulin-like growth factor (IGF)-1, an Akt activator, with or without wortmannin, an inhibitor of PI3K-Akt pathway, was injected into the vitreous before PDT. RESULTS: The number of TUNEL-positive cells in CNV increased at 3 hours after PDT and peaked at 6 hours, showing a dose dependence of verteporfin. Caspase activation was detected in TUNEL-positive cells. Dephosphorylation of Akt in CNV occurred within 1 hour. IGF-1 significantly activated Akt and suppressed the number of TUNEL-positive cells in CNV, and the effects of IGF-1 were diminished by wortmannin. CONCLUSIONS: PDT induced caspase-dependent apoptosis in CNV. These results suggest that PDT leads to dephosphorylation of Akt and subsequent activation of the caspase-dependent pathway. Understanding the intracellular signaling mechanisms of apoptosis in PDT may lead to more selective and effective treatment of CNV secondary to age-related macular degeneration.

Polymorphisms of RDH16 and VEGFR1 influence M. trapezius steatosis in Japanese Black carcass.

The exact cause of steatosis, one of defects in Japanese beef carcasses, has not been elucidated to date, because it is very difficult to diagnose cyclopedically with certain reproducibility due to the bias in the outbreak. Therefore, the objective of this study was to assess the influence of polymorphisms in retinol dehydrogenase 16 (RDH16), myoferlin (MYOF) and vascular endothelial growth factor receptors 1 and 2 (VEGFR1, VEGFR2) on carcass-graded Musculus trapezius steatosis. For logistic regression analysis, 646 carcasses shipped from 29 farms in Miyazaki, Japan, were used. The GG genotype in RDH16 showed significant odds ratios against AA and AG. In VEGFR1, CT had a significant odds ratio against CC. After evaluating for interaction, highly significant odds ratios were observed in the combinations that included the GG risk genotype in RDH16. It is noteworthy that there was no steatosis in the combination GG (RDH16) and CC (VEGFR1). It may be concluded that there is a possibility that steatosis can be suppressed by the CC genotype in VEGFR1. The current study revealed the influence of genetic polymorphisms on M. trapezius steatosis that had not been reported until now, and may help elucidate the cause of steatosis.

[Genetic diagnosis of retinoblastoma by a combination of fluorescence in situ hybridization and restriction fragment length polymorphism].

PURPOSE: It is important to exclude germ line mutation in cases of unilateral retinoblastoma(RB) to estimate hereditary or possible secondary cancer. We investigated whether genetic diagnosis is feasible in a health check screening program. METHODS: Five patients with RB had surgery for enucleation in Keio University Hospital. Tumor cells from enucleated eyes and lymphocytes representing systemic cells were collected and analyzed genetically by fluorescence in situ hybridization(FISH) and restriction fragment length polymorphism (RFLP). RESULTS: One out of three unilateral RB cases could be diagnosed as non-hereditary by the finding of no copies of the RB gene in the tumor cells using the FISH method and no signal in the RFLP method. A decrease of signal in tumor cells to less than 50% in the RFLP method was observed in another case of unilateral RB that seemed to be non-hereditary, but the case ultimately could not be diagnosed as non-hereditary because polycopies were found in the FISH method. No abnormality in tumor cells could be found in another unilateral case or in systemic cells of two bilateral cases. CONCLUSION: A combination of FISH and RFLP methods can be used to diagnose some cases of RB as non-hereditary.

Biological role of lutein in the light-induced retinal degeneration.

Lutein, a xanthophyll of a carotenoid, is anticipated as a therapeutic product to prevent human eye diseases. However, its biological mechanism is still unclear. Here, we show the molecular mechanism of lutein's effect to reduce photodamage of the retina. We analyzed the light-exposed retinas of Balb/c mice given lutein-supplemented or normal diet. Visual function was measured by electroretinogram, and histological changes were observed. Immunohistochemical and immunoblot analyses were performed to analyze molecular mechanism. The reactive oxygen species induced in the retina was evaluated by fluorescent probes. In the mice after light exposure, reduction of a-wave and b-wave amplitudes in electroretinogram, indicating visual impairment, and thinning of the photoreceptor cell layer owing to apoptosis were both attenuated by lutein diet. Interestingly, γ-H2AX, a marker for double-strand breaks (DSBs) in DNA, was up-regulated in the photoreceptor cells after light exposure, but this increase was attenuated by lutein diet, suggesting that DSBs caused by photodamage contributed to the photoreceptor cell death and that this change was suppressed by lutein. Moreover, the expression of eyes absent (EYA), which promotes DNA repair and cell survival, was significantly up-regulated with lutein diet in the light-exposed retina. Therefore, lutein induced EYA for DNA repair, which could suppress DNA damage and photoreceptor cell apoptosis. Lutein reduced light-induced oxidative stress in the retina, which might contribute to promote DNA repair. The lutein-supplemented diet attenuated light-induced visual impairment by protecting the photoreceptor cells' DNA.

GEP100-Arf6-AMAP1-cortactin pathway frequently used in cancer invasion is activated by VEGFR2 to promote angiogenesis.

Angiogenesis and cancer invasiveness greatly contribute to cancer malignancy.Arf6 and its effector, AMAP1, are frequently overexpressed in breast cancer, and constitute a central pathway to induce the invasion and metastasis. In this pathway, Arf6 is activated by EGFR via GEP100. Arf6 is highly expressed also in human umbilical vein endothelial cells (HUVECs) and is implicated in angiogenesis. Here, we found that HUVECs also highly express AMAP1, and that vascular endothelial growth factor receptor-2 (VEGFR2) recruits GEP100 to activate Arf6. AMAP1 functions by binding to cortactin in cancer invasion and metastasis. We demonstrate that the same GEP100-Arf6-AMAP1-cortactin pathway is essential for angiogenesis activities, including cell migration and tubular formation, as well as for the enhancement of cell permeability and VE-cadherin endocytosis of VEGF-stimulated HUVECs. Components of this pathway are highly expressed in pathologic angiogenesis, and blocking of this pathway effectively inhibits VEGF- or tumor-induced angiogenesis and choroidal neovascularization. The GEP100-Arf6-AMAP1-cortactin pathway, activated by receptor tyrosine kinases, appears to be common in angiogenesis and cancer invasion and metastasis, and provides their new therapeutic targets.

Monocyte chemoattractant protein 1 mediates retinal detachment-induced photoreceptor apoptosis.

Photoreceptor apoptosis is a major cause of visual loss in retinal detachment (RD) and several other visual disorders, but the underlying mechanisms remain elusive. Recently, increased expression of monocyte chemoattractant protein 1 (MCP-1) was reported in vitreous humor samples of patients with RD and diabetic retinopathy as well as in the brain tissues of patients with neurodegenerative diseases, including Alzheimer's disease and multiple sclerosis. Here we report that MCP-1 plays a critical role in mediating photoreceptor apoptosis in an experimental model of RD. RD led to increased MCP-1 expression in the Müller glia and increased CD11b+ macrophage/microglia in the detached retina. An MCP-1 blocking antibody greatly reduced macrophage/microglia infiltration and RD-induced photoreceptor apoptosis. Confirming these results, MCP-1 gene-deficient mice showed significantly reduced macrophage/microglia infiltration after RD and very little photoreceptor apoptosis. In primary retinal mixed cultures, MCP-1 was cytotoxic for recoverin+ photoreceptors, and this toxicity was eliminated through immunodepleting macrophage/microglia from the culture. In vivo, deletion of the gene encoding CD11b/CD18 nearly eliminated macrophage/microglia infiltration to the retina after RD and the loss of photoreceptors. Thus, MCP-1 expression and subsequent macrophage/microglia infiltration and activation are critical for RD-induced photoreceptor apoptosis. This pathway may be an important therapeutic target for preventing photoreceptor apoptosis in RD and other CNS diseases that share a common etiology.