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Retinal organoids derived from human-induced pluripotent stem cells (hiPSC) are powerful tools for studying retinal development as they model spatial and temporal differentiation of retinal cell types. Vertebrate retinal development involves a delicate and coordinated process of retinal progenitor cell (RPC) differentiation, and the mammalian target of rapamycin complex 1 (mTORC1) has been reported to play a significant role in this complex process. Herein, using hiPSC-derived retinal organoids, we identify the time-dependent role of mTORC1 in retinal development, specifically in retinal ganglion cell (RGC) differentiation and the retinal lamination process, during the early stages of retinal organoid (RO) development. mTORC1 activity in ROs was the highest at 40 days of differentiation. MHY1485-induced hyperactivation of mTORC1 during this period resulted in a significant increase in the overall size of ROs compared to the untreated controls and rapamycin-treated Ros; there was also a marked increase in proliferative activity within the inner and outer layers of ROs. Moreover, the MHY1485-treated ROs showed a significant increase in the number of ectopic RGCs in the outer layers (indicating disruption of retinal laminar structure), with robust expression of HuC/D-binding proteins in the inner layers. These results demonstrate that mTORC1 plays a critical role in the development of hiPSC-derived ROs, especially during the early stages of differentiation.

// Sung Wook Park 1 , 2 , Sora Im 3 , Hyoung Oh Jun 2 , Kihwang Lee 4 , Young-Jun Park 5 , Jin Hyoung Kim 2 , Woo Jin Park 3 , Young-Hoon Lee 6 and Jeong Hun Kim 1,2,7 1 Department of Biomedical Sciences, Seoul National University College of Medicine, Daehak-Ro, Jongno-Gu, Seoul, Republic of Korea 2 Fight Against Angiogenesis-Related Blindness Laboratory, Biomedical Research Institute, Seoul National University Hospital, Daehak-Ro, Jongno-Gu, Seoul, Republic of Korea 3 Department of Life Sciences, Life Sciences Concentration Gwangju Institute of Science and Technology Cheomdan-Gwagiro, Buk-Gu, Gwangju, Republic of Korea 4 Department of Ophthalmology, Ajou University School of Medicine, World Cup-Ro, Yeongtong-Gu, Suwon-Si, Gyeonggi-Do, Republic of Korea 5 Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea 6 Department of Oral Anatomy, School of Dentistry, Chonbuk National University, Deokjin-Gu, Jeonju-Si, Jeollabuk-Do, Republic of Korea 7 Institute of Oral Biosciences, Chonbuk National University, Deokjin-Gu, Jeonju-Si, Jeollabuk-Do, Republic of Korea Correspondence to Jeong Hun Kim, email: // Keywords : amyloid β, age-related macular degeneration, transmission electron microscopy, retinal pigment epithelium, microarray, Gerotarget Received : September 02, 2016 Accepted : March 29, 2017 Published : April 08, 2017 Abstract Age-related macular degeneration (AMD) is a leading cause of blindness in the elderly. The two types of AMD are: dry and wet AMD. While laser-induced choroidal neovascularization has been used extensively in the studies of wet AMD, there is no established mouse model that fully recapitulates the cardinal features of dry AMD. A lack of appropriate mouse model for dry AMD has hampered the translational research on the pathogenesis of the disease and the development of therapeutic agents. We hypothesized that 5XFAD mice, an animal model for the study of Alzheimer’s disease, can be used as a mouse model for dry AMD with regard to the amyloid beta (Aβ) related pathology. In this study, the ultrastructure of the retinal pigment epithelium (RPE) of 5XFAD mice was analyzed using transmission electron microscopy. Of importance, the aged 5XFAD mice show ultrastructural changes in the RPE and Bruch’s membrane (BM) that are compatible with the cardinal features of human dry AMD, including a loss of apical microvilli and basal infolding of the RPE, increased BM thickness, basal laminar and linear deposits, and accumulation of lipofuscin granules and undigested photoreceptor outer segment-laden phagosomes. In microarray-based analysis, the RPE complex of the aged 5XFAD mice shows differential gene expression profiles consistent with dry AMD in the inflammation response, immune reaction pathway, and decreased retinol metabolism. Taken together, we suggest that aged 5XFAD mice can be used as a mouse model of dry AMD to study Aβ related pathology and develop a new therapeutic approaches.

Ischemic retinopathies and optic neuropathies are important causes of vision loss. The neuroprotective effect of erythropoietin (EPO) in ischemic neuronal injury and the expression of EPO and its receptor in retinal tissue have been well documented. However, the exact regulatory mechanism of EPO expression in retinal ischemia still remains to be elucidated. In this study, we investigated the role of cystine/glutamate antiporter (system xc-) in the regulation of astrocytic EPO expression by using both in vitro and in vivo models. Under hypoxia, the expression of astrocytic system xc- is up-regulated both in vitro and in vivo. Inhibition of system xc- resulted in depletion of intracellular glutathione (GSH) and decrement of GSH disulfide ratios in human brain astrocytes (HBAs). In HBAs, hypoxia-induced stabilization of hypoxia-inducible factor (Hif)-2α is nearly completely abolished by inhibition of system xc-. Hypoxia-induced up-regulation of astrocytic EPO expression is suppressed by both pharmacological inhibition and siRNA-mediated knockdown of system xc-. In contrast, basal EPO expression under normoxia is not affected by system xc- modulation. In summary, under hypoxia, increased system xc- acts as the major source of intracellular GSH, which helps in stabilizing Hif-2α and subsequent up-regulation of EPO in astrocytes.-Lee, B. J., Jun, H. O., Kim, J. H., Kim, J. H. Astrocytic cystine/glutamate antiporter is a key regulator of erythropoietin expression in the ischemic retina.

Ocular diseases featuring pathologic neovascularization are the leading cause of blindness, and anti-VEGF agents have been conventionally used to treat these diseases. Recently, regulating factors upstream of VEGF, such as HIF-1α, have emerged as a desirable therapeutic approach because the use of anti-VEGF agents is currently being reconsidered due to the VEGF action as a trophic factor. Here, we report a novel scaffold discovered through the complete structure–activity relationship of ring-truncated deguelin analogs in HIF-1α inhibition. Interestingly, analog 6i possessing a 2-fluorobenzene moiety instead of a dimethoxybenzene moiety exhibited excellent HIF-1α inhibitory activity, with an IC50 value of 100 nM. In particular, the further ring-truncated analog 34f, which showed enhanced HIF-1α inhibitory activity compared to analog 2 previously reported by us, inhibited in vitro angiogenesis and effectively suppressed hypoxia-mediated retinal neovascularization. Importantly, the heteroatom-substituted ben...

Low oxygen or hypoxia can be observed in the central region of solid tumors. Hypoxia is a strong stimulus for new blood vessel formation or angiogenesis, which is essential for tumor growth and progression. Fibroblast growth factor 11 (FGF11) is an intracellular non-secretory FGF whose function has not yet been fully characterized. In the present study, we demonstrated that FGF11 expression is upregulated under hypoxic conditions in human umbilical vein endothelial cells (HUVECs). FGF11 overexpression stimulated capillary-like tube formation, yet did not affect cell migration. Notably, FGF11 markedly increased the levels of tight junction proteins including occludin, zonula occludens-1 (ZO-1) and claudin-5 in HUVECs. The FGF11 promoter contains hypoxia response elements (HREs), and hypoxia-inducible factor-1 (HIF-1) binds to HREs to activate hypoxia-related genes. We demonstrated that hypoxia or HIF-1 expression under normoxic conditions increased the luciferase activity driven by the FGF11 promoter. However, deletion of the HREs from the FGF11 promoter rendered reporter gene activity unresponsive to hypoxia or HIF-1. Taken together, we propose that FGF11 may be involved in the stabilization of capillary-like tube structures associated with angiogenesis and may act as a modulator of hypoxia-induced pathological processes such as tumorigenesis.

To investigate the effect of protein kinase C (PKC)-ζ inhibition on vascular leakage in diabetic retinopathy, streptozotocin-induced diabetic mice were intravitreously injected with siPKC-ζ. According to the fluorescein angiography of the retinal vessels, suppression of PKC-ζ effectively attenuated vascular leakage in diabetic retina. Further evaluation on the retina with western blot analysis and immunohistochemistry revealed accompanying restoration of tight junction proteins on retinal vessels. As two major contributors to vascular leakage in diabetic retinopathy, vascular endothelial growth factor (VEGF) and advanced glycation end products (AGEs) were investigated on the tight junction protein expression in endothelial cells. Inhibition of PKC-ζ attenuated VEGF-induced decrease of tight junction proteins and accompanying hyperpermeability in human retinal microvascular endothelial cells (HRMECs). PKC-ζ inhibition also attenuated AGE-induced decrease of tight junction proteins in HRMECs. Our findings suggest that inhibition of PKC-ζ could be an alternative treatment option for compromised blood-retinal barrier in diabetic retinopathy.

// Dong Hyun Jo 1, 2, 3, * , Kyungmin Lee 4, 5, * , Jin Hyoung Kim 1, 3 , Hyoung Oh Jun 1, 3 , Younghoon Kim 6 , Young-Lai Cho 7 , Young Suk Yu 1, 8 , Jeong-Ki Min 4, 5 , Jeong Hun Kim 1, 2, 3, 8 1 Fight against Angiogenesis-Related Blindness (FARB) Laboratory, Clinical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea 2 Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, Republic of Korea 3 Tumor Microenvironment Research Center, Global Core Research Center, Seoul National University, Seoul, Republic of Korea 4 Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea 5 Department of Biomolecular Science, University of Science & Technology, Daejeon, Republic of Korea 6 Department of Pathology, College of Medicine, Seoul National University, Seoul, Republic of Korea 7 Department of Chemistry, Dongguk University, Seoul, Republic of Korea 8 Department of Ophthalmology, Seoul National University College of Medicine, Seoul, Republic of Korea * These authors have contributed equally to this work Correspondence to: Jeong-Ki Min, email: jekmin@kribb.re.kr Jeong Hun Kim, email: steph25@snu.ac.kr Keywords: retinoblastoma, L1, cell adhesion molecules, adhesion-mediated proliferation, chemoresistance Received: August 31, 2016 Accepted: December 13, 2016 Published: January 04, 2017 ABSTRACT Retinoblastoma is the most common intraocular cancer in children, affecting 1/20,000 live births. Currently, children with retinoblastoma were treated with chemotherapy using drugs such as carboplatin, vincristine, and etoposide. Unfortunately, if conventional treatment fails, the affected eyes should be removed to prevent extension into adjacent tissues and metastasis. This study is to investigate the roles of L1 in adhesion-mediated proliferation and chemoresistance of retinoblastoma. L1 was differentially expressed in 30 retinoblastoma tissues and 2 retinoblastoma cell lines. Furthermore, the proportions of L1-positive cells in retinoblastoma tumors were negatively linked with the number of Flexner-Wintersteiner rosettes, a characteristic of differentiated retinoblastoma tumors, in each tumor sample. Following in vitro experiments using L1-deleted and -overexpressing cells showed that L1 increased adhesion-mediated proliferation of retinoblastoma cells via regulation of cell cycle-associated proteins with modulation of Akt, extracellular signal-regulated kinase, and p38 pathways. In addition, L1 increased resistance against carboplatin, vincristine, and esoposide through up-regulation of apoptosis- and multidrug resistance-related genes. In vivo tumor formation and chemoresistance were also positively linked with the levels of L1 in an orthotopic transplantation model in mice. In this manner, L1 increases adhesion-mediated proliferation and chemoresistance of retinoblastoma. Targeted therapy to L1 might be effective in the treatment of retinoblastoma tumors, especially which rapidly proliferate and demonstrate resistance to conventional chemotherapeutic drugs.

Reactive oxygen species (ROS) as well as vascular endothelial growth factor (VEGF) play important roles in pathologic retinal neovascularization. We investigated whether betaine inhibits pathologic retinal neovascularization in a mouse model of oxygen induced retinopathy (OIR). Betaine was intravitreally injected in OIR mice at postnatal day (P) 14. At P17, the neovascular tufts area in OIR retina was analyzed. Intravitreal injection of betaine (200μM) effectively reduced the neovascular tufts area in OIR retina (68.0±6.7% of the control eyes, P

Vision loss in diabetic retinopathy is due to macular edema characterized by increased vascular permeability, which involves phosphorylation associated with activation of protein kinase C (PKC) isoforms. Herein, we demonstrated PKC delta inhibition could prevent blood-retinal barrier breakdown in diabetic retinopathy. Increased vascular permeability of diabetic retina was accompanied by a decrease of zonula occludens (ZO)-1 and ZO-2 expression. In diabetic retina and advanced glycation end product-treated human retinal microvascular endothelial cells, vascular leakage and loss of ZO-1 and ZO-2 on retinal vessels were effectively restored or prevented with treatment of rottlerin, transfection of PKC-delta-DN, or siRNA for PKC delta. Interestingly, PKC delta translocated from cytosol to membrane in advanced glycation end product-treated human retinal microvascular endothelial cells, which was blocked by PKC delta inhibition. Taken together, PKC delta activation, related to its subcellular translocation, is involved in vascular permeability in response to diabetes, and inhibition of PKC delta effectively restores loss of tight junction proteins in retinal vessels. Therefore, we suggest that inhibition of PKC delta could be an alternative treatment to blood-retinal barrier breakdown in diabetic retinopathy.

Retinoblastoma is the most common primary intraocular malignancy in children. With the progression of retinoblastoma, retinoblastoma cells lose their ability to differentiate. Regardless of many attempts to identify prognostic factors in retinoblastoma, further investigation for prognostic factors of retinoblastoma progression is still required because of the lack of sensitivity and specificity of these prognostic factors in predicting disease progression. We demonstrated that the differential expression of the neurotrophin receptors TrkA and TrkB is closely related to the differentiation of retinoblastoma cells. While retinoblastoma cells expressed TrkA as well as TrkB, their growth rates were not influenced by the addition of nerve growth factor to the culture medium. In experimental animal models of retinoblastoma, TrkA expression was primarily detected in more differentiated areas with high nm23 immunoreactivity whereas TrkB expression was apparent in more proliferative areas with high Ki67 immunoreactivity. With retinoic-acid-induced differentiation of retinoblastoma cells, TrkA expression significantly increased whereas TrkB significantly decreased. The differential expression of TrkA and TrkB with differentiation of retinoblastoma cells was mediated by extracellular-signal-regulated kinase 1/2 activation, which was confirmed by immunocytochemistry of TrkA. Therefore, our results suggest that the differential expression of TrkA and TrkB could be valuable as a therapeutic target, for instance using specific inhibitors.

Hypoxia regulates a variety of transcription factors including HIF-1, which has been considered a target for anti-angiogenic therapy. While searching for a novel anti-angiogenic agent that would inhibit HIF-1 activity, we identified wondonin, a new bis imidazole purified from an association of the sponges Poecillastra wondoensis. Wondonin significantly decreased hypoxia-induced HIF-1alpha protein and VEGF expression and inhibited angiogenesis in vitro and in vivo. Furthermore, wondonin down-regulated HIF-1alpha protein through the increased interaction with the pVHL in immortalized human keratinocyte cell line. Wondonin also decreased the immunoreactivities of CD31 and VEGF in epidermal hyperplasia in mice. Taken together, these results suggested that wondonin had the potential to become the anti-angiogenic therapeutic agent to target HIF-1alpha.

Vascular integrity is important in maintaining homeostasis of brain microenvironments. In various brain diseases including Alzheimer’s disease, stroke and multiple sclerosis, increased paracellular permeability due to breakdown of blood-brain barrier is linked with initiation and progression of pathological conditions. We developed a capacitance sensor array to monitor dielectric responses of cerebral endothelial cell monolayer, which could be utilized to evaluate the integrity of brain microvasculature. Our system measured real-time capacitance values which demonstrated frequency- and time-dependent variations. With the measurement of capacitance at the frequency of 100 Hz, we could differentiate the effects of vascular endothelial growth factor (VEGF), a representative permeability-inducing factor, on endothelial cells and quantitatively analyse the normalized values. Interestingly, we showed differential capacitance values according to the status of endothelial cell monolayer, confluent or sparse, evidencing that the integrity of monolayer was associated with capacitance values. Another notable feature was that we could evaluate the expression of molecules in samples in our system with the reference of real-time capacitance values. We suggest that this dielectric spectroscopy system could be successfully implanted as a novel in vitro assay in the investigation of the roles of paracellular permeability in various brain diseases.

Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and thus contributes to many vasoproliferative retinopathies including retinopathy of prematurity. Based on the importance of canonical transient receptor potential (TRPC) channels in VEGF signaling, we firstly evaluated the expression of TRPC channels in mouse retina by reverse transcriptase-polymerase chain reaction. All seven TRPC channels were expressed in mouse retina. TRPC4 channels were chosen for further analysis based on their upregulation on hypoxic retina according to the GEO database under the identifier GSE19886. Interestingly, TRPC4 suppression by intravitreal injection of siRNA against mTRPC4 significantly inhibited retinal neovascularization. To further investigate the effect of TRPC4 suppression on neovascularization, human retina microvascular endothelial cells (HRMECs) that are responsible for initiating neovascularization in response to increased VEGF in OIR retina were transfected with siRNA against TRPC4. As we have expected, suppression of TRPC4 effectively inhibited VEGF-induced migration and tube formation as well. Further evaluation on VEGF signaling pathway by western blot analysis of signaling molecules discovered that VEGF-induced activation of ERK, p38 MAPK and AKT signaling pathways were inhibited by suppression of TRPC4. These findings suggest that suppression of TRPC4 could be an alternative therapeutic option for VEGF-induced retinal neovascularization.

Ocular toxoplasmosis is mediated by monocytes infected with Toxoplasma gondii that are disseminated to target organs. Although infected monocytes can easily access to outer blood-retinal barrier due to leaky choroidal vasculatures, not much is known about the effect of T. gondii-infected monocytes on outer blood-retinal barrier. We prepared human monocytes, THP-1, infected with T. gondii and human retinal pigment epithelial cells, ARPE-19, grown on transwells as an in vitro model of outer blood-retinal barrier. Exposure to infected monocytes resulted in disruption of tight junction protein, ZO-1, and decrease in transepithelial electrical resistance of retinal pigment epithelium. Supernatants alone separated from infected monocytes also decreased transepithelial electrical resistance and disrupted tight junction protein. Further investigation revealed that the supernatants could activate focal adhesion kinase (FAK) signaling in retinal pigment epithelium and the disruption was attenuated by FAK inhibitor. The disrupted barrier was partly restored by blocking CXCL8, a FAK activating factor secreted by infected monocytes. In this study, we demonstrated that monocytes infected with T. gondii can disrupt outer blood-retinal barrier, which is mediated by paracrinely activated FAK signaling. FAK signaling can be a target of therapeutic approach to prevent negative influence of infected monocytes on outer blood-retinal barrier.

In diabetic retinopathy (DR), visual deterioration is related with retinal neovascularization and vascular hyperpermeability. Anti-vascular endothelial growth factor (VEGF) agents are currently utilized to suppress retinal neovascularization and macular edema (ME); however, there are still concerns on the widespread use of them because VEGF is a trophic factor for neuronal and endothelial cells in the retina. As an alternative treatment strategy for DR, it is logical to address hypoxia-related molecules to treat DR because the retina is in relative hypoxia as DR progresses. In this study, we demonstrate that destabilization of hypoxia-inducible factor-1α (HIF-1α) by SH-1242 and SH-1280, novel heat shock protein 90 (hsp90) inhibitors, leads to suppression of hypoxia-mediated retinal neovascularization and vascular leakage in diabetic retina. In vitro experiments showed that these inhibitors inhibited hypoxia-induced upregulation of target genes of HIF-1α and further secretion of VEGF. Furthermore, these inhibitors effectively suppressed expression of target genes of HIF-1α including vegfa in the retina of oxygen-induced retinopathy (OIR) mice. Interestingly, despite hsp90 inhibition, these inhibitors do not induce definite toxicity at the level of gene expression, cellular viability, and histologic integrity. We suggest that SH-1242 and SH-1280 can be utilized in the treatment of DR, as an alternative treatment of direct VEGF inhibition. Key message: SH-1242 and SH-1280 are novel hsp90 inhibitors similar to deguelin. HIF-1α destabilization by hsp90 inhibition leads to anti-angiogenic effects. Despite hsp90 inhibition, both inhibitors do not induce definite toxicity. HIF-1α modulation can be a safer therapeutic option than direct VEGF inhibition.

Clusterin is a cytoprotective chaperone protein that is known to protect various retinal cells. It was also reported to be overexpressed in several types of malignant tumors, whose chemoresistance correlates with the expression of clusterin. Herein, we investigated the effect of clusterin on cisplatin-induced cell death of retinoblastoma cells. Firstly, evaluation of clusterin expression demonstrated that it was highly expressed in human retinoblastoma tissues and cell lines (SNUOT-Rb1 and Y79) particularly in the area between viable cells around vessels and necrotic zones in the relatively avascular area in human retinoblastoma tissues. Furthermore, the effects of cisplatin on retinoblastoma cells were evaluated. Cisplatin (1 µg/ml) significantly affected cell viability of SNUOT-Rb1 cells by inducing caspase-3-dependent apoptosis. Notably, the cell death due to cisplatin was prevented by 5 µg/ml of clusterin administered 4 h prior to cisplatin treatment by inhibiting cisplatin-induced apoptosis. Furthermore, overexpression of clusterin exerted its anti-apoptotic effect on cisplatin-induced apoptosis, and effectively prevented cisplatin-induced cell death. These data suggest that clusterin, found to be expressed in human retinoblastoma, may exert anti-apoptotic effects on cisplatin-induced apoptosis and prevent cell death. Therefore, clusterin can contribute to cisplatin resistance of retinoblastoma.

Effective and validated animal models are valuable to investigate the pathogenesis and potential therapeutics for human diseases. There is much concern for diabetic retinopathy (DR) in that it affects substantial number of working population all around the world, resulting in visual deterioration and social deprivation. In this review, we discuss animal models of DR based on different species of animals from zebrafish to monkeys and prerequisites for animal models. Despite criticisms on imprudent use of laboratory animals, we hope that animal models of DR will be appropriately utilized to deepen our understanding on the pathogenesis of DR and to support our struggle to find novel therapeutics against catastrophic visual loss from DR.

Retinoblastoma is the most common intraocular malignant tumor in children, affecting approximately 1 in 20,000 live births. In this study, we investigated the role of L1, a transmembrane protein, in proliferation and chemoresistance of retinoblastoma to figure out the potential for the novel therapeutic target. Retinoblastoma tissues demonstrated varying degrees of L1 positivity in 86.6% (26/30) of samples. In particular, the degree of L1 positivity was inversely related with that of Flexner-Wintersteiner rosette formation. Further in vitro studies using stable cell lines which showed up- or down-regulation of L1 demonstrated that L1 was associated with cell-cell adhesion and proliferation of retinoblastoma cells. Retinoblastoma cells with low expression of L1 showed decreased tumor formation in vivo. In addition, L1 expression was related with resistance to carboplatin, one of the most-widely utilized chemotherapeutic agents against retinoblastoma. In line with these results, retinoblastoma cells with higher expression of L1 demonstrated increased resistance to carboplatin in vivo. We also observed diffuse and dense expression of L1 in retinoblastoma tissues from 4 patients who underwent enucleation despite intensive chemotherapy based on carboplatin. Taken together, L1 was related with proliferation and chemoresistance of retinoblastoma and might be a potential therapeutic target of retinoblastoma. Citation Format: Dong Hyun Jo, Kyungmin Lee, Jin Hyoung Kim, Hyoung Oh Jun, Young Hoon Kim, Young-Lai Cho, Young Suk Yu, Jeong-Ki Min, Jeong Hun Kim. The role of L1 in proliferation and chemoresistance of retinoblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2469.

The fifth author's affiliations are incomplete. Dong Hun Kim is affiliated with both 3 Department of Radiology, College of Medicine, Sooncheonhyang University, Bucheon, Korea, and 5 Department of Radiology, College of Medicine, Chosun University, Gwangju, Korea

Hydraulic flood routing is advantageous for computational accuracy; however, it requires individual calculations of an extensive pipe network in the case of a two-dimensional analysis. In this study, a method for considering the characteristics of a two-dimensional pipe network has been developed using the meta-channel concept to simplify the detailed calculations involved in the estimation of runoff from urban catchments. In essence, the meta-channel concept turns a two-dimensional pipe network into a one-dimensional pipe with an effective hydraulic geometry. Once such geometry has been identified, the flood routing can then be performed for an urban drainage system. A nonlinear diffusion wave equation, derived from the Saint-Venant equation, was used for flood routing, with an explicit method used for the numerical solution. The celerity and diffusion coefficients, which are two parameters of the diffusion wave equation, were estimated for the Goonja drainage from a two-dimensional pipe network using the meta-channel concept. A comparison of the results of the meta-channel-based pipe routing with the distributed SWMM simulation and observed data, showed close similarities, and identified the applicability of the meta-channel concept in an urban drainage setting.

Osteopontin (OPN, SPP1) is a secretory extracellular matrix protein that has been implicated in cancer-associated mechanisms such as metastasis, invasion and angiogenesis. Three OPN isoforms (OPN-a, -b and -c) derived from alternative splicing are known to exist, but their functional specificity remains unclear. Here, we found that the expression profile of OPN isoforms in hepatocellular carcinoma (HCC) cell lines and patient tissues were correlated with specific cellular phenotypes and tumorigenicity of HCC. Thus, SK-Hep1 cells with a robust migratory capacity dominantly expressed both OPN-a and -b, but non-migratory cell lines such as Hep3B and PLC/PRF/5 mainly expressed OPN-c. Moreover, tumor tissues predominantly expressed OPN-a and -b, whereas normal liver tissues mainly expressed OPN-c. Transwell infiltration and wound-induced migration assays revealed that both OPN-a and -b induced Hep3B cell migration, while OPN-c had no significant effects. By contrast, OPN-c suppressed the migratory activity of SK-Hep1 cells although no significant changes were induced by OPN-a. Consistently, OPN isoforms differentially activated migration-associated signaling pathways such that OPN-a and -b increased the expression of urokinase type plasminogen activator and the phosphorylation of p42/p44 MAP kinase, but these pathways were not activated by OPN-c. Thus, the findings of the present study suggest that OPN splice variants differentially couple to signaling pathways to modulate the migratory property of HCC cells and that this is one of the mechanisms underlying the pathological heterogeneity of HCC progression.

Purpose Age-related macular degeneration (AMD) is the leading cause of blindness in elderly. The detailed mechanism of choroidal neovascularization (CNV) leads to severe vision loss in patients with AMD. This study was undertaken to evaluate the inhibitory effect of homoisoflavanone on CNV. Methods Antiangiogenic activity of homoisoflavanone was evaluated by in vitro tube formation assay of human umbilical vein endothelial cells (HUVECs) and cell migration assay of HUVECs., Homoisoflavanone or PBS was injected intravitreously into a mouse model of laser-photocoagulation-induced CNV. Fluorescein angiography and vessel counting in cross sections were employed to examine CNV lesions. The toxicity of homoisoflavanone was evaluated through 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT) assay in HUVECs as well as histological examination and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining in the retina. Results Homoisoflavanone effectively inhibited in vitro tube formation and cell migration of HUVECs. Interestingly, homoisoflavanone significantly reduced CNV and its leakage in a mouse model of laser-photocoagulation-induced CNV. In addition, homoisoflavanone showed no effect on cell viability of HUVECs and no retinal toxicity in a concentration range of 1-10 μM. Conclusions Our data suggest that homoisoflavanone is a potent inhibitor of CNV and may be applied in the treatment of other vasoproliferative retinopathies and tumor.

AKAP12 (A-Kinase anchoring protein 12) is a protein kinase C substrate and a potential tumor suppressor. AKAP12 is down-regulated by several oncogenes and strongly suppressed in various cancers including prostate, ovarian and breast cancers. AKAP12 acts as a regulator of mitogenesis by anchoring key signal proteins such as PKA, PKC, and cyclins. In this study, AKAP12 was found to suppress tumor cell viability by inducing apoptosis via caspase-3 in HT1080 cells. This AKAP12-induced apoptosis was associated with a decreased expression of Bcl-2 and increased expression of Bax. Moreover, AKAP12-transfectant strongly induced the expression of Cip1/p21 and Kip1/p27, but resulted in a decrease in cyclin D1 involved in G(1) progression. Accordingly, these results suggest that AKAP12 may play an important role in tumor growth suppression by inducing apoptosis with the regulation of multiple molecules in the cell cycle progression.

Brain microvessels possess barrier structures comprising tight junctions which are critical for the maintenance of central nervous system homeostasis. Brain vascular diseases, such as ischemic stroke damage to blood-brain barrier, increase the vascular permeability, and then lead to vasogenic brain edema. Herein, we examined whether angiopoietin-1 (Ang-1) could regulate zonula occludens-2 (ZO-2) expression and counteract vascular endothelial growth factor (VEGF)-induced vascular permeability. When we treated brain microvascular endothelial cells with Ang-1, Ang-1 caused a time- and dose-dependent increase of ZO-2 and down-regulation in endothelial permeability. VEGF, one of the key regulators of ischemia-induced vascular permeability, increased endothelial cell permeability in vitro, whereas, Ang-1 reversed this VEGF effect by up-regulating ZO-2 expression. Additionally, the recovery effect of Ang-1 on permeability was strongly blocked by siRNA against ZO-2. Collectively, our results suggest that Ang-1 shows anti-permeability activity through up-regulation of ZO-2.

Purpose Oxidative stress-induced vascular endothelial growth factor (VEGF) is thought to play a critical role in the pathogenesis of retinopathy of prematurity (ROP). This study was performed to investigate the anti-angiogenic effect of luteolin against reactive oxygen species (ROS)-induced retinal neovascularization. Methods The toxicity of luteolin was evaluated through modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in human retinal microvascular endothelial cells (HRMECs) as well as TUNEL staining in the retina of C57BL/6J mice. After intravitreal injection of luteolin in the mouse model of ROP, retinal neovascularization was examined by fluorescence angiography and vessel counting. Anti-angiogenic activity of luteolin was evaluated by VEGF-induced migration and tube formation assay. The effect of luteolin on tertiary-butylhydroperoxide (t-BH)-induced ROS production was measured with 2'7'-dichlorofluorescein diacetate. The effect of luteolin on t-BH-induced and hypoxia-induced VEGF transcription and expression were evaluated by RT-PCR and Western blot, respectively. Results Luteolin never affected the viability of HRMECs up to 10 μM, where luteolin never induced any structural change in all retinal layers. Luteolin inhibited retinal neovascularization in the mouse model of ROP. Moreover, VEGF-induced migration and tube formation were significantly decreased by cotreatment of luteolin. Luteolin attenuated VEGF transcription via blockade of t-BH-induced ROS production. Luteolin suppressed hypoxia-induced VEGF expression via attenuating hypoxia inducible factor 1 α expression. Conclusions Our results suggest that luteolin could be a potent anti-angiogenic agent for retinal neovascularization, which is related to anti-oxidative activity to block ROS production and to subsequently suppress VEGF expression and the pro-angiogenic effect of VEGF.

Vascular endothelial growth factor (VEGF) is a major regulator in retinal and choroidal angiogenesis, which are common causes of blindness in all age groups. Recently anti-VEGF treatment using anti-VEGF antibody has revolutionarily improved the visual outcome in patients with vaso-proliferative retinopathies. Herein, we demonstrated that bevacizumab as an anti-VEGF antibody could inhibit differentiation of retinoblastoma cells without affection to cellular viability, which would be mediated via blockade of extracellular signal-regulated kinase (ERK) 1/2 activation. The retinoblastoma cells expressed VEGFR-2 as well as TrkA which is a neurotrophin receptor associated with differentiation of retinoblastoma cells. TrkA in retinoblastoma cells was activated with VEGF treatment. Interestingly even in the concentration of no cellular death, bevascizumab significantly attenuated the neurite formation of differentiated retinoblastoma cells, which was accompanied by inhibition of neurofilament and shank2 expression. Furthermore, bevacizumab inhibited differentiation of retinoblastoma cells by blockade of ERK 1/2 activation. Therefore, based on that the differentiated retinoblastoma cells are mostly photoreceptors, our results suggest that anti-VEGF therapies would affect to the maintenance or function of photoreceptors in mature retina.

Clusterin is a secretory glycoprotein, which is highly up-regulated in a variety of normal and injury tissues undergoing apoptosis including infarct region of the myocardium. Here, we report that clusterin protects H9c2 cardiomyocytes from H2O2-induced apoptosis by triggering the activation of Akt and GSK-3β. Treatment with H2O2 induces apoptosis of H9c2 cells by promoting caspase cleavage and cytochrome c release from mitochondria. However, co-treatment with clusterin reverses the induction of apoptotic signaling by H2O2, thereby recovers cell viability. The protective effect of clusterin on H2O2-induced apoptosis is impaired by PI3K inhibitor LY294002, which effectively suppresses clusterin-induced activation of Akt and GSK-3β. In addition, the protective effect of clusterin is independednt on its receptor megalin, because inhibition of megalin has no effect on clusturin-mediated Akt/GSK-3β phosphoylation and H9c2 cell viability. Collectively, these results suggest that clusterin has a role protecting cardiomyocytes from oxidative stress and the Akt/GSK-3β signaling mediates anti-apoptotic effect of clusterin.

PURPOSE To investigate the differential roles of matrix metalloproteinase (MMP)-9 and MMP-2 in the proliferation or differentiation of retinoblastoma cells. METHODS Cell proliferation assay with an MMP-9 inhibitor and cell viability assay with an MMP-2 inhibitor were performed in retinoblastoma cells with 5 ng/mL fibroblast growth factor 2 for proliferation, 0.1% bovine serum albumin for differentiation, or reverse transcriptase-polymerase chain reaction (RT-PCR) for MMP-9, MMP-2, and their tissue inhibitors TIMP-1 and TIMP-2. Immunohistochemistry for MMP-2 and nm23 was performed using an experimental model of retinoblastoma. With the use of an MMP-2 inhibitor, Western blot analysis was performed for neurofilament, extracellular signal-regulated kinases 1 and 2 (ERK 1/2), and phospho-ERK 1/2, and neurite length was measured in differentiated retinoblastoma cells. RESULTS With the proliferation of retinoblastoma cells, MMP-9 expression was upregulated without alteration of MMP-2, TIMP-1, or TIMP-2. However, proliferation was not affected by the inhibition of MMP-9 activity. Interestingly, only MMP-2 expression, colocalized with differentiated cells in retinoblastoma tissue, was significantly increased in the differentiation of retinoblastoma cells. Inhibition of MMP-2 activity did not affect cellular viability but attenuated neurite outgrowth and neurofilament expression of differentiated retinoblastoma cells, which was mediated through the suppression of ERK 1/2 activation. CONCLUSIONS The authors suggest that differential expression of MMP-9 and -2 could reflect biological features, such as proliferation and differentiation, of retinoblastoma cells. In particular, MMP-2 could be directly involved in the regulation of differentiation of retinoblastoma cells. Therefore, therapeutic targeting to MMP-2 may prove useful for reducing malignancy through the differentiation of retinoblastoma cells.

Purpose Oxidative stress to retinal pigment epithelial (RPE) cells is thought to play a critical role in the pathogenesis of age-related macular degeneration (AMD). This study was conducted to investigate whether clusterin protects human RPE cells from ROS-induced apoptosis through a PI3K/Akt survival pathway. Methods The preventive effect of clusterin on reactive oxygen species (ROS) production and RPE cell death induced by hydrogen peroxide was determined in ARPE-19 cells. The ability of clusterin to protect RPE cells against ROS-mediated apoptosis was assessed by caspase-3 activity and DAPI staining. Furthermore, the protective effect of clusterin via the PI3K/Akt pathway was determined by Western blot analysis. Results Clusterin prevented ARPE-19 cells from H(2)O(2)-induced cell death and ROS production. H(2)O(2)-induced oxidative stress increased caspase-3 activity, which was significantly inhibited by clusterin, as determined by the abrogation of apoptotic bodies. Interestingly, clusterin induced Akt phosphorylation in human RPE cells under oxidative stress, which contributed to cell viability in ARPE-19 cells. This cell survival by clusterin was blocked by a PI3K inhibitor. Conclusions Clusterin may play a protective role in responding to the local redox environment of human RPE cells, which contributes to the cell survival via the PI3K/Akt pathway. Therefore, clusterin could be considered for the preventive approach to AMD.