Hyoung Oh Jun

Protective effect of clusterin from oxidative stress-induced apoptosis in human retinal pigment epithelial 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.

Hypoxia-mediated retinal neovascularization and vascular leakage in diabetic retina is suppressed by HIF-1α destabilization by SH-1242 and SH-1280, novel hsp90 inhibitors

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 messageSH-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.

Inhibitory Activity of Bevacizumab to Differentiation of Retinoblastoma Cells

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.

Antiangiogenic effect of betaine on pathologic retinal neovascularization via suppression of reactive oxygen species mediated vascular endothelial growth factor signaling.

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<0.05). Even in a high concentration (2mM), betaine never induced any retinal toxicity or cytotoxicity. Betaine significantly inhibited VEGF-induced proliferation, migration, and tube formation in human retinal microvascular endothelial cells (HRMECs). Betaine suppressed VEGF-induced VEGFR-2, Akt and ERK phosphorylation in HRMECs. In human brain astrocytes, betaine reduced tBH-induced ROS production, and subsequently attenuated tBH-induced VEGFA mRNA transcription via suppression of ROS. Our data suggest that betaine has an anti-angiogenic effect on pathologic retinal neovascularization via suppression of ROS mediated VEGF signaling. Betaine could be a potent anti-angiogenic agent to treat pathologic retinal neovascularization.

Anti-apoptotic effect of clusterin on cisplatin-induced cell death of retinoblastoma cells

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.

L1 increases adhesion-mediated proliferation and chemoresistance of retinoblastoma

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.

Novel Hypoxia-Inducible Factor 1α (HIF-1α) Inhibitors for Angiogenesis-Related Ocular Diseases: Discovery of a Novel Scaffold via Ring-Truncation Strategy

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 benzene ring as a key structural feature of analog 34f was identified as a novel scaffold for HIF-1α inhibitors that can be used in lieu of a chromene ring.

Dry age-related macular degeneration like pathology in aged 5XFAD mice: Ultrastructure and microarray analysis

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.