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1.
Int J Mol Sci ; 24(15)2023 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-37569444

RESUMO

Increasing evidence suggests that exosomes are involved in retinal cell degeneration, including their insufficient release; hence, they have become important indicators of retinopathies. The exosomal microRNA (miRNA), in particular, play important roles in regulating ocular and retinal cell functions, including photoreceptor maturation, maintenance, and visual function. Here, we generated retinal organoids (ROs) from human induced pluripotent stem cells that differentiated in a conditioned medium for 60 days, after which exosomes were extracted from ROs (Exo-ROs). Subsequently, we intravitreally injected the Exo-RO solution into the eyes of the Royal College of Surgeons (RCS) rats. Intravitreal Exo-RO administration reduced photoreceptor apoptosis, prevented outer nuclear layer thinning, and preserved visual function in RCS rats. RNA sequencing and miRNA profiling showed that exosomal miRNAs are mainly involved in the mitogen-activated protein kinase (MAPK) signaling pathway. In addition, the expression of MAPK-related genes and proteins was significantly decreased in the Exo-RO-treated group. These results suggest that Exo-ROs may be a potentially novel strategy for delaying retinal degeneration by targeting the MAPK signaling pathway.


Assuntos
Exossomos , Células-Tronco Pluripotentes Induzidas , MicroRNAs , Degeneração Retiniana , Cirurgiões , Ratos , Humanos , Animais , Degeneração Retiniana/tratamento farmacológico , Degeneração Retiniana/metabolismo , Proteínas Quinases Ativadas por Mitógeno , Exossomos/metabolismo , Espécies Reativas de Oxigênio , Células-Tronco Pluripotentes Induzidas/metabolismo
2.
Cell Death Discov ; 8(1): 56, 2022 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-35136019

RESUMO

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.

3.
FASEB J ; 33(5): 6045-6054, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30742774

RESUMO

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.


Assuntos
Sistema y+ de Transporte de Aminoácidos/metabolismo , Astrócitos/metabolismo , Eritropoetina/metabolismo , Degeneração Retiniana/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células Cultivadas , Eritropoetina/genética , Glutationa/metabolismo , Humanos , Isquemia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Retina/metabolismo , Retina/patologia , Vasos Retinianos/metabolismo
4.
J Med Chem ; 61(20): 9266-9286, 2018 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-30252468

RESUMO

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.


Assuntos
Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Desenho de Fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Neovascularização Retiniana/tratamento farmacológico , Inibidores da Angiogênese/uso terapêutico , Animais , Benzeno/química , Benzeno/farmacologia , Benzeno/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Camundongos , Solubilidade , Relação Estrutura-Atividade , Água/química
5.
Oncotarget ; 8(25): 40006-40018, 2017 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-28467791

RESUMO

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 (Aß) 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 Aß related pathology and develop a new therapeutic approaches.


Assuntos
Envelhecimento/genética , Doença de Alzheimer/genética , Modelos Animais de Doenças , Degeneração Macular/genética , Envelhecimento/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Lâmina Basilar da Corioide/metabolismo , Lâmina Basilar da Corioide/patologia , Lâmina Basilar da Corioide/ultraestrutura , Perfilação da Expressão Gênica/métodos , Humanos , Degeneração Macular/metabolismo , Camundongos Endogâmicos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Epitélio Pigmentado da Retina/ultraestrutura
6.
PLoS One ; 12(4): e0175159, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28406972

RESUMO

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.


Assuntos
Barreira Hematorretiniana , Quinase 1 de Adesão Focal/imunologia , Monócitos , Comunicação Parácrina/imunologia , Transdução de Sinais/imunologia , Toxoplasma/imunologia , Toxoplasmose/imunologia , Barreira Hematorretiniana/imunologia , Barreira Hematorretiniana/parasitologia , Linhagem Celular , Humanos , Interleucina-8/imunologia , Monócitos/imunologia , Monócitos/parasitologia , Epitélio Pigmentado da Retina/imunologia , Epitélio Pigmentado da Retina/parasitologia , Proteína da Zônula de Oclusão-1/imunologia
7.
Oncotarget ; 8(9): 15441-15452, 2017 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-28061460

RESUMO

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.


Assuntos
Proliferação de Células/genética , Resistencia a Medicamentos Antineoplásicos/genética , Molécula L1 de Adesão de Célula Nervosa/genética , Neoplasias da Retina/genética , Retinoblastoma/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Adolescente , Adulto , Animais , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Criança , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Interferência de RNA , Neoplasias da Retina/tratamento farmacológico , Neoplasias da Retina/metabolismo , Retinoblastoma/tratamento farmacológico , Retinoblastoma/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto Jovem
8.
Vascul Pharmacol ; 90: 19-26, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27473515

RESUMO

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.


Assuntos
Inibidores da Angiogênese/farmacologia , Betaína/farmacologia , Células Endoteliais/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Neovascularização Retiniana , Vasos Retinianos/efeitos dos fármacos , Retinopatia da Prematuridade/prevenção & controle , Fator A de Crescimento do Endotélio Vascular/metabolismo , Inibidores da Angiogênese/administração & dosagem , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Betaína/administração & dosagem , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Células Endoteliais/metabolismo , Células Endoteliais/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Hiperóxia/complicações , Injeções Intravítreas , Camundongos Endogâmicos C57BL , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia , Retinopatia da Prematuridade/etiologia , Retinopatia da Prematuridade/metabolismo , Retinopatia da Prematuridade/patologia , Transdução de Sinais/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
9.
Oncol Rep ; 34(5): 2745-51, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26323829

RESUMO

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.


Assuntos
Células Endoteliais/citologia , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Sítios de Ligação , Hipóxia Celular , Movimento Celular , Claudina-5/metabolismo , Células Endoteliais/metabolismo , Fatores de Crescimento de Fibroblastos/química , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Regiões Promotoras Genéticas , Regulação para Cima , Proteína da Zônula de Oclusão-1/metabolismo
10.
Cell Calcium ; 57(2): 101-8, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25605522

RESUMO

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.


Assuntos
Retina/efeitos dos fármacos , Neovascularização Retiniana/patologia , Canais de Cátion TRPC/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Hipóxia Celular , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Retina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Canais de Cátion TRPC/antagonistas & inibidores , Canais de Cátion TRPC/genética , Regulação para Cima , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
11.
Oncotarget ; 5(22): 11513-25, 2014 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-25359779

RESUMO

Retinoblastoma, the most common intraocular malignant tumor in children, is characterized by the loss of both functional alleles of RB1 gene, which however alone cannot maintain malignant characteristics of retinoblastoma cells. Nevertheless, the investigation of other molecular aberrations such as matrix metalloproteinases (MMPs) and miRNAs is still lacking. In this study, we demonstrate that STAT3 is activated in retinoblastoma cells, Ki67-positive areas of in vivo orthotopic tumors in BALB/c nude mice, and human retinoblastoma tissues of the advanced stage. Furthermore, target genes of STAT3 including BCL2, BCL2L1, BIRC5, and MMP9 are up-regulated in retinoblastoma cells compared to other retinal constituent cells. Interestingly, STAT3 inhibition by targeted siRNA suppresses the proliferation of retinoblastoma cells and the formation of in vivo orthotopic tumors. In line with these results, STAT3 siRNA effectively induces down-regulation of target genes of STAT3. In addition, miRNA microarray analysis and further real-time PCR experiments with STAT3 siRNA treatment show that STAT3 activation is related to the up-regulation of miR-17-92 clusters in retinoblastoma cells via positive feedback loop between them. In conclusion, we suggest that STAT3 inhibition could be a potential therapeutic approach in retinoblastoma through the suppression of tumor proliferation.


Assuntos
Regulação Neoplásica da Expressão Gênica , MicroRNAs/metabolismo , Retinoblastoma/metabolismo , Fator de Transcrição STAT3/metabolismo , Alelos , Animais , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Humanos , Antígeno Ki-67/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Família Multigênica , RNA Longo não Codificante , RNA Interferente Pequeno/metabolismo
12.
J Mol Med (Berl) ; 92(10): 1083-92, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24875598

RESUMO

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.


Assuntos
Benzopiranos/uso terapêutico , Diabetes Mellitus Experimental/tratamento farmacológico , Retinopatia Diabética/tratamento farmacológico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neovascularização Retiniana/tratamento farmacológico , Animais , Astrócitos , Benzopiranos/farmacologia , Permeabilidade Capilar/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Retinopatia Diabética/metabolismo , Células Endoteliais , Expressão Gênica/efeitos dos fármacos , Humanos , Hipóxia/tratamento farmacológico , Hipóxia/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Neovascularização Retiniana/metabolismo
13.
Biochem Biophys Res Commun ; 444(1): 63-8, 2014 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-24434146

RESUMO

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.


Assuntos
Retinopatia Diabética/metabolismo , Proteína Quinase C/antagonistas & inibidores , Vasos Retinianos/metabolismo , Proteínas de Junções Íntimas/metabolismo , Animais , Barreira Hematorretiniana , Permeabilidade Capilar , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Retinopatia Diabética/genética , Células Endoteliais/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , RNA Interferente Pequeno/genética , Junções Íntimas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
14.
Oncol Rep ; 30(6): 2713-8, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24085287

RESUMO

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.


Assuntos
Apoptose/efeitos dos fármacos , Cisplatino/administração & dosagem , Clusterina/genética , Retinoblastoma/tratamento farmacológico , Caspase 3/biossíntese , Linhagem Celular Tumoral , Clusterina/biossíntese , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos
15.
J Biomed Sci ; 20: 38, 2013 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-23786217

RESUMO

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.


Assuntos
Retinopatia Diabética/patologia , Retinopatia Diabética/terapia , Modelos Animais de Doenças , Animais , Humanos
16.
Invest Ophthalmol Vis Sci ; 53(12): 7718-26, 2012 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-23099493

RESUMO

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.


Assuntos
Regulação para Baixo , Luteolina/uso terapêutico , RNA Mensageiro , Espécies Reativas de Oxigênio/antagonistas & inibidores , Retina/patologia , Neovascularização Retiniana/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Apoptose , Western Blotting , Sobrevivência Celular , Células Cultivadas , Modelos Animais de Doenças , Angiofluoresceinografia , Fundo de Olho , Humanos , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Estresse Oxidativo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/metabolismo , Retina/efeitos dos fármacos , Retina/metabolismo , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia , Fator A de Crescimento do Endotélio Vascular/biossíntese
17.
PLoS One ; 7(3): e33456, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22457763

RESUMO

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.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Diferenciação Celular/efeitos dos fármacos , Retinoblastoma/patologia , Sequência de Bases , Bevacizumab , Linhagem Celular Tumoral , Primers do DNA , Humanos , Imuno-Histoquímica , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Neurofilamentos/metabolismo , Receptor trkA/metabolismo , Retinoblastoma/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
18.
Exp Mol Med ; 43(1): 53-61, 2011 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-21270507

RESUMO

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-3ß. 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-3ß. In addition, the protective effect of clusterin is independent on its receptor megalin, because inhibition of megalin has no effect on clusterin-mediated Akt/GSK-3ß phosphoylation and H9c2 cell viability. Collectively, these results suggest that clusterin has a role protecting cardiomyocytes from oxidative stress and the Akt/GSK-3ß signaling mediates anti-apoptotic effect of clusterin.


Assuntos
Apoptose , Clusterina/farmacologia , Miócitos Cardíacos/metabolismo , Estresse Oxidativo , Transdução de Sinais , Animais , Western Blotting , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular , Cromonas/farmacologia , Clusterina/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Peróxido de Hidrogênio/farmacologia , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno , Ratos , Espécies Reativas de Oxigênio/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos
19.
Am J Pathol ; 176(3): 1517-24, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20110406

RESUMO

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.


Assuntos
Barreira Hematorretiniana/enzimologia , Barreira Hematorretiniana/patologia , Retinopatia Diabética/enzimologia , Retinopatia Diabética/patologia , Proteína Quinase C-delta/antagonistas & inibidores , Animais , Barreira Hematorretiniana/efeitos dos fármacos , Barreira Hematorretiniana/fisiopatologia , Permeabilidade Capilar/efeitos dos fármacos , Citosol/efeitos dos fármacos , Citosol/enzimologia , Retinopatia Diabética/fisiopatologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Células Endoteliais/patologia , Genes Dominantes/genética , Humanos , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microvasos/patologia , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Proteína Quinase C-delta/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Vasos Retinianos/patologia , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia
20.
Invest Ophthalmol Vis Sci ; 51(3): 1783-8, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19933182

RESUMO

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.


Assuntos
Diferenciação Celular , Proliferação de Células , Metaloproteinase 2 da Matriz/fisiologia , Metaloproteinase 9 da Matriz/fisiologia , Neoplasias da Retina/enzimologia , Retinoblastoma/enzimologia , Animais , Western Blotting , Sobrevivência Celular , Modelos Animais de Doenças , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Inibidores de Metaloproteinases de Matriz , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase 6 Ativada por Mitógeno/metabolismo , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Neuritos/fisiologia , Neoplasias da Retina/patologia , Retinoblastoma/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Soroalbumina Bovina/farmacologia , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Células Tumorais Cultivadas
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