Yosuke Inagaki

Advanced glycation end product-induced apoptosis and overexpression of vascular endothelial growth factor and monocyte chemoattractant protein-1 in human-cultured mesangial cells.

Advanced glycation end products (AGE) have been implicated in the pathogenesis of glomerulosclerosis in diabetes. However, their involvement in the development of the early phase of diabetic nephropathy has not been fully elucidated. We investigated the effects of AGE on growth and on vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein-1 (MCP-1) expression in human cultured mesangial cells. We prepared three immunochemically distinct AGE by incubating bovine serum albumin (BSA) with glucose, glyceraldehyde, or glycolaldehyde. When human mesangial cells were cultured with various types of AGE-BSA, viable cell numbers as well as DNA syntheses were significantly decreased. All of the AGE-BSA were found to significantly increase p53 and Bax protein accumulations and subsequently induce apoptotic cell death in mesangial cells. An antioxidant, N-acetylcysteine, significantly prevented the AGE-induced apoptotic cell death in mesangial cells. Human mesangial cells stimulated prostacyclin production by co-cultured glomerular endothelial cells. Furthermore, various types of AGE-BSA were found to up-regulate the levels of mRNAs for VEGF and stimulate the secretion of VEGF and MCP-1 proteins in mesangial cells. The results suggest that AGE disturbed glomerular homeostasis by inducing apoptotic cell death in mesangial cells and elicited hyperfiltration and microalbuminuria by stimulating the secretion of VEGF and MCP-1 proteins, thereby being involved in the pathogenesis of the early phase of diabetic nephropathy.

Angiogenesis induced by advanced glycation end products and its prevention by cerivastatin

We previously have found that advanced glycation end products (AGE), senescent macroproteins formed at an accelerated rate in diabetes, arise in vivo not only from glucose but also from reducing sugars. Furthermore, we recently have shown that glyceraldehyde‐ and glycolaldehyde‐derived AGE (glycer‐ and glycol‐AGE) are mainly involved in loss of pericytes, the earliest histopathological hallmark of diabetic retinopathy. However, the effects of these AGE proteins on angiogenesis, another vascular derangement in diabetic retinopathy, remain to be elucidated. In this study, we investigated whether these AGE proteins elicit changes in cultured endothelial cells that are associated with angiogenesis. When human skin microvascular endothelial cells (EC) were cultured with glycer‐AGE or glycol‐AGE, growth and tube formation of EC, the key steps of angiogenesis, were significantly stimulated. The AGE‐induced growth stimulation was significantly enhanced in AGE receptor (RAGE)‐overexpressed EC. Furthermore, AGE increased transcriptional activity of nuclear factor‐κB (NF‐κB) and activator protein‐1 (AP‐1) and then up‐regulated mRNA levels of vascular endothelial growth factor (VEGF) and angiopoietin‐2 (Ang‐2) in EC. Cerivastatin, a hydroxymethylglutaryl CoA reductase inhibitor; pyrrolidinedithiocarbamate; or curcumin was found to completely prevent the AGE‐induced increase in NF–κB and AP‐1 activity, VEGF mRNA up‐regulation, and the resultant increase in DNA synthesis in microvascular EC. These results suggest that the AGE‐RAGE interaction elicited angiogenesis through the transcriptional activation of the VEGF gene via NF‐κB and AP‐1 factors. By blocking AGE‐RAGE signaling pathways, cerivastatin might be a promising remedy for treating patients with proliferative diabetic retinopathy.

Advanced Glycation End-Products Attenuate Human Mesenchymal Stem Cells and Prevent Cognate Differentiation Into Adipose Tissue, Cartilage, and Bone†‡

The impact of AGEs on human MSCs was studied. AGEs inhibited the proliferation of MSCs, induced apoptosis, and prevented cognate differentiation into adipose tissue, cartilage, and bone, suggesting a deleterious effect of AGEs in the pathogenesis of musculoskeletal disorders in aged and diabetic patients.

Pigment epithelium-derived factor inhibits advanced glycation end product-induced retinal vascular hyperpermeability by blocking reactive oxygen species-mediated vascular endothelial growth factor expression

Pigment epithelium-derived factor (PEDF) is the most potent inhibitor of angiogenesis, suggesting that loss of PEDF contributes to proliferative diabetic retinopathy. However, the role of PEDF against retinal vascular hyperpermeability remains to be elucidated. We investigated here whether and how PEDF could inhibit the advanced glycation end product (AGE) signaling to vascular hyperpermeability. Intravenous administration of AGEs to normal rats not only increased retinal vascular permeability by stimulating vascular endothelial growth factor (VEGF) expression but also decreased retinal PEDF levels. Simultaneous treatments with PEDF inhibited the AGE-elicited VEGF-mediated permeability by down-regulating mRNA levels of p22phox and gp91phox, membrane components of NADPH oxidase, and subsequently decreasing retinal levels of an oxidative stress marker, 8-hydroxydeoxyguanosine. PEDF also inhibited the AGE-induced vascular hyperpermeability evaluated by transendothelial electrical resistance by suppressing VEGF expression. Furthermore, PEDF decreased reactive oxygen species (ROS) generation in AGE-exposed endothelial cells by suppressing NADPH oxidase activity via down-regulation of mRNA levels of p22PHOX and gp91PHOX. This led to blockade of the AGE-elicited Ras activation and NF-κB-dependent VEGF gene induction in endothelial cells. These results indicate that the central mechanism for PEDF inhibition of the AGE signaling to vascular permeability is by suppression of NADPH oxidase-mediated ROS generation and subsequent VEGF expression. Substitution of PEDF may offer a promising strategy for halting the development of diabetic retinopathy.

Pigment epithelium-derived factor protects cultured retinal pericytes from advanced glycation end product-induced injury through its antioxidative properties

Pigment epithelium-derived factor (PEDF) has recently been shown to be the most potent inhibitor of angiogenesis in the mammalian eye, suggesting that loss of PEDF is involved in the pathogenesis of proliferative diabetic retinopathy. However, a protective role for PEDF in pericyte loss in early diabetic retinopathy remains to be elucidated. In this study, we investigated whether PEDF proteins could protect against advanced glycation end product (AGE)-induced injury in retinal pericytes. Ligand blot analysis revealed that pericytes possessed a membrane protein with binding affinity for PEDF. PEDF proteins were found to significantly inhibit AGE-induced reactive oxygen species (ROS) generation and the subsequent decrease in DNA synthesis and apoptotic cell death in pericytes. Further, PEDF proteins completely restored the down-regulation of bcl-2 gene expression in AGE-exposed pericytes. The results demonstrated that PEDF proteins protected cultured pericytes from AGE-induced cytotoxicity through its anti-oxidative properties. Our present study suggests that substitution of PEDF proteins may be a promising strategy in treatment of patients with early diabetic retinopathy.

Overexpression of Pigment Epithelium-Derived Factor Decreases Angiogenesis and Inhibits the Growth of Human Malignant Melanoma Cells in Vivo

Pigment epithelium-derived factor (PEDF) has recently been shown to be the most potent inhibitor of angiogenesis in the mammalian eye, and is involved in the pathogenesis of angiogenic eye disease such as proliferative diabetic retinopathy. However, a functional role for PEDF in tumor growth and angiogenesis remains to be determined. In this study, we have investigated both the in vitro and in vivo growth characteristics of human malignant melanoma G361 cell lines, stably transfected to overexpress human PEDF. Expression levels of PEDF proteins in melanoma cell lines G361 and A375 were comparable with that of human cultured melanocytes, whereas vascular endothelial growth factor levels in two tumor cell lines were much stronger than that in normal melanocytes. Overexpression of PEDF was found to significantly inhibit tumor growth and vessel formation in G361 nude mice xenografts. Furthermore, in vitro proliferation rates of G361 cells were decreased in PEDF-transfected cells. PEDF proteins showed dose-dependent induced growth retardation and apoptotic cell death in nontransfected G361 cells, which were completely prevented by treatment with antibodies against the Fas ligand. Our present study highlights two beneficial effects of PEDF treatment on melanoma growth and expansion; one is the suppression of tumor angiogenesis, and the other is induction of Fas ligand-dependent apoptosis in tumor cells. PEDF therefore might be a promising novel therapeutic agent for treatment of patients with melanoma.

Pigment epithelium-derived factor prevents advanced glycation end products-induced monocyte chemoattractant protein-1 production in microvascular endothelial cells by suppressing intracellular reactive oxygen species generation

Aims/hypothesisMonocytes and macrophages accumulate in the lesion of the diabetic retina, which are most likely involved in the progression of diabetic retinopathy. The levels of monocyte chemoattractant protein-1 (MCP-1) in vitreous fluids were associated with the severity of proliferative diabetic retinopathy. Recently, pigment epithelium-derived factor has been shown to be involved in the pathogenesis of proliferative diabetic retinopathy. However, a role of pigment epithelium-derived factor in monocyte recruitments in diabetic retinopathy remains to be elucidated. In this study, we investigated effects of purified pigment epithelium-derived factor on AGE-induced reactive oxygen species generation, MCP-1 mRNA up-regulation and protein production in human cultured microvascular endothelial cells.MethodsThe intracellular formation of reactive oxygen species was measured using the fluorescent probe CM-H2DCFDA. MCP-1 gene expression was analysed in quantitative reverse transcription-polymerase chain reaction. Monocyte chemoattractant protein-1 production by microvascular endothelial cells was measured with an ELISA system.ResultsAGE increased intracellular reactive oxygen species generation in microvascular endothelial cells. Pigment epithelium-derived factor inhibited the AGE-induced reactive oxygen species generation in a dose-dependent manner. An anti-oxidant, N-acetylcysteine, or pigment epithelium-derived factor completely prevented the AGE-induced up-regulation of MCP-1 mRNA contents as well as protein production in microvascular endothelial cells.Conclusions/interpretationsPigment epithelium-derived factor inhibits the AGE-induced reactive oxygen species generation and the subsequent increase in MCP-1 production in microvascular endothelial cells. Our study suggests that substitution of pigment epithelium-derived factor could prevent the progression of diabetic retinopathy by attenuating the deleterious effects of AGE.

SOX6 suppresses cyclin D1 promoter activity by interacting with β-catenin and histone deacetylase 1 and its down-regulation induces pancreatic β-cell proliferation

Sex-determining region Y-box (SOX) 6 negatively regulates glucose-stimulated insulin secretion from β-cells and is a down-regulated transcription factor in the pancreatic islet cells of hyperinsulinemic obese mice. To determine the contribution of SOX6 to insulin resistance, we analyzed the effects of SOX6 on cell proliferation. Small interfering RNA-mediated attenuation of SOX6 expression stimulated the proliferation of insulinoma INS-1E and NIH-3T3 cells, whereas retroviral overexpression resulted in inhibition of cell growth. Quantitative real time-PCR analysis revealed that the levels of cyclin D1 transcripts were markedly decreased by SOX6 overexpression. Luciferase-reporter assay with β-catenin showed that SOX6 suppresses cyclin D1 promoter activities. In vitro binding experiments showed that the LZ/Q domain of SOX6 physically interacts with armadillo repeats 1-4 of β-catenin. Furthermore, chromatin immunoprecipitation assay revealed that increased SOX6 expression significantly reduced the levels of acetylated histones H3 and H4 at the cyclin D1 promoter. By using a histone deacetylase (HDAC) inhibitor and co-immunoprecipitation analysis, we showed that SOX6 suppressed cyclin D1 activities by interacting withβ-catenin and HDAC1. The data presented suggest that SOX6 may be an important factor in obesity-related insulin resistance.

Pigment epithelium-derived factor inhibits leptin-induced angiogenesis by suppressing vascular endothelial growth factor gene expression through anti-oxidative properties

Leptin, a circulating hormone secreted mainly from adipose tissues, is involved in the control of body weight. Recently, leptin was found to be an angiogenic factor, and its vitreous levels are associated with angiogenic eye diseases such as proliferative diabetic retinopathy. However, the molecular mechanism for leptin-elicited angiogenesis remains to be elucidated. Pigment epithelium-derived factor (PEDF) has been shown to be the most potent natural inhibitor of angiogenesis in the mammalian eye, and its levels in the vitreous were decreased in angiogenic eye diseases. In this study, we investigated whether and how PEDF could inhibit the leptin-induced DNA synthesis in microvascular endothelial cells (EC), a key step of angiogenesis. Leptin significantly increased intracellular reactive oxygen species (ROS) generation in microvascular EC. PEDF was found to inhibit the leptin-induced ROS generation in EC. An anti-oxidant, N-acetylcysteine, or PEDF completely prevented the leptin-induced upregulation of vascular endothelial growth factor (VEGF) mRNA levels as well as any increase in DNA synthesis in microvascular EC. Polyclonal antibodies against human VEGF were also found to completely inhibit DNA synthesis in leptin-exposed EC. The present study suggests that leptin could elicit angiogenesis through autocrine VEGF production via intracellular ROS generation. PEDF may block the angiogenic effects of leptin through its anti-oxidative properties.

Minodronate, a Newly Developed Nitrogen- Containing Bisphosphonate, Suppresses Melanoma Growth and Improves Survival in Nude Mice by Blocking Vascular Endothelial Growth Factor Signaling

Angiogenesis, a process by which new vascular networks are formed from pre-existing capillaries, is required for tumors to grow, invade, and metastasize. Vascular endothelial growth factor (VEGF), a specific mitogen to endothelial cells, is a crucial factor for tumor angiogenesis. In this study, we investigated whether minodronate, a newly developed nitrogen-containing bisphosphonate, could inhibit melanoma growth and improve survival in nude mice by suppressing the VEGF signaling. We found here that minodronate inhibited melanoma growth and improved survival in nude mice by suppressing the tumor-associated angiogenesis and macrophage infiltration. Minodronate completely inhibited the VEGF-induced increase in DNA synthesis and tube formation in endothelial cells by suppressing NADPH oxidase-mediated reactive oxygen species generation and Ras activation. Furthermore, minodronate inhibited the VEGF-induced expression of intercellular adhesion molecule-1 and monocyte chemoattractant protein-1 in endothelial cells. Minodronate decreased DNA synthesis and increased apoptotic cell death of cultured melanoma cells as well. Our present study suggests that minodronate might suppress melanoma growth and improve survival in nude mice by two independent mechanisms; one is by blocking the VEGF signaling in endothelial cells, and the other is by inducing apoptotic cell death of melanoma. The present study provides a novel potential therapeutic strategy for the treatment of melanoma.