Biomedical subjects

Zenji Makita

Publications and source records attributed to Zenji Makita.

At least 19 recordsLinked to original sources

PubMed2004

N epsilon-carboxymethyllysine in brain aging, diabetes mellitus, and Alzheimer's disease.

Oxidative stress has been implicated in the pathophysiology of Alzheimer's disease (AD) and diabetes mellitus (DM). N epsilon-carboxymethyllysine (CML) is an advanced glycation end product (AGE) recently found to be associated with oxidative stress mechanisms. Using immunocytochemical methods we examined the distribution of CML in brain tissue from AD and DM subjects and aging controls. CML reactivity was present in the cytoplasm of neurons, but there were marked differences in the intensity of expression, number of cells, and topographical distribution. CML expression was higher in hippocampus than in frontal and temporal cortex. In the hippocampus, neuronal and, to an extent, glial expression was more marked in CA3 and CA4 than in CA1 and CA2. In AD, CML was found to be coexpressed with tau protein, showing the similar neurofibrillary tangle shape, as well as in neuritic plaques but not in the core of amyloid plaques. We noted an increasing degree of CML expression such that the highest reactivity was evident in those with both AD and DM, followed by AD, DM, and aging controls. There was an inverse relationship between Braak staging and topography of CML expression. Although DM cases did not show Abeta deposition or neurofibrillary tangles, these findings suggest increased CML expression is not limited to AD. Nonetheless, high CML expression in AD with coexistent DM suggests there are additive effects compared with AD alone. It is plausible that the microangiopathy also associated with DM could worsen AD pathogenesis.

Aged↗

PubMed2003

Novel splice variants of the receptor for advanced glycation end-products expressed in human vascular endothelial cells and pericytes, and their putative roles in diabetes-induced vascular injury.

The binding of advanced glycation end-products (AGE) to the receptor for AGE (RAGE) is known to deteriorate various cell functions and is implicated in the pathogenesis of diabetic vascular complications. In the present study, we show that the cellular constituents of small vessels, endothelial cells (EC) and pericytes express novel splice variants of RAGE mRNA coding for the isoforms that lack the N-terminal V-type immunoglobulin-like domain (N-truncated) or the C-terminal transmembrane domain (C-truncated), as well as the known full-length mRNA. The ratio of the expression of the three variants was different between EC and pericytes; the content of the C-truncated form was highest in EC, whereas the full-length form was the most abundant in pericytes. Transfection experiments with COS-7 cells demonstrated that those variant mRNAs were translated into proteins as deduced; C-truncated RAGE was efficiently secreted into the culture media, and N-truncated RAGE was located mainly on the plasma membrane. The three isoforms were also detected in primary cultured human EC and pericytes. Further, full-length and C-truncated forms of RAGE bound to an AGE-conjugated column, whereas N-truncated RAGE did not. The AGE induction of extracellular-signal-related kinase phosphorylation and vascular endothelial growth factor in EC and of the growth and cord-like structure formation of EC was abolished completely by C-truncated RAGE, indicating that this endogenous secretory receptor (endogenous secretory RAGE) is cytoprotective against AGE. The results may contribute to our understanding of the molecular basis for the diversity of cellular responses to AGE and for individual variations in the susceptibility to diabetic vascular complications.

Alternative Splicing↗

PubMed2003

Effect of proteasome inhibitor on cultured mesencephalic dopaminergic neurons.

Proteasomal dysfunction has been implicated in the pathogenesis of Parkinson's disease (PD). We examined the effect of a selective proteasomal inhibitor, epoxomicin, on primary cultured mesencephalic neurons. Exposing rat cultured mesencephalic neurons to epoxomicin for 24 h resulted in neurotoxicity in a dose-dependent manner. Epoxomicin caused mitochondrial dysfunction, reduction in reduced glutathione (GSH), and increased generation of free radicals. Neuronal damage was significantly blocked by antioxidative/GSH-augmenting agents. Epoxomicin also increased the expression of Bax and decreased that of Bcl-2, which may cause mitochondrial dysfunction and release of free radicals. Dopaminergic neurons were preferentially resistant to the toxicity of epoxomicin. Inhibiting the synthesis of tetrahydrobiopterin (BH(4)), which has been reported to have antioxidative function, increased the susceptibility of dopaminergic neurons, whereas increasing BH(4) levels protected non-dopaminergic neurons. These findings suggest that BH(4) is at least in part a contributing factor to grand the resistance to dopaminergic neurons against epoxomicin neurotoxicity. Our results suggest that proteasome inhibition causes the neurotoxicity in mesencephalic neurons, but that is not sufficient to reproduce the selective damage to dopaminergic neurons, such as that seen in PD.

Animals↗

PubMed2003

Glycation--a sweet tempter for neuronal death.

Glycation, one of the post-translational modifications of proteins, is a nonenzymatic reaction initiated by the primary addition of a sugar aldehyde or ketone to the amino groups of proteins. In the early stage of glycation, the synthesis of intermediates leading to the formation of Amadori compounds occurs. In the late stage, advanced glycation end products (AGE) are irreversibly formed after a complex cascade of reactions. Several AGEs have been characterized chemically, while other new compounds remain to be identified. To date, studies of the contribution of glycation to diseases have been primarily focused on its relationship to diabetes and diabetes-related complications. However, glucose-induced damage is not limited to diabetic patients. Although it does not cause rapid or remarkable cell damage, glycation advances slowly and accompanies every fundamental process of cellular metabolism. It has recently become clear that glycation also affects physiological aging and neurodegenerative diseases such as Alzheimer's disease and amyotrophic lateral sclerosis. Glycation alters the biological activity of proteins and their degradation processes. Protein cross-linking by AGE results in the formation of detergent-insoluble and protease-resistant aggregates. Such aggregates may interfere with both axonal transport and intracellular protein traffic in neurons. In addition, glycation reactions lead to the production of reactive oxygen species. Conversely, glycation is promoted by oxidative stress. We speculate on the presence of synergism between glycation and oxidative stress. In this review, we provide an outline of glycation and propose some possible mechanisms of its cytotoxicity and defense systems against it.

Animals↗

PubMed2002

High expression of inducible 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (iPFK-2; PFKFB3) in human cancers.

Tumor cells maintain an especially high glycolytic rate to supply the anabolic precursors essential for de novo nucleotide synthesis. We recently cloned an inducible isozyme of 6-phosphofructo-2 kinase (iPFK-2) that bears an oncogene-like regulatory element in its mRNA and functions to produce fructose-2,6-bisphosphate, which is a powerful allosteric activator of glycolysis. Rapidly proliferating cancer cells constitutively express iPFK-2 in vitro, and inhibition of iPFK-2 expression decreases tumor growth in experimental animal models. We report herein that the expression of iPFK-2 mRNA and protein, as assessed by in situ hybridization and immunohistochemistry, is increased in many human cancers when compared with corresponding normal tissues. In particular, iPFK-2 expression was found to be markedly elevated in multiple aggressive primary neoplasms, including colon, breast, ovarian, and thyroid carcinomas. iPFK-2 mRNA and protein expression were induced by hypoxia in cultured human colon adenocarcinoma cells, and an examination of normal lung fibroblasts showed that iPFK-2 and fructose-2,6-bisphosphate levels increased specifically during the S phase of the cell cycle. These data indicate that iPFK-2 is abundantly expressed in human tumors in situ and may serve as an essential regulator of glycolysis during cell cycle progression and growth in an hypoxic microenvironment.

Cell Hypoxia↗

PubMed2002

Interferon-gamma-induced apoptosis and activation of THP-1 macrophages.

Apoptotic macrophages are frequently observed in human atherosclerotic lesions, and are considered to be involved in plaque instability in atherosclerosis. However, the molecular mechanism that promotes programmed cell death of macrophages in atherosclerosis remains to be elucidated. In this study, we investigated the effects of interferon-gamma (IFN-gamma), a cytokine secreted by activated T helper 1 (Th1) lymphocytes, on apoptotic cell death of THP-1 macrophages. Further we studied whether these apoptotic macrophages could be simultaneously activated in vitro and subsequently overgenerate monocyte chemoattractant protein-1 (MCP-1). When THP-1 macrophages were cultured with various concentrations of IFN-gamma, DNA synthesis was significantly decreased. IFN-gamma was found significantly to induce apoptotic cell death in THP-1 macrophages. RNase protection assay revealed that IFN-gamma up-regulated the mRNA levels of two pro-apoptotic molecules, tumor necrosis factor-alpha receptor 1 (TNFR1) and caspase-8, in THP-1 cells. Furthermore, TNF-alpha antibodies were found completely to neutralize the IFN-gamma-induced inhibition in DNA synthesis as well as apoptotic cell death in macrophages. IFN-gamma was found to activate these macrophages to stimulate MCP-1 production. The results suggest that IFN-gamma not only exerted apoptotic effects on macrophages, but also activated them and subsequently overgenerated MCP-1, and was thus involved in the development and progression of atherosclerosis.

Antibodies, Blocking↗

PubMed2002

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-kB (NF-kB) 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-kB 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-kB and AP-1 factors. By blocking AGE-RAGE signaling pathways, cerivastatin might be a promising remedy for treating patients with proliferative diabetic retinopathy.

Angiogenesis Inducing Agents↗

PubMed2002

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.

Antioxidants↗

PubMed2002

Advanced glycation end products (AGE) and their receptor (RAGE) in the brain of patients with Creutzfeldt-Jakob disease with prion plaques.

The objective of this study was to assess the pathological role of advanced glycation end products (AGE) and the receptor for AGE (RAGE) in Creutzfeldt-Jakob disease (CJD). We immunohistochemically investigated the occipital lobe of three patients with CJD containing with prion protein (PrP) plaques using anti-AGE and RAGE antibodies. Many PrP-positive plaques were observed in these patients, and the PrP-positive prion plaques also showed immunoreactivity for the anti-AGE antibody. Furthermore, many astrocytes contained-PrP positive granules, and the same astrocytes also contained many AGE- and RAGE-immunopositive granules. The staining pattern of these granules showed good concordance with that of PrP. These findings suggest that there may be a RAGE-mediated PrP degradation pathway in CJD as is the case for beta-amyloid protein in Alzheimer's disease.

Astrocytes↗

PubMed2002

Immunohistochemical demonstration of -(carboxymethyl)lysine protein adducts in normal and osteoarthritic cartilage.

N(epsilon)-(carboxymethyl)lysine (CML) is an advanced glycation end product formed by non-enzymatic glycation and oxidation of proteins. The distribution pattern of CML-modified proteins in normal and osteoarthritic (OA) cartilage was investigated using specific antibodies. In healthy articular cartilage, immunoreactivity for CML was preferably found in the extracellular matrix (ECM) of the superficial layer. In OA samples, CML immunoreactivity was not restricted to the ECM of the superficial layer. Interestingly, OA chondrocytes showed a remarkable cytoplasmic immunoreactivity for CML. With the help of a western blot analysis CML-modified proteins between 68 and 39 kDa could be demonstrated in OA cartilage samples. These results suggest that the accumulation of CML adducts contributes to the matrix damage in osteoarthritis. Therefore, the inhibition of CML accumulation may represent an effective therapeutic strategy to prevent severe OA cartilage injury.

Blotting, Western↗

PubMed2002

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.

Acetylcysteine↗

PubMed2002

Advanced glycation end products-induced apoptosis and overexpression of vascular endothelial growth factor in bovine retinal pericytes.

The influence of advanced glycation end products (AGEs) on apoptotic cell death and vascular endothelial growth factor (VEGF) gene expression in cultured bovine retinal pericytes was investigated. When pericytes were incubated with three immunochemically distinct AGEs, which were prepared in vitro by incubating bovine serum albumin with glucose, glyceraldehyde, or glycolaldehyde, apoptotic cell death and DNA ladder formation were significantly induced. The cytopathic effects of glyceraldehyde- or glycolaldehyde-derived AGEs were significantly enhanced in AGE receptor-transfected pericytes. Furthermore, all of these AGEs were found to upregulate the secretory forms of VEGF mRNA levels in retinal pericytes. These results suggest that AGEs disturbed retinal microvascular homeostasis by inducing pericyte apoptosis and VEGF overproduction and thus were involved in the pathogenesis of early phase diabetic retinopathy.

Acetaldehyde↗

PubMed2002

Advanced glycation endproduct-induced calcium handling impairment in mouse cardiac myocytes.

Long-standing diabetes causes cardiovascular complications including direct cardiac muscle weakening known as diabetic cardiomyopathy. This is characterized by disturbances in both cardiac contraction and relaxation, which are maintained by calcium homeostasis in cardiac cells. Our recent in vitro and in vivo studies have shown that advanced glycation endproducts (AGE) account for diabetic vasculopathy through their engagement of the receptor for AGE (RAGE). Here we show that AGE and RAGE may directly affect the myocardial Ca(2+) homeostasis. We created transgenic mice that overexpressed human RAGE in the heart and analyzed the Ca(2+) transients in cultivated cardiac myocytes (CM) from the RAGE-transgenic and non-transgenic control fetuses. RAGE overexpression was found to reduce the systolic and diastolic intracellular calcium concentration ([Ca(2+)](i)). Exposure to AGE caused a significant prolongation of the decay time of [Ca(2+)](i) in CM from control mice, and this response was augmented in CM from the RAGE transgenic mice. The results suggest that the AGE and RAGE could play an active role in the development of diabetes-induced cardiac dysfunction.

Animals↗

PubMed2002

Advanced glycation end products induce angiogenesis in vivo.

Advanced glycation end products (AGEs) have been thought to participate in diabetic microangiopathy. However, the effects of AGEs on angiogenesis have so far been mainly examined either in vitro or by using cultured cells. In the present study, we have analyzed whether AGEs induce angiogenesis in vivo by using the chorioallantoic membrane (CAM) assay. The CAM assay was carried out in embryonated hen eggs to determine the effects of AGEs. Following generation of AGEs based on bovine serum albumin (BSA), either AGE-BSA or nonglycated BSA was administered to the CAM and their effects on angiogenesis were assessed, together with an inhibitory effect of an anti-AGE antibody against AGE-BSA-induced angiogenesis. The histological features of AGE-induced vascular lumens were examined by immunohistochemical analysis for Factor VIII and smooth muscle alpha-actin. AGE-BSA induced angiogenesis in CAM in a dose- and time-dependent manner. AGE-induced angiogenesis on CAM was neutralized by the anti-AGE antibody. Immunohistochemical analysis demonstrated that AGE-induced vascular lumens were devoid of pericytes. Our data demonstrated that AGEs are an angiogenetic factor and that our system of AGE-induced abnormal vessels in CAMs is useful in further investigations of the mechanism of diabetic retinal angiogenesis and can also be used to provide a therapeutic model for diabetic angiopathy.

Animals↗

PubMed2002

Detection of N epsilon-(carboxymethyl)lysine (CML) and non-CML advanced glycation end-products in the anterior horn of amyotrophic lateral sclerosis spinal cord.

INTRODUCTION: The involvement of glycation in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis (ALS) was recently indicated. We previously reported the existence of an Amadori product, 1-hexitol-lysine (1-HL), which is formed in the early glycation reaction, in axonal spheroids of the anterior horn of the ALS spinal cord. OBJECTIVE: The purpose of the present study was to confirm the occurrence of the later-stage glycation reaction that follows the early glycation reaction and leads to the formation of advanced glycation end products (AGEs). METHOD: We examined whether N(epsilon)-(carboxymethyl)lysine (CML) and non-CML AGE are present in ALS spinal cords. RESULTS: Immunohistochemical staining with anti-CML antibody revealed intense positivity in the cell bodies of the remaining atrophic motor neurons and in microglia. Microglia were also positive on staining with anti-non-CML antibody. Axonal spheroids were also positive on anti-non-CML-antibody staining. Vascular endothelial cells were slightly stained by both antibodies. CONCLUSIONS: The presence of non-CML AGE in the anterior horn of the ALS spinal cord indicates that the later stage of the glycation reaction is involved in the pathology of ALS. The presence of CML in the anterior horn was also confirmed, and this may reflect augmented oxidative stress.

Aged↗

PubMed2002

Palmitate-induced apoptosis of microvascular endothelial cells and pericytes.

BACKGROUND: Recent observations in the EURODIAB Complications Study demonstrated that markers of insulin resistance are strong risk factors for retinopathy incidence in patients with diabetes. However, the molecular mechanism underlying this remains to be elucidated. In this study, we investigated the influence of palmitate, a major saturated free fatty acid in plasma, on the apoptotic cell death of cultured microvascular endothelial cells (EC) and retinal pericytes. MATERIALS AND METHODS: The intracellular formation of reactive oxygen species (ROS) was detected using the fluorescent probe CM-H(2)DCFDA. DNA synthesis was determined by measuring [(3) H]-thymidine incorporation into cells. DNA fragmentations of EC were quantitatively analyzed in an enzyme-linked immunosorbent assay, and DNA laddering was evaluated on agarose gel electrophoresis. RESULTS: Palmitate increased ROS generation in microvascular EC. Furthermore, palmitate significantly inhibited DNA synthesis and induced apoptotic cell death in EC, which were completely prevented by an antioxidant, N-acetylcysteine. Palmitate up-regulated pericyte mRNA levels of a receptor for advanced glycation end products (AGE), and thereby potentiated the apoptotic effects of AGE on pericytes. CONCLUSIONS: The results suggest that palmitate could induce apoptotic cell death in microvascular EC and pericytes through the overgeneration of intracellular ROS, and thus be involved in the development of diabetic retinopathy.

Apoptosis↗

PubMed2002

[OPB-9195].

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Animals↗

PubMed2002

Beraprost sodium, a prostaglandin I2 analogue, protects against advanced gycation end products-induced injury in cultured retinal pericytes.

BACKGROUND: Beraprost sodium, a prostaglandin I2 analogue, has been recently reported to exhibit beneficial effects on atherosclerosis in patients with diabetes. However, effects of beraprost sodium on microvascular injury in diabetes remain to be elucidated. We have previously shown that advanced glycation end products (AGE), senescent macroproteins formed at an accelerated rate in diabetes, caused pericyte apoptosis, thus being involved in the pathogenesis of the early phase of diabetic retinopathy. In this study, we examined whether beraprost sodium can protect against AGE-induced cytotoxicity in cultured retinal pericytes. MATERIALS AND METHODS: Intracellular formation of reactive oxygen species (ROS) was detected using a fluorescent probe. DNA synthesis was determined by measuring [3H]thymidine incorporation into cells. Apoptosis was determined by DNA fragmentations, which were quantitatively measured in an enzyme-linked immunosorbent assay. RESULTS: Beraprost sodium or forskolin, a stimulator of adenylate cyclase, was found to significantly inhibit AGE-induced ROS generation and the subsequent decrease in DNA synthesis in pericytes. Both treatments significantly prevented AGE-induced apoptotic cell death in pericytes. Furthermore, beraprost sodium was found to down-regulate AGE receptor mRNA levels in pericytes. CONCLUSION: The results demonstrated that cyclic AMP-elevating agents such as beraprost sodium and forskolin protected retinal pericytes from AGE-induced cytotoxicity through its anti-oxidative properties. Our present study suggests that beraprost sodium may have therapeutic potentials in treatment of patients with early diabetic retinopathy.

Animals↗