Advanced glycation end products inhibit de novo protein synthesis and induce TGF-beta overexpression in proximal tubular cells.

BACKGROUND: We have shown previously that OPB-9195, a novel inhibitor of advanced glycation end products (AGE), significantly prevented renal tubular injury and tubulointerstitial fibrosis in spontaneous diabetic rats. However, the molecular mechanisms underlying this have not been fully elucidated. METHODS: Three immunochemically distinct AGE were prepared by incubating bovine serum albumin (BSA) with glucose, glyceraldehyde, or methylglyoxal. Then, the effects of AGE on human proximal tubular epithelial cells were examined. The intracellular formation of reactive oxygen species (ROS) was detected using the fluorescent probe CM-H2DCFDA. DNA synthesis was evaluated by thymidine uptake, and de novo protein synthesis was determined by [3H]leucine incorporation. Prostaglandin E2 (PGE2) and transforming growth factor-beta (TGF-beta) released into media were quantitatively analyzed in an enzyme-linked immunosorbent assay. TGF-beta gene expression was analyzed by quantitative reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: When these AGE-BSA were administered to tubular cells, each of them increased generation of intracellular ROS. All of the AGE-BSA, but not non-glycated BSA, were found to induce statistically significant decreases in de novo protein synthesis and PGE2 secretion by tubular cells. Furthermore, AGE-BSA up-regulated the levels of mRNAs for TGF-beta in tubular cells. The structural epitope designated glucose-derived AGE was found to have the greatest cytopathic effects on tubular cells. These AGE-induced inhibition of protein synthesis and PGE2 secretion as well as the up-regulation of TGF-beta mRNA were found to be completely prevented by N-acetylcysteine. Furthermore, H2O2 was shown to inhibit protein synthesis and PGE2 secretion by proximal tubular cells in a dose-dependent manner. CONCLUSION: The results suggest that AGE inhibits de novo protein synthesis and stimulates TGF-beta mRNA expression in proximal tubular epithelial cells through overgeneration of intracellular ROS. Thus, AGE are involved in the pathogenesis of tubular injury in diabetic nephropathy.

CS-886, a new angiotensin II type 1 receptor antagonist, ameliorates glomerular anionic site loss and prevents progression of diabetic nephropathy in Otsuka Long-Evans Tokushima fatty rats.

BACKGROUND: Diabetic nephropathy is a leading cause of end-stage renal disease in industrialized countries. Previous studies have documented that angiotensin converting enzyme (ACE) inhibitors consistently reduce albuminuria and retard the progression of diabetic nephropathy. However, the involvement of angiotensin II in diabetic nephropathy is not fully understood. MATERIALS AND METHODS: In this study we compared the effects of CS-866, a new angiotensin II type 1 receptor antagonist, to that of an ACE inhibitor, temocapril hydrochloride, on the development and progression of diabetic nephropathy using Otsuka Long-Evans Tokushima fatty rats, a type II diabetes mellitus model animal. RESULTS: High doses of CS-866 or temocapril treatment were found to significantly improve urinary protein and beta(2)-microglobulin excretions in diabetic rats. In electron microscopic analysis, loss of glomerular anionic sites, one of the causes of glomerular hyperpermeability in diabetic nephropathy, was found to be significantly prevented by CS-866 treatment. Light microscopic examinations revealed that both treatments ameliorated glomerular sclerosis and tubulointerstitial injury in diabetic rats. Furthermore, high doses of CS-866 or temocapril treatment significantly reduced the positive stainings for transforming growth factor-beta (TGF-beta), vascular endothelial growth factor, and type IV collagen in glomeruli of diabetic rats. CONCLUSIONS: These results indicate that intrarenal angiotensin II type 1 receptor activation plays a dominant role in the development and progression of diabetic nephropathy. Our study suggests that CS-866 represents a valuable new drug for the treatment of diabetic patients with 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-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.

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.

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.

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.

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.