Sho-ichi Yamagishi

The Receptor for Advanced Glycation End Products Is Induced by the Glycation Products Themselves and Tumor Necrosis Factor-α through Nuclear Factor-κB, and by 17β-Estradiol through Sp-1 in Human Vascular Endothelial Cells

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. Here we show that AGE, tumor necrosis factor-α (TNF-α), and 17β-estradiol (E2) up-regulated RAGE mRNA and protein levels in human microvascular endothelial cells and ECV304 cells, with the mRNA stability being essentially invariant. Transient transfection experiments with human RAGE promoter-luciferase chimeras revealed that the region from nucleotide number −751 to −629 and the region from −239 to −89 in the RAGE 5′-flanking sequence exhibited the AGE/TNF-α and E2 responsiveness, respectively. Site-directed mutation of an nuclear factor-κB (NF-κB) site at −671 or of Sp-1 sites at −189 and −172 residing in those regions resulted in an abrogation of the AGE/TNF-α- or E2-mediated transcriptional activation. Electrophoretic mobility shift assays revealed that ECV304 cell nuclear extracts contained factors which retarded the NF-κB and Sp-1 elements, and that the DNA-protein complexes were supershifted by anti-p65/p50 NF-κB and anti-Sp-1/estrogen receptor α antibodies, respectively. These results suggest that AGE, TNF-α, and E2 can activate the RAGE gene through NF-κB and Sp-1, causing enhanced AGE-RAGE interactions, which would lead to an exacerbation of diabetic microvasculopathy.

Advanced Glycation End Products-driven Angiogenesis in Vitro INDUCTION OF THE GROWTH AND TUBE FORMATION OF HUMAN MICROVASCULAR ENDOTHELIAL CELLS THROUGH AUTOCRINE VASCULAR ENDOTHELIAL GROWTH FACTOR

This study was undertaken to determine whether and how advanced glycation end products (AGE), senescent macroproteins accumulated in various tissues under hyperglycemic states, cause angiogenesis, the principal vascular derangement in diabetic microangiopathy. We first prepared AGE-bovine serum albumin (BSA) and anti-AGE antiserum using AGE-RNase A. Then AGE-BSA was administered to human skin microvascular endothelial cells in culture, and their growth was examined. The AGE-BSA, but not nonglycated BSA, was found to induce a statistically significant increase in the number of viable endothelial cells as well as their synthesis of DNA. The increase in DNA synthesis by AGE-BSA was abolished by anti-AGE antibodies. AGE-BSA also stimulated the tube formation of endothelial cells on Matrigel. We obtained the following evidence that it is vascular endothelial growth factor (VEGF) that mainly mediates the angiogenic activities of AGE. (1) Quantitative reverse transcription-polymerase chain reaction analysis of poly(A)+ RNA from microvascular endothelial cells revealed that AGE-BSA up-regulated the levels of mRNAs for the secretory forms of VEGF in time- and dose-dependent manners, while endothelial cell expression of the genes encoding the two VEGF receptors, kinase insert domain-containing receptor and fms-like tyrosine kinase 1, remained unchanged by the AGE treatment. Immunoprecipitation analysis revealed that AGE-BSA did increase de novo synthesis of VEGF. (2) Monoclonal antibody against human VEGF completely neutralized both the AGE-induced DNA synthesis and tube formation of the endothelial cells. The results suggest that AGE can elicit angiogenesis through the induction of autocrine vascular VEGF, thereby playing an active part in the development and progression of diabetic microangiopathies.

Induction of various blood-brain barrier properties in non-neural endothelial cells by close apposition to co-cultured astrocytes

Vascular endothelial cells (EC) exhibit organ‐to‐organ heterogeneity in their functions and morphologies. In particular, brain capillary EC have unique characteristics exemplified by the blood‐brain barrier (BBB). The formation and the maintenance of BBB have been ascribed to EC responses to inductive signal(s) or factor(s) from astrocytes that encircle microvessels in the central nervous system. These EC responses were demonstrated in numerous in vivo studies, exemplified by those of Janzer and Raff (Nature 325:253, 1987) and Tout et al. (Neuroscience 55:291, 1993) showing that transplanted astrocytes induced BBB properties in non‐neural vascular EC. In this study, we constructed a heterologous co‐culture system, in which rat fetal brain astrocytes were cultivated on one surface of a porous membrane and human umbilical vein EC on the opposite surface. Electron microscopic examination revealed that astrocytes passed their endfeet through the pores, making contact with EC. In this system, γ‐glutamyltranspeptidase (γ‐GTP) activity in EC was found to be significantly increased by contacting astrocytes in a density‐and time‐dependent manner, but not when the astrocyte feeder layer was apart from EC or replaced by COS cells; astrocyte‐derived extracellular matrix partially activated γ‐GTP. mRNAs for some of the representative BBB markers, including transferrin receptor, P‐glycoprotein, brain‐type glucose transporter(GLUT‐1), and γ‐GTP were also demonstrated by reverse transcription‐polymerase chain reaction to be upregulated in EC co‐cultured with astrocytes. Astrocyte inductions of close membrane apposition resembling a zonula occludens and of an increase in the content of mitochondria in EC were also noted in electron micrographs. Furthermore, an increased barrier activity against inulin was conferred on EC when they were lined with astrocytes. The results obtained with this heterologous co‐culture system thus indicate that through contact with their feet, astrocytes are capable of transdifferentiating non‐neural EC into the brain type, endowing them with the BBB properties. Glia 19:13–26, 1997

Advanced glycation endproducts inhibit prostacyclin production and induce plasminogen activator inhibitor-1 in human microvascular endothelial cells

Summary Several thrombogenic abnormalities are associated with diabetes. To investigate the underlying molecular mechanisms, we examined the effects of advanced glycation endproducts (AGE), non-enzymatically glycated protein derivatives, on the production of prostacyclin (PGI2), an anti-thrombogenic prostanoid, and of plasminogen activator inhibitor-1 (PAI-1), a fast-acting serine protease inhibitor of fibrinolysis, in human microvascular endothelial cells (EC). Firstly, AGE-bovine serum albumin (BSA) but not non-glycated BSA, was found to considerably decrease the production of PGI2 to about two-thirds of the control value. Secondly, quantitative reverse transcription-polymerase chain reaction showed that AGE-BSA increased the EC levels of mRNA coding for PAI-1, this being associated with a concomitant increase in the immunoreactive PAI-1 contents and the anti-fibrinolytic activity. Thirdly, the effects of AGE on PGI2 and PAI-1 syntheses in EC were found to be mediated by a receptor for AGE (RAGE) because antisense DNA against RAGE mRNA could reverse the AGE effects. Further, it was found that AGE decreased the intracellular cyclic AMP concentrations in EC and that cyclic AMP agonists such as dibutyryl cyclic AMP, forskolin and PGI2 analogue reduced the AGE-stimulated PAI-1 production, suggesting the involvement of cyclic AMP in the AGE-signalling pathway. The results thus suggest that AGE have the ability to cause platelet aggregation and fibrin stabilization, resulting in a predisposition to thrombogenesis and thereby contributing to the development and progression of diabetic vascular complications. [Diabetologia (1998) 41: 1435–1441]

Deficient cotinine formation from nicotine is attributed to the whole deletion of the CYP2A6 gene in humans

Nicotine is mainly metabolized to cotinine by cytochrome P450 (CYP) 2A6. Large interindividual differences in nicotine metabolism have been reported in humans. The purpose of this study was to clarify the relationship between the poor metabolism of nicotine and the existence of the CYP2A6v1 and CYP2A6v2 alleles, and a whole deletion allele of the CYP2A6 gene. The plasma concentrations of nicotine and cotinine were measured in 10 healthy subjects after each smoked one cigarette or chewed one piece of nicotine gum. One subject showed no detectable cotinine level in plasma when smoking and the lowest cotinine level when receiving nicotine gum. The subject was regarded as a poor metabolizer of nicotine by a probit analysis and was found to carry a homozygous whole deletion allele of the CYP2A6 gene. This is the first report to show that deficient cotinine formation in humans is attributed to the whole deletion of the CYP2A6 gene.

Placenta growth factor (PlGF) mRNA expression in brain tumors

To investigate the relationship between placenta growth factor (PlGF) and brain tumor angiogenesis, we screened 36 primary and 3 metastatic brain tumors. We examined the expression of PlGF mRNA with respect to vasculature of various tumors which was determined by preoperative angiography. The expression of genes of the other angiogenic factors, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) was also tested, and compared to that of PlGF. The primary tumors consisted of 16 meningiomas, 7 gliomas, 7 schwannomas, 4 pituitary adenomas, 1 germinoma, and 1 choriocarcinoma. Using a quantitative reverse transcription-polymerase chain reaction, the mRNA for PlGF149 and PlGF170 were detected in 25 out of 39 (64.1%) brain tumors. In primary brain tumors, PlGF mRNA was expressed in all the hypervascular tumors, but only in 5 of 16 hypovascular tumors (31.3%). None of the 3 metastatic hypervascular tumors expressed PlGF mRNA. The VEGF and bFGF mRNA expression was both detected in 87.2% of the tumors examined. We conducted hypoxic experiments with cultured U-251MG human glioma cells to determine the mechanism of PlGF gene regulation. As the atmospheric oxygen concentration was decreased, the PlGF mRNA level in the U-251MG cells was markedly increased. These results suggest that PlGF may contribute to the pathogenesis of brain tumor angiogenesis.

Roles of the AGE-RAGE system in vascular injury in diabetes

Abstract: This study concerns whether advanced glycation endproducts (AGE) are related to microvascular derangement in diabetes, exemplified by pericyte loss and angiogenesis in retinopathy and by mesangial expansion in nephropathy. AGE caused a decrease in viable pericytes cultivated from bovine retina. On the other hand, AGE stimulated the growth and tube formation of human microvascular endothelial cells (EC), this being mediated by autocrine vascular endothelial growth factor. In AGE‐exposed rat mesangial cells, type IV collagen synthesis was induced. Those AGE actions were dependent on a cell surface receptor for AGE (RAGE), because they were abolished by RAGE antisense or ribozyme. The AGE‐RAGE system may thus participate in the development of diabetic microangiopathy. This proposition was supported by experiments with animal models; several indices characteristic of retinopathy were correlated with circulating AGE levels in OLETF rats. The predisposition to nephropathy was augmented in RAGE transgenic mice when they became diabetic.

Upregulation of Retinal Vascular Endothelial Growth Factor mRNAs in Spontaneously Diabetic Rats without Ophthalmoscopic Retinopathy

Vascular endothelial growth factor (VEGF) has recently been shown to be involved in the pathogenesis of proliferative diabetic retinopathy. However, its involvement in the development of the early phase of diabetic retinopathy is not fully understood. In this study we investigated the retinal VEGF mRNA level in spontaneously diabetic Otsuka Long-Evans Tokushima fatty (OLETF) rats, a model of non-insulin-dependent diabetes, without overt retinopathy, using quantitative reverse-transcription polymerase chain reaction. The retinal VEGF mRNA level was 2.2 times higher (p < 0.0005) in OLETF rats than in control rats at the age of 60 weeks. Moreover, their retinal mRNA level was positively correlated with serum concentration of advanced glycation end products (AGEs) but not to serum glucose concentration. Furthermore, the peak latency of the oscillatory potentials in the electroretinogram, one of the most sensitive markers for the early phase of diabetic retinopathy, was significantly prolonged in OLETF rats (p < 0.05), being also correlated with the serum AGE concentration. The results thus suggest that AGEs, which are formed acceleratedly in diabetic conditions, are involved in the development of the early phase of diabetic retinopathy probably through the induction of retinal VEGF mRNAs.

Advanced glycosylation end products stimulate the growth but inhibit the prostacyclin-producing ability of endothelial cells through interactions with their receptors

The influence of advanced glycosylation end products (AGE) on endothelial cells was investigated. When human umbilical endothelial cells were cultured with AGE‐bovine serum albumin, viable cell number as well as DNA synthesis was significantly stimulated, whereas prostacyclin production by the endothelial cells was decreased. Antisense oligodeoxyribonucleotides against mRNA coding for AGE receptor were found to reverse both the AGE‐induced growth stimulation and the inhibition of prostacyclin production in endothelial cells. These results thus suggest that AGE ligand‐receptor interactions in endothelial cells can promote angiogenesis and thrombogenesis, leading to the development of diabetic vascular complications.

Serum 1,5-anhydro-D-glucitol levels in liver cirrhosis.

Abstract We studied the serum 1,5-anhydro-d-glucitol (AG) levels, a marker of glycemic control, in liver cirrhotic patients who had no evidence of glycosuria in 24-h urine samples in order to clarify the effects of impaired liver function on serum AG metabolism. We showed first that serum AG concentrations were significantly lower in cirrhotic patients than in age- and sex-matched healthy controls (17.6±1.6 vs 26.3±1.7 µg/ml, P<0.05). Moreover, serum AG levels were found to be positively correlated with both serum cholinesterase and albumin levels. The observations indicate that serum AG levels were decreased in liver cirrhosis, especially in cases of severely reduced hepatic functions, suggesting the possibility of altered AG synthesis in liver cirrhosis.