Department of Biochemistry and Molecular Vascular Biology, and *Department of Ophthalmology, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan.
Correspondence to Dr. Hiroshi Yamamoto, Department of Biochemistry and Molecular Vascular Biology, Division of Cardiovascular Medicine, Kanazawa University Graduate School of Medical Science, 13-1 Takara-machi, Kanazawa 920-8640, Japan. Phone: 81-76-265-2180; Fax: 81-76-234-4226;
ABSTRACT. As is diabetes itself, diabetic vasculopathy is amultifactor disease. Studies revealed advanced glycation endproducts (AGE) as the major environmental account for vascularcell derangement characteristic of diabetes and the receptorfor AGE (RAGE) as the major genic factor that responds to them.AGE fractions that caused the vascular derangement were provedto be RAGE ligands. When made diabetic, RAGE transgenic miceexhibited the exacerbation of the indices of nephropathy andretinopathy, and this was prevented by the inhibition of AGEformation. Extracellular signals and nuclear factors that inducethe transcription of human RAGE gene were also identified, whichwould be regarded as risk factors of diabetic complications.Through an analysis of vascular polysomal poly(A)+RNA, a novelsplice variant coding for a soluble RAGE protein was found andwas named endogenous secretory RAGE. Endogenous secretory RAGEwas able to capture AGE ligands and to neutralize the AGE actionon endothelial cells, suggesting that this variant has a potentialto protect blood vessels from diabetes-induced injury. The AGE-RAGEsystem, therefore, should be a candidate molecular target forovercoming this life- and quality-of-life–threateningdisease. E-mail: yamamoto@med.kanazawa-u.ac.jp
In 1912, Maillard (1) reported the generation of brown-coloredsubstances by a nonenzymatic reaction between reducing sugarsand amino acids. It begins with linkage between the carbonylgroup and the amino group to form Schiff bases and then Amadoricompounds, finally yielding irreversibly cross-linked productstermed "mélanoïdine." The series of the chemicalreactions was named after the discoverer and has been one ofthe major themes in food chemistry, because melanoidines constitutean essential component of colors, odors, and tastes of a widevariety of foods. The Maillard reaction, however, does not occurmerely on kitchen ranges. In 1981, Monnier and Cerami (2) documentedthat it can take place within our bodies. Because it proceedsas we age, the final products were then termed "AGE." The AGEformation and accumulation are most accelerated under diabetes.
The major sources of the carbonyl group in the glycation reactionin vivo include glucose and carbonyl compounds, such as glyceraldehyde,glyoxal, glycolaldehyde, methylglyoxal, and 3-deoxyglucosone,which are derived from glucose, Schiff bases, and Amadori compounds(3) (Figure 1). Long half-lived proteins, such as serum albumin,lens crystallin, and collagen in the extracellular matrix, areakin to be glycated.
Microvessels, which are first deranged in diabetic retinopathyand nephropathy, are composed of endothelial cells (EC) andpericytes. By co-culture experiments, Yamagishi et al. (4,5)showed that pericytes not only regulate the growth of neighboringEC but also preserve EC-specific functions, including the productionof prostacyclin, an antithrombogenic prostanoid. This indicatesthat when the pericyte–EC interaction is impaired, angiogenesisand thrombogenesis should result. Such a state does occur inthe early phase of retinopathy and is known as "pericyte loss."It was AGE that we noticed as a cause of pericyte loss. We preparedAGE-BSA by incubating BSA with glucose and administered theresultant material to bovine retinal pericytes in culture. AGEwere found to retard the growth of pericytes and also to exertan acute toxicity to this cell type (6). Yonekura et al. (7)also prepared AGE-BSA with glyceraldehydes, glyoxal, glycolaldehyde,methylglyoxal, and 3-deoxyglucosone and examined their effectson pericytes. Among those AGE fractions, glyceraldehyde- andglycolaldehyde-derived AGE strongly retarded the pericyte growth.
AGE also act on EC. Contrasting with the case of pericytes,AGE-BSA supershifted upward the growth curve of human microvascularEC (8,9). EC synthesis of DNA was also stimulated by the exposureto AGE-BSA but not to nonglycated BSA. These results indicatethat AGE are potentially angiogenic. A thrombogenic activityof AGE was also noted. AGE-BSA inhibited EC ability to synthesizedprostacyclin on one hand (8,9) and stimulated the productionof plasminogen activator inhibitor-1 on the other (10). AGE,therefore, can cause angiogenesis and thrombogenesis by dualmechanisms: first by impairing EC-pericyte interactions andsecond by the direct action on EC.
The angiogenic activity of AGE is mediated by autocrine vascularendothelial growth factor (VEGF) (11,12), as evidenced by thefacts that AGE induced the expression of VEGF gene in EC andthat AGE-induced EC proliferation and tube formation were abolishedby the anti-VEGF neutralizing antibody (9).
Receptor for AGE (RAGE) is a multi-ligand cell surface receptorinitially isolated from bovine lung by the group of Stern (13).Endogenous ligands, such as amphoterin, calgranulin, amyloid proteins, and transthyretin, have also been identified. Yamamotoet al. (manuscript submitted) conducted a surface plasmon resonanceassay using purified human RAGE proteins and glucose- and aldehyde-derivedAGE. As a result, in addition to glucose-derived AGE, glyceraldehydes-and glycolaldehyde-derived AGE fractions were found to bindto RAGE. These ligands were those that were observed to elicitpericyte and EC derangement, suggesting that certain AGE structureseffect vascular cells through interactions with RAGE (Figure 2).This idea seems to be supported by the findings that theAGE-induced decrease in pericyte proliferation, increase inEC proliferation, inhibition of EC synthesis of prostacyclin,and stimulation of EC production of plasminogen activator inhibitor-1all were abolished by RAGE antisense (6,8,10). Tsuji el al.(14) showed that AGE induction of collagen synthesis is blockedby RAGE ribozyme.
From these lessons, a hypothesis has been drawn that the AGE-RAGEsystem may participate in the development of diabetic vascularcomplications. To evaluate this hypothesis in vivo, Yamamotoet al. (15,16) created transgenic mice that overexpress humanRAGE proteins in vascular cells and cross-bred them with anothertransgenic mouse line that develops insulin-dependent diabetesearly after birth. The resultant double transgenic mice showedstatistically significant increases in kidney weight, albuminuria,glomerulosclerosis index, and serum creatinine compared withthe diabetic control (Figure 3), whereas blood glucose, hemoglobinA1c, and serum AGE levels were essentially invariant betweenthe two groups. The increases in serum creatinine and sclerosisindex were effectively prevented with (±)-2-isopropylidenehydrazono-4-oxo-thiazolidin-5-ylacetanilide,an inhibitor of AGE formation (15,16). Indices diagnostic ofdiabetic retinopathy were also most prominent in double transgenicmice (Figure 3). Thus, this transgenic approach has supportedthe concept that the AGE-RAGE system plays an active role inthe development of diabetic complications and has developeda useful animal model for testing remedies.
In light of the findings with RAGE-overexpressing transgenicmice, it is reasonable to assume that upregulation of the endogenousRAGE gene would aggravate diabetic vascular derangement. Accordingly,it seemed important to know the mechanism of RAGE gene regulation,so we screened factors that can influence the EC level of RAGEmRNA. Tanaka et al. (17) identified three inducers of RAGE genetranscription: AGE ligands themselves, TNF- and 17-estradiol.The former two shared the same cis-element for induction, whichwas located around nucleotide -671 in the human RAGE 5'-flankingsequence, whereas estradiol-responsible elements resided at-189 and at -172. Electromobility shift assays revealed thatnuclear factor-B is the trans-factor that binds to the AGE-and TNF-–responsible element and that Sp-1/estrogen receptor-complex is the factor acting on the estradiol-responsive elementsestradiol (17) (Figure 3). TNF- has been known to be responsiblefor insulin resistance (18). The action on RAGE gene unveiledanother side of this cytokine; that is, an increased TNF- levelin diabetic patients may worsen diabetic complications throughRAGE induction. The finding that AGE themselves can activatethe RAGE gene seems to be consistent with the observation thatthe AGE-rich vasculature exhibits enhanced RAGE immunoreactivity(19). Such a positive feedback loop may exacerbate diabeticvasculopathy. The RAGE gene activation by estradiol may providea biochemical basis for the well-known fact that pregnancy worsensdiabetic complications (20).
Yonekura et al. (21) analyzed poly(A)+RNA isolated from polysomesof human EC and pericytes and isolated previously undescribedsplice variants of RAGE mRNA. Three major variants were identified:the known full-length membrane-bound form, a novel N-terminallytruncated membrane-bound form, and a novel C-terminally truncatedsoluble form (Figure 4). The ratio of expression of these variantsdiffered from one cell type to another; C-truncated > full-length= N-truncated in EC; full-length > N-truncated > C-truncatedin pericytes. Each cDNA directed synthesis of the protein productof the expected size in COS cells. Both the full-length andthe N-truncated forms mainly resided on the plasma membrane,whereas the C-truncated form was liberated into the media. Furthermore,the full-length and C-truncated forms bound to an AGE-immobilizedcolumn, whereas the N-truncated form was recovered in the pass-throughfractions, thus confirming that the ligand-binding site is locatedin the amino-terminal V-region–like domain of RAGE proteins.We named the C-truncated form endogenous secretory RAGE (esRAGE).esRAGE would be cytoprotective, because it is able to captureAGE outside cells (Figure 5). In effect, this variant was foundto neutralize effectively the AGE action on EC and does existin human circulation (21). An ELISA system for esRAGE has beendeveloped, and, with it, diabetic subjects with or without complicationsare now being screened.
This work was supported by "the Research for the Future Program"of the Japan Society for the Promotion of Science and grants-in-aidfrom the Ministry of Education, Culture, Sports, Science andTechnology, Japan.
The authors thank Professor Hi Bahl Lee, Soon Chun Hyang University,the President of the 5th Hyonam Kidney Laboratory InternationalSymposium for providing an opportunity to submit this paper;Professor Toshio Doi, Tokushima University; Professor HiroshiOkamoto; Dr. Shin Takasawa and Dr. Ichiro Kato, Tohoku University;Dr. Masayoshi Takeuchi, Hokuriku University; Professor ZenjiMakita and Dr. Sho-ichi Yamagishi, Kurume University, for collaboration;and Shin-ichi Matsudaira, Reiko Kitamura, and Yoshie Yamamotofor assistance.
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Top
Abstract
Introduction
proteins, and transthyretin, have also been identified. Yamamoto et al. (manuscript submitted) conducted a surface plasmon resonance assay using purified human RAGE proteins and glucose- and aldehyde-derived AGE. As a result, in addition to glucose-derived AGE, glyceraldehydes- and glycolaldehyde-derived AGE fractions were found to bind to RAGE. These ligands were those that were observed to elicit pericyte and EC derangement, suggesting that certain AGE structures effect vascular cells through interactions with RAGE (Figure 2). This idea seems to be supported by the findings that the AGE-induced decrease in pericyte proliferation, increase in EC proliferation, inhibition of EC synthesis of prostacyclin, and stimulation of EC production of plasminogen activator inhibitor-1 all were abolished by RAGE antisense (6,8,10). Tsuji el al. (14) showed that AGE induction of collagen synthesis is blocked by RAGE ribozyme. 
and 17
B is the trans-factor that binds to the AGE- and TNF-
