Sulforaphane reduces advanced glycation end products (AGEs)-induced inflammation in endothelial cells and rat aorta.

BACKGROUND AND AIMS: Advanced glycation end products (AGEs)-receptor RAGE interaction evokes oxidative stress and inflammatory reactions, thereby being involved in endothelial cell (EC) damage in diabetes. Sulforaphane is generated from glucoraphanin, a naturally occurring isothiocyanate found in widely consumed cruciferous vegetables, by myrosinase. Sulforaphane has been reported to protect against oxidative stress-mediated cell and tissue injury. However, effects of sulforaphane on AGEs-induced vascular damage remain unclear. METHODS AND RESULTS: In this study, we investigated whether and how sulforaphane could inhibit inflammation in AGEs-exposed human umbilical vein ECs (HUVECs) and AGEs-injected rat aorta. Sulforaphane treatment for 4 or 24 h dose-dependently inhibited the AGEs-induced increase in RAGE, monocyte chemoattractant protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecular-1 (VCAM-1) gene expression in HUVECs. AGEs significantly stimulated MCP-1 production by, and THP-1 cell adhesion to, HUVECs, both of which were prevented by 1.6 µM sulforaphane. Sulforaphane significantly suppressed oxidative stress generation and NADPH oxidase activation evoked by AGEs in HUVECs. Furthermore, aortic RAGE, ICAM-1 and VCAM-1 expression in AGEs-injected rats were increased, which were suppressed by simultaneous infusion of sulforaphane. CONCLUSION: The present study demonstrated for the first time that sulforaphane could inhibit inflammation in AGEs-exposed HUVECs and AGEs-infused rat aorta partly by suppressing RAGE expression through its anti-oxidative properties. Inhibition of the AGEs-RAGE axis by sulforaphane might be a novel therapeutic target for vascular injury in diabetes.

Linagliptin blocks renal damage in type 1 diabetic rats by suppressing advanced glycation end products-receptor axis.

Advanced glycation end products (AGEs) and their receptor (RAGE) play a role in diabetic nephropathy. We have recently found that linagliptin, an inhibitor of dipeptidyl peptidase-4 (DPP-4) suppresses the AGE-induced oxidative stress generation and intercellular adhesion molecule-1 (ICAM-1) gene expression in endothelial cells. However, whether linagliptin could have beneficial effects on experimental diabetic nephropathy in a glucose-lowering independent manner remains unknown. To address the issue, this study examined the effects of linagliptin on renal damage in streptozotocin-induced diabetic rats. Serum levels of DPP-4 were significantly elevated in diabetic rats compared with control rats. Although linagliptin treatment for 2 weeks did not improve hyperglycemia in diabetic rats, linagliptin significantly reduced AGEs levels, RAGE gene expression, and 8-hydroxy-2'-deoxyguanosine, a marker of oxidative stress in the kidney of diabetic rats. Furthermore, linagliptin significantly reduced albuminuria, renal ICAM-1 mRNA levels, and lymphocyte infiltration into the glomeruli of diabetic rats. Our present study suggests that linagliptin could exert beneficial effects on diabetic nephropathy partly by blocking the AGE-RAGE-evoked oxidative stress generation in the kidney of streptozotocin-induced diabetic rats. Inhibition of DPP-4 by linagliptin might be a promising strategy for the treatment of diabetic nephropathy.

Positive association of serum levels of pigment epithelium-derived factor with high-sensitivity C-reactive protein in apparently healthy unmedicated subjects.

Pigment epithelium-derived factor (PEDF), a glycoprotein with neuronal differentiating activity, possesses anti-inflammatory properties both in cell culture and animal models. However, the relationship between serum levels of PEDF and high-sensitivity C-reactive protein (hs-CRP), one of the representative biomarkers for inflammation in humans, is largely unknown. This study investigated whether serum PEDF levels were associated with hs-CRP in 120 apparently healthy unmedicated Japanese subjects (93 males, 27 females; mean age 58.0 years). All subjects underwent a complete history and physical examination, including blood chemistries, anthropometric and metabolic variables. Multiple regression analysis found that serum hs-CRP, creatinine and triglyceride levels, and the homeostasis model assessment of insulin resistance (HOMA-IR) and body mass index were significantly and independently associated with serum PEDF levels. It is concluded that, serum levels of PEDF are associated with serum levels of hs-CRP independently of anthropometric, metabolic and renal function variables. The results also suggest that serum PEDF levels may be elevated as a counter-system against subclinical inflammation.

Hyperglycemia potentiates collagen-induced platelet activation through mitochondrial superoxide overproduction.

Alteration of platelet function contributes to microthrombus formation and may play an important role in the pathogenesis of diabetic micro- and macroangiopathies. However, the molecular mechanism for platelet dysfunction observed in patients with diabetes has not been fully elucidated. In this study, the direct effects of hyperglycemia on platelet function in vitro were investigated. Hyperglycemia increased reactive oxygen species generation in human platelets, and this effect was additive with that of collagen. Thenoyltrifluoroacetone (TTFA), an inhibitor of mitochondrial electron transport chain complex II, and carbonyl cyanide m-chlorophenylhydrazone (CCCP), an uncoupler of oxidative phosphorylation, completely prevented the effects of hyperglycemia, suggesting that reactive oxygen species arise from the mitochondrial electron transport chain. Hyperglycemia potentiated both platelet aggregation and the subsequent release of platelet-derived growth factor AB induced by a nonaggregating subthreshold concentration of collagen, which were also completely inhibited by TTFA or CCCP. Furthermore, hyperglycemia was found to inhibit protein tyrosine phosphatase (PTP) activity and increase phosphorylation of the tyrosine kinase Syk in platelets exposed to collagen. Hyperglycemia-induced PTP inhibition and Syk phosphorylation were found to be completely prevented by TTFA, CCCP, or Mn(III)tetrakis (4-benzoic acid) porphyrin, a stable cell-permeable superoxide dismutase mimetic. These results suggest that hyperglycemia-induced mitochondrial superoxide generation may play an important role in platelet dysfunction observed in patients with diabetes.

Elevations of AGE and vascular endothelial growth factor with decreased total antioxidant status in the vitreous fluid of diabetic patients with retinopathy.

BACKGROUND/AIMS: Advanced glycation end product (AGE) induces vascular endothelial growth factor (VEGF) expression in cell culture and animal models, being considered to be involved in development of diabetic retinopathy; oxidative stress also has a part in diabetic retinopathy. However, the interrelations between AGE, VEGF, and oxidative stress remain to be elucidated. In this study, vitreous AGE, VEGF, and total antioxidant levels in were determined in diabetic patients with retinopathy, and the relations among them investigated. METHODS: ELISA (enzyme linked immunosorbent assay) was used to determine the vitreous levels of AGE and VEGF in 41 patients with diabetic retinopathy and 28 non-diabetic control subjects. Total antioxidant levels in vitreous of 20 diabetic patients and 18 controls were also analysed by ELISA. RESULTS: The vitreous levels of AGE and VEGF were significantly higher in diabetic patients than in control subjects (p<0.01 for both). There was a significant correlation between the vitreous AGE and VEGF levels (p<0.001). Total antioxidant status was decreased in vitreous in patients with diabetes compared with the controls (p<0.01). Furthermore, both AGE and VEGF levels were inversely correlated with the total antioxidant status (p<0.01 and p<0.05, respectively). CONCLUSION: This study suggests that AGE and decreased total antioxidant status may contribute to the pathogenesis of diabetic retinopathy via induction of VEGF.

Decrease in the glyceraldehyde derived advanced glycation end products in the sera of patients with Vogt-Koyanagi-Harada disease.

BACKGROUND/AIMS: Advanced glycation end products (AGEs) are considered to act as mediators of both age related pathologies and diabetic complications. It was recently reported that glyceraldehyde derived AGE (AGE-2) has a strong biological effect on various diseases. The aim of this study was to investigate the serum AGE-2 levels in Vogt-Koyanagi-Harada (VKH) disease. METHODS: Sera were obtained from 31 patients with active VKH. 20 of these 31 patients were treated with systemic corticosteroids. As controls, 33 healthy volunteers were also examined. The serum AGE-2 levels were determined with a competitive enzyme linked immunosorbent assay using AGE-2 polyclonal antibody. RESULTS: The mean AGE-2 level in the sera of patients with VKH disease was 4.91 (SD 2.23) U/ml, which was significantly lower than that of the healthy control subjects (8.32 (2.94), p<0.001). The average serum AGE-2 level significantly increased to 13.49 (2.17) U/ml after the patients were treated with systemic corticosteroids (p<0.001). CONCLUSIONS: These results suggest that AGE-2 may be involved in the onset of VKH disease.

Ezetimibe reduces urinary albumin excretion in hypercholesterolaemic type 2 diabetes patients with microalbuminuria.

OBJECTIVE: This study investigated the effects of ezetimibe, an inhibitor of intestinal cholesterol absorption, on early phase diabetic nephropathy. METHODS: A total of 32 hypercholesterolaemic type 2 diabetes patients with microalbuminuria, defined as a urinary albumin excretion (UAE) 30 but < 300 mg/g creatinine, were enrolled. Various clinical and laboratory parameters were determined at baseline and after 6 months of treatment with 10 mg/day ezetimibe. RESULTS: Ezetimibe treatment significantly decreased glycated haemoglobin (HbA(1c)), low-density lipoprotein-cholesterol (LDL-C), triglycerides and UAE, and significantly increased high-density lipoprotein-cholesterol and albumin. It also decreased the serum level of monocyte chemoattractant protein-1 (MCP-1), but this difference was not statistically significant. Univariate analyses showed a correlation between UAE and body mass index, systolic and diastolic blood pressures, HbA(1c), LDL-C, estimated glomerular filtration rate (inverse), creatinine and MCP-1. Since these parameters may be closely correlated with each other, multiple stepwise regression analysis was performed and demonstrated that HbA(1c) and MCP-1 were independent determinants of UAE. CONCLUSIONS: Ezetimibe may be a promising therapeutic strategy for improving albumin excretion, partly through its anti-inflammatory properties, and for reducing LDL-C in hypercholesterolaemic type 2 diabetes patients with microalbuminuria.

PEDF and septic shock.

Septic shock is one of the leading causes of morbidity and mortality. Endotoxin plays an important role in the pathogenesis of septic shock. Lack of clinical success with anti-endotoxin or anti-cytokine therapies has shifted interest to extracorporeal therapies to reduce circulating levels of various mediators for septic shock patients. Polymyxin B -immobilized polystyrene fiber (PMX-F) is a medical device that aims to remove circulating endotoxin by adsorption. Since 1994, PMX-F column has been available in Japan, and many investigators have reported that PMX-F treatment is safe and effective in patients with septic shock. Pigment epithelium-derived factor (PEDF) is a glycoprotein that belongs to the superfamily of serine protease inhibitors. PEDF induces macrophages apoptosis and necrosis through the activation of peroxisome proliferator-activated receptor-gamma by which PEDF could modulate inflammatory reactions in septic shock. Further, given the fact that PEDF possesses anti-oxidative and anti-inflammatory properties in vivo, serum PEDF level may be a biomarker of septic shock. However, little is known about the relationship between serum level of PEDF and inflammatory biomarkers such as endotoxin and high mobility group box 1 (HMGB1) in septic shock and the effects of PMX-F treatment on these markers. This review aims to provide current knowledge about the pathogenesis of septic shock and the clinical utility of PMX-F treatment. We also discuss here the pathophysiological role of PEDF in this devastating disorder.

Anti-atherothrombogenic properties of PEDF.

Cardiovascular disease (CVD) such as myocardial infarction and stroke is a leading cause of death in developed countries. Atherothrombosis, characterized by atherosclerotic lesion disruption with superimposed thrombus formation, is thought to be the major cause of CVD. Although remarkable therapeutic advances in the treatment of atherothrombosis have been made with anti-platelet and anti-thrombotic therapy, these therapeutic options may be limited by considerable side effects. In addition, they may not protect the endothelium and thus could not stabilize culprit lesions. Therefore, to develop a novel therapeutic strategy is needed for the prevention of atherothrombosis in high-risk patients. Recently, we, along with others, have shown that pigment epithelium-derived factor (PEDF), a glycoprotein with potent neuronal differentiating activity, exerts anti-oxidative and anti-inflammatory anti-thrombogenic and vasculoprotective properties in various cell types. In addition, PEDF not only suppresses neointimal hyperplasia after balloon angioplasty, but also blocks occlusive thrombus formation in a rat arterial thrombosis model. These observations suggest that substitution of PEDF may be a novel therapeutic strategy for atherothrombosis. This article summarizes the pathophysiological role of PEDF in atherothrombosis and its potential therapeutic implication in CVD. We also discuss here the kinetics and regulation of PEDF in high-risk patients for atherothrombosis.

Pathophysiological role of pigment epithelium-derived factor (PEDF) in hepatic disorders.

Pigment epithelium-derived factor (PEDF) is a glycoprotein that belongs to the superfamily of serine protease inhibitors with a potent neuronal differentiating activity. Recently, PEDF is found to be a highly effective inhibitor of pathological angiogenesis in both cell culture and animal models. Further, it has also been shown to have neuroprotective, anti-oxidative and anti-inflammatory properties, any of which could potentially be exploited as a therapeutic option for the treatment of cardiometabolic disorders, neurodegenerative disease and cancers. However, as far as we know, there are few comprehensive reviews to deal with the involvement of PEDF in hepatic disease. This article summarizes the pathophysiological role of PEDF for various liver diseases such as hepatic insulin resistance, alcoholic and nonalcoholic liver disease, and hepatocellular carcinoma, and its potential therapeutic implication in these devastating disorders.

Development of enzyme-linked immunosorbent assay system for PEDF and its clinical utility.

Pigment epithelium-derived factor (PEDF) is reported to play a protective role against diabetic vascular complications through its anti-oxidative properties. However, since a commercially available kit is not suitable for measurement of serum PEDF in humans, kinetics and regulation of serum PEDF are not known in these devastating disorders. Therefore, we developed a simple, specific and reliable method for measurement of serum PEDF in humans using a competitive enzyme-linked immunosorbent assay (ELISA) system. Assay linearity was shown intact with 50~300-fold dilution of urea-pretreated serum by phosphate-buffered saline. The recovery ratio of added recombinant human PEDF in serum was 94.2 +/- 1.7 %. Inter- and intra-assay coefficient of variations of the ELISA were 4.7 and 7.3 %, respectively. When we measured serum PEDF levels in a general population, PEDF levels were elevated in proportion to the accumulation of the number of the components of the metabolic syndrome. Further, the percent changes in serum levels of PEDF during 1-year observational periods were positively correlated with those of body mass index (BMI) in patients with type 2 diabetes. In addition, PEDF mRNA levels in cultured adipocytes were increased in parallel to the BMI values of subjects from which adipocytes were derived, especially in omental adipocytes. These observations suggest that PEDF is generated from adipose tissues and could be increased as a counter-system against vascular cell damage in humans. PEDF may be one of the useful biomarkers for vascular injury in high-risk patients.

The role of PEDF in tumor growth and metastasis.

Pigment epithelium-derived factor (PEDF), one of the non-inhibitory serpines, is widely expressed throughout the body. Although PEDF was initially identified as a neuronal differentiation factor, more attention has been paid to its anti-angiogenic activity. Additionally, recent researches have demonstrated that PEDF has an anti-tumor effect against several human neoplasms. This review focuses on the pathological role of PEDF in tumors, especially tumor growth and metastasis. PEDF is an endogenous anti-tumor factor and its clinical application seems quite promising, although there is much to be further investigated.

Structure-function relationships of PEDF.

Pigment epithelial-derived factor (PEDF) is a 50-kDa secreted glycoprotein that belongs to the noninhibitory serpin. It has an alpha/beta core serine-protease inhibitor domain, 3 major beta-sheets, and 10 alpha-helices. Although PEDF does not inhibit either serine or cysteine proteinases, PEDF exerts diverse physiological activities including anti-angiogenesis, anti-vasopermeability, anti-tumor, and neurotrophic activities. Recent studies have shown that a variety of peptides derived from PEDF possess activities similar to those of the parent molecule through interactions with the extracellular matrix, binding to PEDF receptors, nuclear localization and phosphorylation. Thus, peptides derived from PEDF have therapeutic potential for various diseases and therefore, it is important to clarify the structure-function relationship of PEDF. In this review, we summarize structural features of PEDF that could affect various target organs such as blood vessels, tumors, and the central nervous system. In addition, since PEDF is recently identified as a regulator for glucose and lipid metabolism, we also discuss PEDF structures specially related to insulin-sensitizing and triglyceridereducing properties.

Anti-vasopermeability effects of PEDF in retinal-renal disorders.

The vascular system has an important function of supplying tissues with oxygen and nutrients and clearing waste products. Therefore, the microvasculature must be sufficiently permeable to allow the free, bidirectional passage of small molecules and gases and, to a lesser extent, of plasma proteins. It is well recognized that vascular endothelial growth factor (VEGF) can increase vascular permeability, thus playing important roles in variety of disorders, including diabetic retinopathy, nephrotic syndrome, brain edema, acute respiratory distress syndrome, and sepsis-associated hypotension. However, how vascular permeability is controlled by anti-permeable factors is not fully understood. We have recently found that pigment epithelium derived factor (PEDF), a 50 kD glycoprotein, inhibits retinal, renal and brain hyperpermeability by counteracting the biological actions of VEGF. In this review, we discuss about the pathophysiological role of PEDF in vascular permeability, especially focusing on retinal-renal disorders.

Possible molecular mechanisms by which angiotensin II type 1 receptor blockers (ARBs) prevent the development of atrial fibrillation in insulin resistant patients.

Atrial fibrillation (AF) is the most common disorder of cardiac rhythm and is responsible for substantial morbidity and mortality in general population. A recent community-based observational study revealed that diabetes and/or hypertension were associated with the development of AF. However, there is no definite evidence to show that patients with type 1 diabetes have an increased risk for the development of AF. These findings suggest that hyperglycemia per se may not explain the positive association between diabetes and AF. Growing body of evidence supports the presence of insulin resistance as the fundamental pathophysiological disturbance responsible for the metabolic syndrome, a constellation of metabolic disorders such as hypertension, dyslipidemia, and obesity that raise the risk for diabetes mellitus and cardiovascular diseases. Further, several clinical trials have shown that the renin-angiotensin system (RAS) plays an important role in the pathogenesis of insulin resistance. These observations suggest that insulin resistance could account for the increased risk for AF in the patients with diabetes and/or hypertension and that the interruption of the RAS may be a promising therapeutic strategy for preventing the development of AF. In the first part of this paper, we review clinical studies to support the concept that angiotensin II type 1 receptor blockers (ARBs) could prevent the development of AF in insulin resistant patients and discuss the possible underlying mechanisms. In the second part, we discuss the potential utility of telmisartan, a unique ARB with peroxisome proliferator-activated receptor-gamma (PPAR-gamma)-modulating activity, for blocking the development of AF in patients with insulin resistance.

Role of postprandial hyperglycaemia in cardiovascular disease in diabetes.

Diabetes is associated with a marked increase in the risk of atherosclerotic vascular disorders, including coronary, cerebrovascular and peripheral artery disease. Cardiovascular disease (CVD) could account for disabilities and high mortality rates in patients with diabetes. Conventional risk factors, including hyperlipidaemia, hypertension, smoking, obesity, lack of exercise and a positive family history, contribute similarly to macrovascular complications in type 2 diabetic patients and non-diabetic subjects. The levels of these factors in diabetic patients are certainly increased, but not enough to explain the exaggerated risk for macrovascular complications in diabetic population. Therefore, specific diabetes-related risk factors should be involved in the excess risk in diabetic patients. In this paper, we review the molecular mechanisms for accelerated atherosclerosis in diabetes, especially focusing on postprandial hyperglycaemia. We also discuss here the potential therapeutic strategy that specifically targets CVD in patients with diabetes.

Ossifying gastric carcinoid tumor containing bone morphogenetic protein, osteopontin and osteonectin.

We report a case of gastric carcinoid tumor with ossification. A 47-yr-old man complaining of abdominal discomfort underwent gastrointestinal endoscopic examination, which revealed a submucosal tumor in his stomach. The tumor was extirpated by endoscopic enucleation. Histologically, the tumor was widely occupied by mature bone tissues, where scattered carcinoid tumor cell nests surrounded bone tissues or located in stromal areas. Immunohistochemically, the tumor cells were strongly positive for cytokeratin, chromogranin A, synaptophysin, neurofilaments and neuron-specific enolase, underscoring the diagnosis of carcinoid tumor. They also stained positive with markers of bone formation and differentiation, such as bone morphogenetic protein, osteopontin and osteonectin. There are only four cases in the world literature, including a current case of ossifying gastric carcinoid tumor, in which the excessive production of peptides promoting ossification was considered to be implicated in the unusual appearance of the bone.

Neuropathy in diabetic mice overexpressing human aldose reductase and effects of aldose reductase inhibitor.

The present study was designed to examine the effect of aldose reductase (AR) overexpression on the development of diabetic neuropathy by using mice transgenic for human AR. At 8 weeks of age, transgenic mice (Tg) and non-transgenic littermates (Lm) were made diabetic with streptozotocin. After 8 weeks of untreated diabetes, plasma glucose levels and the reduction in body weight were similar between the groups of diabetic animals. Despite the comparable levels of hyperglycaemia, levels of sorbitol and fructose were significantly greater in the peripheral nerve of diabetic Tg than in diabetic Lm (both P < 0.01). Ouabain sensitive Na(+),K(+)-ATPase activity was similarly decreased in both diabetic Tg and Lm. Protein kinase C activity in the sciatic nerve membrane fraction was unaffected by diabetes in Lm, but was reduced by nearly 40% in the diabetic Tg. Although both groups of diabetic animals exhibited a significant decrease in tibial nerve motor nerve conduction velocity (MNCV), this decrease was significantly more severe (P < 0.01) in diabetic Tg than in diabetic Lm. Consistent with these findings, nerve fibre atrophy was significantly more severe in diabetic Tg than in diabetic Lm (P < 0.01). These findings implicate increased polyol pathway activity in the pathogenesis of diabetic neuropathy. In support of this hypothesis, treating diabetic Tg with an aldose reductase inhibitor (WAY121-509, 4 mg/kg/day) for 8 weeks significantly prevented the accumulation of sorbitol, the decrease in MNCV and the increased myelinated fibre atrophy in diabetic Tg.

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.