Reduced monocyte adhesion to aortae of diabetic plasminogen activator inhibitor-1 knockout mice.

OBJECTIVE AND DESIGN: To determine the requirement of plasminogen activator inhibitor-1-knockout (PAI-1) for monocyte adhesion in animals and cells under diabetic conditions. METHODS AND SUBJECTS: Monocyte adhesion assay, enzyme-linked immunosorbent assay, and Western blotting were used in analyzing samples from PAI-1-knockout (PAI-1-KO) mice or cultured human umbilical vein endothelial cells (HUVEC). TREATMENTS: Diabetes in PAI-1-KO and wild-type mice was induced by intraperitoneal injection of streptozotocin (STZ). HUVEC was transfected with short interference RNA (siRNA) against PAI-1, tumor necrosis factor-α (TNFα), or toll-like receptor (TLR4), and then was treated with glycated low-density lipoproteins (glyLDL). RESULTS: The adhesion of monocytes to aortic intima was reduced in PAI-1-KO mice, which was associated with decreased levels of TNFα and monocyte chemotactic protein-1 (MCP-1) in plasma and cardiovascular tissue, and increased abundances of urokinase plasminogen activator (uPA) and uPA receptor (uPAR) in cardiovascular tissue compared to wild-type mice. Significant reductions in monocyte adhesion, inflammatory, and fibrinolytic regulators were detected in cardiovascular tissue or plasma in diabetic PAI-1-KO mice compared to wild-type diabetic mice. Transfection of PAI-1, TNFα or TLR4 siRNA to HUVEC inhibited glyLDL-induced monocyte adhesion to EC. PAI-1 siRNA inhibited the abundances of TLR4 and TNFα in EC. CONCLUSION: The findings suggest that PAI-1 is required for diabetes-induced monocyte adhesion via interactions with uPA/uPAR, and it also regulates TLR4 and TNFα expression in vascular EC. Inhibition of PAI-1 potentially reduces vascular inflammation under diabetic condition.

Effects of glycated low-density lipoprotein on cell viability, proliferation, and growth factors of mouse embryo fibroblasts.

The predominant cause of death in diabetic patients is atherosclerotic coronary artery disease (CAD). Major gross cellular changes in the vascular wall of persons with CAD include endothelial injury and foam cell formation, as well as smooth muscle cell and fibroblast proliferation. This study examined the effects of glycated low density lipoprotein (glyLDL), a biochemical marker of diabetes, on cell viability, proliferation, and the expression of multiple growth factors in mouse embryo fibroblasts (MEF). The results demonstrated that exposure to ≥150 µg/mL of glyLDL for 24 h or 100 µg/mL of glyLDL for ≥48 h either significantly reduced cell viability or increased DNA fragmentation in MEF. GlyLDL treatment (25-100 µg/mL for up to 12 h) significantly increased the abundance of proliferating cell nuclear antigen (PCNA) and achieved a peak after 4 h exposure to glyLDL. Abundances of fibroblast growth factor-basic (FGF), transforming growth factor-ß (TGF), and platelet-derived growth factor-A (PDGF) in MEF reached maximal levels after 2 h exposure to 50 µg/mL of glyLDL. The maximal increase of vascular endothelial growth factor (VEGF) was detected in MEF after 4 h of exposure to 50 µg/mL of glyLDL. Inhibitors for FGF (AZD4547), VEGF, or PDGF receptors (Axitinib), but not that for TGF receptor (LY364947), significantly decreased the abundance of (PCNA) in endothelial cells. The findings suggest that early exposure to a low dosage of glyLDL transiently increases the proliferation of MEF through the upregulation of FGF, VEGF, and (or) PDGF, and prolonged exposure to high concentrations of glyLDL reduced cell viability, which possibly accelerates atherogenesis under diabetic condition.

Impact of anthocyanin component and metabolite of Saskatoon berry on gut microbiome and relationship with fecal short chain fatty acids in diet-induced insulin resistant mice.

Previous studies demonstrated that oral administration of Saskatoon berry powder (SBp) reduced fasting plasma glucose (FPG), insulin resistance, lipids, and inflammatory markers in diet-induced insulin resistant rodents. Mechanism for the beneficial effects of SB remains unclear. The present study examined the effects of high fat-high sucrose (HFHS) diet supplemented with or without 5% SBp, cyanidin-3-glucoside (C3G, an anthocyanin rich in SBp) at a dosage of C3G in 5% SBp, or equimolar concentration of protocatechuic acid (PCA, a relatively stable metabolite of C3G) for 11 weeks on FPG, cholesterol, triglycerides, insulin, homeostatic model assessment of insulin resistance (HOMA-IR), circulatory inflammatory markers, fecal microbiota, and short chain fatty acids in mice. HFHS diet significantly enhanced FPG, insulin, HOMA-IR, lipids and inflammatory markers, but reduced the abundance of fecal Bacteroidetes, Muribaculaceae and propionate compared to low fat diet. Supplementation of SBp, C3G or PCA significantly attenuated HFHS diet induced metabolic and inflammatory markers, and increased the abundances of fecal Muribaculaceae and propionate compared to HFHS diet alone. The abundances of fecal Muribaculaceae negatively correlated with FPG, lipids, HOMA-IR and inflammatory markers in the mice. The abundances of fecal propionate positively correlated with fecal Muribaculaceae and negatively correlated with the metabolic and inflammatory markers. The findings suggest that C3G in SBp and PCA contribute to the metabolic and anti-inflammatory effect of SBp in mice. The increases in fecal Muribaculaceae and propionate may play important regulatory roles in the anti-diabetic and anti-inflammatory benefits of SBp, C3G, and PCA in mice.

Influence of Brown or Germinated Brown Rice Supplementation on Fecal Short-Chain Fatty Acids and Microbiome in Diet-Induced Insulin-Resistant Mice.

Intake of whole grain foods is associated with improving metabolic profile compared to refined grain products, but the underlying mechanism remains unclear. The present study examined the effects of brown rice (BRR) or germinated brown rice (GBR) supplementation on fecal short-chain fatty acids (SCFAs), and relationship with gut microbiota, metabolism and inflammation in high fat (HF)-diet-fed mice. The results demonstrated that an HF diet supplemented with BRR or GBR comparably increased the abundance of fecal isobutyric acid compared to that in mice receiving HF+white rice (WHR) diet (p < 0.01). The abundance of valeric acid in HF+GBR-diet-fed mice was higher than those receiving HF+WHR diet (p < 0.05). The abundances of fecal isobutyric acid negatively correlated with fasting plasma glucose, insulin, cholesterol, triglycerides, tumor necrosis factor-α, plasminogen activator inhibit-1, monocyte chemotactic protein-1 and homeostatic model assessment of insulin resistance (p < 0.01). The abundance of valeric acids negatively correlated with insulin resistance (p < 0.05). The abundances of isobutyric acid positively correlated with Lactobacillus, but negatively correlated with Dubosiella genus bacteria (p < 0.05). The findings demonstrated that the increases in SCFAs in the feces of BRR and GBR-treated mice were associated with improvements in gut microbiome, metabolic and inflammatory profile, which may contribute to the antidiabetic and anti-inflammatory effects of the whole grains in HF-diet-fed mice.

Impact of cyanidin-3-glucoside on glycated LDL-induced NADPH oxidase activation, mitochondrial dysfunction and cell viability in cultured vascular endothelial cells.

Elevated levels of glycated low density lipoprotein (glyLDL) are frequently detected in diabetic patients. Previous studies demonstrated that glyLDL increased the production of reactive oxygen species (ROS), activated NADPH oxidase (NOX) and suppressed mitochondrial electron transport chain (mETC) enzyme activities in vascular endothelial cells (EC). The present study examined the effects of cyanidin-3-glucoside (C3G), a type of anthocyanin abundant in dark-skinned berries, on glyLDL-induced ROS production, NOX activation and mETC enzyme activity in porcine aortic EC (PAEC). Co-treatment of C3G prevented glyLDL-induced upregulation of NOX4 and intracellular superoxide production in EC. C3G normalized glyLDL-induced inhibition on the enzyme activities of mETC Complex I and III, as well as the abundances of NADH dehydrogenase 1 in Complex I and cytochrome b in Complex III in EC. Blocking antibody for the receptor of advanced glycation end products (RAGE) prevented glyLDL-induced changes in NOX and mETC enzymes. Combination of C3G and RAGE antibody did not significantly enhance glyLDL-induced inhibition of NOX or mETC enzymes. C3G reduced glyLDL-induced RAGE expression with the presence of RAGE antibody. C3G prevented prolonged incubation with the glyLDL-induced decrease in cell viability and the imbalance between key regulators for cell viability (cleaved caspase 3 and B cell Lyphoma-2) in EC. The findings suggest that RAGE plays an important role in glyLDL-induced oxidative stress in vascular EC. C3G may prevent glyLDL-induced NOX activation, the impairment of mETC enzymes and cell viability in cultured vascular EC.

Impact of Germinated Brown Rice and Brown Rice on Metabolism, Inflammation, and Gut Microbiome in High Fat Diet-Induced Insulin Resistant Mice.

The constituents of germinated brown rice (GBR), brown rice (BRR), and white rice (WHR) and their impact on metabolism, inflammation, and gut microbiota in high fat (HF) diet-fed mice were examined. The contents of total fiber and γ-aminobutyric acid in BRR and GBR were higher than that in WHR (p < 0.05). Male C57 BL/6J mice received HF diet+26 g% of WHR, BRR, or GBR for 12 weeks. BRR and GBR comparably reduced HF diet-induced increases in fasting plasma glucose, lipids, insulin resistance, and inflammatory markers compared to WHR (p < 0.01). The abundance of fecal Bacteroidetes in mice fed HF+GBR or HF+BRR was higher than in HF+WHR-fed mice (p < 0.05). The abundance of fecal Lactobacillus gasseri in GBR-fed mice was greater than that in WHR- or BRR-fed mice (p < 0.05). The results indicated that GBR or BRR attenuated hyperglycemia, insulin resistance, and inflammation in mice. HF+GBR, but not HF+BRR, increased a probiotic bacteria in the gut.

Dose-Responses Relationship in Glucose Lowering and Gut Dysbiosis to Saskatoon Berry Powder Supplementation in High Fat-High Sucrose Diet-Induced Insulin Resistant Mice.

Administration of freeze-dried powder of Saskatoon berry (SB), a popular fruit enriched with antioxidants, reduced glucose level, inflammatory markers and gut microbiota disorder in high fat-high sucrose (HFHS) diet-induced insulin resistant mice. The present study examined the dose-response relationship in metabolic, inflammatory and gut microbiotic variables to SB power (SBp) supplementation in HFHS diet-fed mice. Male C57 BL/6J mice were fed with HFHS diet supplemented with 0, 1%, 2.5% or 5% SBp for 11 weeks. HFHS diet significantly increased the levels of fast plasma glucose (FPG), cholesterol, triglycerides, insulin, homeostatic model assessment of insulin resistance (HOMA-IR), tumor necrosis factor-α, monocyte chemotactic protein-1 and plasminogen activator inhibitor-1, but decreased fecal Bacteroidetes phylum bacteria and Muribaculaceae family bacteria compared to low fat diet. SBp dose-dependently reduced metabolic and inflammatory variables and gut dysbiosis in mice compared with mice receiving HFHS diet alone. Significant attenuation of HFHS diet-induced biochemical disorders were detected in mice receiving ≥1% SBp. The abundances of Muribaculaceae family bacteria negatively correlated with body weights, FPG, lipids, insulin, HOMA-IR and inflammatory markers in the mice. The results suggest that SBp supplementation dose-dependently attenuated HFHS diet-induced metabolic and inflammatory disorders, which was associated with the amelioration of gut dysbiosis in the mice.

Saskatoon berry powder reduces hepatic steatosis and insulin resistance in high fat-high sucrose diet-induced obese mice.

Non-alcoholic fatty liver disease is a common metabolic disorder associated with insulin resistance and lacks a specific treatment. Our previous studies demonstrated that freeze-dried Saskatoon berry powder (SBp) reduced high fat-high sucrose (HFHS) diet-induced hyperglycemia and insulin resistance in mice. The present study examined the effect of SBp and one of its active components, cyanidin-3-glucoside (C3G), on hepatic steatosis in mice fed with HFHS diet for 10 weeks. HFHS diet significantly increased fasting plasma glucose, cholesterol, triglycerides, insulin resistance, inflammatory markers (tumor necrosis factor-α, monocyte chemotactic protein-1, plasminogen activator inbitor-1), alanine aminotransferase activity, and monocyte adhesion compared to control diet. In the liver, HFHS diet increased steatosis, lipid accumulation, collagen deposition, and the abundance of patatin-like phospholipase domain-containing 3, CCAAT-enhancer-binding protein homologous protein, toll-like receptor-4, and macrophage marker. Supplementation with SBp (5%) or C3G in an amount corresponding to that in 5% SBp to HFHS diet had similar effects to reduced fasting plasma glucose, liver steatosis, enzyme activity, lipid, collagen and macrophage deposition, hyperglycemia, hyperlipidemia, insulin resistance, monocyte adhesion, markers related to liver steatosis, inflammation, oxidative or endoplasmic reticulum stress in the peripheral circulation and/or liver compared to mice fed with HFHS diet alone. No significant difference in the studied variables was detected between mice treated with HFHS+SBp and C3G diet. The results suggest that SBp or C3G administration attenuates HFHS diet-induced liver steatosis in addition to insulin resistance and chronic inflammation in mice. C3G may contribute to the beneficial effects of SBp.

Impact of Cyanidin-3-Glucoside on Gut Microbiota and Relationship with Metabolism and Inflammation in High Fat-High Sucrose Diet-Induced Insulin Resistant Mice.

The present study assessed the effects of freeze-dried cyanidin-3-glucoside (C3G), an anthocyanin enriched in dark-red berries, compared to Saskatoon berry powder (SBp) on metabolism, inflammatory markers and gut microbiota in high fat-high sucrose (HFHS) diet-induced insulin-resistant mice. Male C57 BL/6J mice received control, HFHS, HFHS + SBp (8.0 g/kg/day) or HFHS + C3G (7.2 mg/kg/day, equivalent C3G in SBp) diet for 11 weeks. The HFHS diet significantly increased plasma levels of glucose, cholesterol, triglycerides, insulin resistance and inflammatory markers. The HFHS + SBp diet increased the Bacteroidetes/Firmicutes (B/F) ratio and relative abundance of Muriculaceae family bacteria in mouse feces detected using 16S rRNA gene sequencing. The HFHS + SBp or HFHS + C3G diet attenuated glucose, lipids, insulin resistance and inflammatory markers, and increased the B/F ratio and Muriculaceae relative abundance compared to the HFHS diet alone. The relative abundances of Muriculaceae negatively correlated with body weight, glucose, lipids, insulin resistance and inflammatory mediators. Functional predication analysis suggested that the HFHS diet upregulated gut bacteria genes involved in inflammation, and downregulated bacteria involved in metabolism. C3G and SBp partially neutralized HFHS diet-induced alterations of gut bacteria. The results suggest that C3G is a potential prebiotic, mitigating HFHS diet-induced disorders in metabolism, inflammation and gut dysbiosis, and that C3G contributes to the metabolic beneficial effects of SBp.

North American Wild Rice-Attenuated Hyperglycemia in High-Fat-Induced Obese Mice: Involvement of AMP-Activated Protein Kinase.

Previous studies indicated that North American wild rice (WIR) reduced atherosclerosis and vascular inflammation in low-density lipoprotein receptor knockout mice. The effects of WIR on hyperglycemia in diabetic animal models have not been documented. The present study aims to determine the impact of WIR on glucose metabolism in high-fat (HF)-induced diabetic mice and a key modulator. Male C57 BL/J6 mice were treated with a control diet and a HF diet supplemented with 26% (weight/weight, a substitute for carbohydrates in the diet) of WIR or white rice (WHR) (n = 8/group) for 11 weeks. HF + WHR diet significantly increased fasting plasma glucose, cholesterol, triglycerides, insulin, insulin resistance, monocyte adhesion, and the levels of relevant inflammatory mediators (tumor necrotic factor-α, plasminogen activator inhibitor-1, and monocyte chemotactic protein-1) in mice compared to the control diet (p < 0.01). HF + WIR significantly reduced HF diet-induced metabolic and inflammatory changes compared to the HF + WHR diet (p < 0.01). Metabolomics analysis indicated that an array of metabolites related to glucose metabolism was significantly more abundant in WIR than in WHR, including adenosine 5'-monophosphate (AMP), a potent agonist for AMP-activated protein kinase or AMPK. WIR normalized HF diet-induced reduction in the abundance of phospho-AMPKα in skeletal muscle, liver, and adipose tissue from the mice. The findings for the first time demonstrated that WIR decreased HF diet-induced hyperglycemia in mice compared to WHR. The metabolic benefits of WIR may result, at least in part, from the activation of AMPKα in insulin-sensitive tissue in the mice.

Involvement of NADPH oxidase in oxidized LDL-induced upregulation of heat shock factor-1 and plasminogen activator inhibitor-1 in vascular endothelial cells.

Plasminogen activator inhibitor-1 (PAI-1) is implicated in thrombogenesis, inflammation, and extracellular matrix remodeling. Previous studies indicated that oxidized low-density lipoprotein (LDL) stimulated the generation of PAI-1 in vascular endothelial cells (EC). The present study demonstrated that LDL oxidized by copper, iron, or 3-morpholinosydnonimine increased the expression of NADPH oxidase (NOX) 2, PAI-1, and heat shock factor-1 (HSF1) in human umbilical vein EC or coronary artery EC compared with LDL or vehicle. Diphenyleneiodonium, a NOX inhibitor, prevented the increases of the expression of HSF1 and PAI-1 in EC induced by oxidized LDLs. Small-interference RNA (siRNA) for p22(phox), an essential subunit of NOX, prevented oxidized LDL-induced expression of NOX2, HSF1, and PAI-1 in EC. HSF1 siRNA inhibited oxidized LDL-induced expression of PAI-1 and HSF1, but not NOX2, in EC. The binding of HSF1 to PAI-1 promoter and the activity of PAI-1 promoter in EC were enhanced by oxidized LDL. Butylated hydroxytulene, a potent antioxidant, inhibited oxidized LDL-induced release of hydrogen peroxide (H(2)O(2)) and the expression of NOX2, HSF1, and PAI-1 in EC. Treatment with H(2)O(2) increased the abundance of NOX2, HSF1, and PAI-1 in EC. The results of the present study indicate that oxidized LDL-induced expression of NOX may lead to the elevated release of reactive oxygen species, the activation of HSF1, and the enhancement of the transcription of PAI-1 gene in cultured vascular EC.

Impact of Saskatoon berry powder on insulin resistance and relationship with intestinal microbiota in high fat-high sucrose diet-induced obese mice.

The present study examined the impact of Saskatoon berry powder (SBp) on insulin resistance, inflammation and intestinal microbiota in diet-induced obese mice. Male C57 BL/6 J mice were fed control diet, high fat-high sucrose (HFHS) diet or HFHS+5% SBp (HFHS+B) diet for 15 weeks. The composition of fecal bacterial community was characterized using the Illumina sequencing of V4 region of 16S rRNA gene. HFHS diet increased body weight, fasting plasma glucose, cholesterol, triglycerides, insulin, homeostatic model assessment-insulin resistance, monocyte adhesion, tumor necrosis factor-α, plasminogen activator inhibitor-1, monocyte chemotactic protein-1, intracellular cell adhesion molecule-1, urokinase plasminogen activator and its receptor in plasma or aortae compared to the control diet. HFHS+B diet postponed the increase in body weight, suppressed HFHS diet-induced disorders in the metabolic and inflammatory variables. The ratio of Firmicutes/Bacteroidetes in the HFHS group was higher than that in the control group (P<.01), and that in the HFHS+B group was lower than that in the HFHS group (P<.05). The abundances of S24-7 family negatively correlated with body weight and tested metabolic or inflammatory variables. The results suggest that SBp attenuated HFHS diet-induced metabolic disorders and vascular inflammation in gut microbiota in mice.

Germinated Brown Rice Attenuates Atherosclerosis and Vascular Inflammation in Low-Density Lipoprotein Receptor-Knockout Mice.

The present study investigates the impact of germinated brown rice (GBR) on atherosclerosis and the underlying mechanism in low-density lipoprotein receptor-knockout (LDLr-KO) mice. The intensity of atherosclerosis in aortas of LDLr-KO mice receiving diet supplemented with 60% GBR (weight/weight) was significantly less than that in mice fed with 60% white rice (WR) or control diet ( p < 0.05); all diets contained 0.06% cholesterol. WR or GBR diet did not significantly alter plasma total or LDL-cholesterol, fecal sterols, or glucose, or the activities of antioxidant enzymes, compared to the control diet. The adhesion of monocytes to aortas from LDLr-KO mice fed with WR diet was significantly more than that from mice receiving the control diet ( p < 0.01). GBR diet decreased monocyte adhesion to aortas compared to WR diet ( p < 0.01). GBR diet also reduced the levels of plasminogen activator inhibitor-1 (PAI-1), monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor-α (TNF-α) in plasma, and the abundances of MCP-1, PAI-1, TNF-α, intracellular cell adhesion molecule-1, toll-like receptor-4, PAI-1, LDLr-like protein, and urokinase plasminogen activator and its receptor in aortas or hearts from LDLr-KO mice in comparison to the WR diet ( p < 0.05, 0.01, respectively). The findings suggest that GBR administration attenuated atherosclerosis and vascular inflammation in LDLr-KO mice compared to WR. The anti-atherosclerotic effect of GBR in LDLr-KO mice at least in part results from its anti-inflammatory activity.

Inhibitory Effects of North American Wild Rice on Monocyte Adhesion and Inflammatory Modulators in Low-Density Lipoprotein Receptor-Knockout Mice.

The present study examined the effects of wild rice on monocyte adhesion, inflammatory and fibrinolytic mediators in low-density lipoprotein receptor-knockout (LDLr-KO) mice. Male LDLr-KO mice received a cholesterol (0.06%, w/w)-supplemented diet with or without white or wild rice (60%, w/w) for 20 weeks. White rice significantly increased monocyte adhesion and abundances of monocyte chemoattractant protein-1, tissue necrosis factor-α, intracellular cell adhesion molecule-1, plasminogen activator inhibitor-1, urokinase plasminogen activator (uPA), and uPA receptor in aortae and hearts of LDLr-KO mice compared to the control diet. Wild rice inhibited monocyte adhesion to the aorta, atherosclerosis, and abundances of the inflammatory and fibrinolytic regulators in the cardiovascular tissue of LDLr-KO mice compared to white rice. White or wild rice did not significantly alter the levels of cholesterol, triglycerides, or antioxidant enzymes in plasma. The anti-atherosclerotic effect of wild rice may result from its inhibition on monocyte adhesion and inflammatory modulators in LDLr-KO mice.

Effects of oxidized and glycated low-density lipoproteins on transcription and secretion of plasminogen activator inhibitor-1 in vascular endothelial cells.

Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor of fibrinolysis. Elevated levels of PAI-1 were frequently detected in patients with coronary artery disease (CAD) or diabetes. Low-density lipoprotein (LDL) is a classical risk factor of CAD. Oxidation and glycation increase the atherogenecity of LDL. Previous studies demonstrated that oxidized LDL (oxLDL) or glycated LDL (gly-LDL) increased the release of PAI-1 from endothelial cells (ECs). The present study examined the effects of oxLDL and gly-LDL on the transcription, expression, secretion, and subcellular distribution of PAI-1 in cultured human ECs. Treatment with LDL significantly increased the promoter activity, mRNA level, and the release of PAI-1 from ECs by two- to threefold compared to controls. Oxidation or glycation significantly enhanced the effects of LDL on PAI-1 production in ECs compared to LDL (four- to fivefold vs. controls). No significant differences were detected between the effects of oxLDL and gly-LDL. Abundant PAI-1 antigens were detected in the perinuclear region of ECs and overlapped with giantin, a marker of Golgi apparatus. Treatment with brefeldin A disturbed the stack structure of Golgi apparatus and almost completely inhibited the release of PAI-1 from ECs induced by the lipoproteins and at basal conditions. The results suggest that oxidation and glycation enhanced the effects of LDL on the production of PAI-1 in ECs through increasing the transcription of PAI-1. Intact Golgi apparatus is required for PAI-1 generation from ECs induced by LDL or its modified forms.

Impact of glycated LDL on endothelial nitric oxide synthase in vascular endothelial cells: involvement of transmembrane signaling and endoplasmic reticulum stress.

Cardiovascular diseases are the major cause of mortality in diabetes patients. Increased levels of glycated low density lipoprotein (glyLDL) are detected in diabetic patients. Endothelial nitric oxide synthase (eNOS) generates nitric oxide, which is responsible to endothelium-dependent vasodilation. The impact of glyLDL on the expression and activity of eNOS in vascular endothelial cells (EC) remains unknown. The present study investigated the effect of glyLDL on the levels of protein, mRNA and activity of eNOS in cultured human umbilical vein EC. The results demonstrated that incubation of EC with physiological concentrations of glyLDL significantly reduced the abundances of eNOS protein in EC with the maximal inhibition at 100µg/ml for 24h. At the optimized condition, glyLDL decreased eNOS mRNA and reduced its activity in EC. Blocking antibody against the receptor for advanced glycation end products (RAGE) prevented glyLDL-induced downregulation of eNOS in EC. GlyLDL increased the translocation of H-Ras from cytoplasm to membrane in EC. Farnesyl-transferase inhibitor-276, an H-Ras antagonist, normalized glyLDL-induced downregulation of eNOS and prevented glyLDL-induced upregulation of H-Ras in EC membrane. Treatment with 4-phenylbutyric acid, an endoplasmic reticulum (ER) stress antagonist, prevented glyLDL-induced eNOS downregulation in EC. The results suggest that diabetes-associated metabolic stress inhibits the production and activity of eNOA in cultured human vascular EC through the activation of RAGE/H-Ras mediated upstream signaling pathway. ER stress induced by glyLDL is possibly involved in eNOS downregulation.

Functional modulation of antioxidant enzymes in vascular endothelial cells by glycated LDL.

Reactive oxygen species (ROS) have been implicated in atherogenesis. Previous studies demonstrated that oxidized LDL (oxLDL) or glycated LDL (gly-LDL) increased the generation of superoxide from vascular endothelial cells (EC). The present study examined the effects of gly-LDL on the activation of antioxidant enzymes for the metabolism of ROS in cultured human vascular endothelial cells in comparison to oxLDL and LDL without chemical modification. Treatment with LDL, oxLDL or gly-LDL significantly increased the release of hydrogen peroxide (H(2)O(2)) from EC following 2h of incubation and the release of superoxide after 24 h of treatment. The increased release of H(2)O(2), but not superoxide, was normalized in EC treated with LDL or its modified forms. Elevated activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase in EC were detected following a 24 h-treatment with the LDLs. The levels of GR activity and reduced/oxidized glutathione (GSH/GSSG) in EC treated with the lipoproteins were increased after 2 h, but were reduced after > or =24 h of incubation. Gly-LDL caused less increases in SOD, GPx or catalase activity, but more evident changes in GR activity and H(2)O(2) release compared to oxLDL or LDL. The findings suggest that exposure to glucose-modified LDL altered the activities of multiple antioxidant enzymes in cultured EC, which partially normalizes the excess generation of ROS, but reduced the intracellular reservoir of GSH.

Impact of simvastatin on hemostatic and fibrinolytic regulators in Type 2 diabetes mellitus.

Simvastatin, a widely used 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor, effectively reduced cardiac death and ischemic events in patients with coronary heart disease (CHD) and diabetes mellitus (DM). The mechanism of cardiovascular benefits of statins in DM remains unclear. We examined how simvastatin influences the levels of several in vivo markers for coagulation and fibrinolysis in 26 Type 2 DM patients. The diabetic patients received 20 mg/day of simvastatin up to 12 months. The levels of total cholesterol (TC), low density lipoprotein-cholesterol (LDL-c) and triglycerides in peripheral circulation of patients were significantly reduced after > or =6 weeks of simvastatin treatment. Levels of prothrombin fragment 1+2 (F1+2), factor VII, plasminogen activator inhibitor-1 (PAI-1) and tissue factor pathway inhibitor (TFPI) antigens, but not tissue plasminogen activator (tPA) antigen, in the pre-simvastatin plasmas of the diabetic patients were significantly higher than the levels found in plasmas of healthy subjects. Significant reductions in F1+2 and PAI-1 levels were evident > or =6 weeks after the diabetic patients received simvastatin. Levels of total tPA, TFPI, FVII, hemoglobin A1c, fasting blood glucose, and insulin in the diabetic patients' plasma were not significantly altered by simvastatin treatment. Positive correlations were found between PAI-1 versus TC, PAI-1 versus LDL-c, and FVII versus triglycerides in the plasmas of simvastatin-treated patients. The results suggest that simvastatin reduces in vivo prothrombinase activity and PAI-1 levels in type 2 DM patients. These actions may contribute to the protective properties of simvastatin against ischemic events in diabetic patients.

Endoplasmic reticulum stress in diabetic mouse or glycated LDL-treated endothelial cells: protective effect of Saskatoon berry powder and cyanidin glycans.

Endoplasmic reticulum (ER) stress is associated with insulin resistance and diabetic cardiovascular complications, and mechanism or remedy for ER stress remains to be determined. The results of the present study demonstrated that the levels of ER stress or unfolded protein response (UPR) markers, the intensity of thioflavin T (ThT) fluorescence and the abundances of GRP78/94, XBP-1 and CHOP proteins were elevated in cardiovascular tissue of diabetic leptin receptor-deficient (db/db) mice. Cyanidin-3-glucoside (C3G) and cyanidin-3-galactoside (C3Ga) are major anthocyanins in Saskatoon berry (SB) powder. The administration of 5% SB powder for 4 weeks attenuated ThT fluorescence and the UPR markers in hearts and aortae of wild-type and db/db mice. Treatment with glycated low-density lipoprotein (gLDL) increased ThT intensity in human umbilical vein endothelial cells (ECs). Elevated UPR markers were detected in gLDL-treated EC compared to control cultures. The involvement of ER stress in gLDL-treated EC was supported by that the addition of 4-phenyl butyrate acid (a known ER stress antagonist) inhibited gLDL-induced increases in ER stress or UPR markers. C3G at 30 µM or C3Ga at 100 µM reached their maximal inhibition on gLDL-induced increases in ThT, GRP78/94, XBP-1 and CHOP in EC. The results demonstrated that ER stress was enhanced in cardiovascular tissue of db/db mice or gLDL-treated EC. SB powder or cyanidin glycans prevented the abnormal increases in ER stress and UPR markers in cardiovascular tissue of diabetic db/db mice or gLDL-treated EC.

Effects of Saskatoon berry powder on monocyte adhesion to vascular wall of leptin receptor-deficient diabetic mice.

HYPOTHESIS: Atherosclerotic cardiovascular complications are the leading cause of death in diabetic patients. Monocyte adhesion is an early event for atherogenesis. Previous studies demonstrated that dark-skin berries had cardiovascular protective effects. We hypothesize that Saskatoon berry (SB) powder may reduce monocyte adhesion in leptin receptor-deficient (db/db) diabetic mice. METHODS: Wild-type and db/db mice were fed with chow or supplemented with SB powder. Anthocyanins in SB powder were identified using mass spectrometry. Mouse monocytes were incubated with mouse aorta. Monocyte adhesion was counted under microscopy. Inflammatory or metabolic markers in blood or tissue were analyzed using immunological or biochemical methods. RESULTS: SB powder significantly reduced monocyte adhesion to aorta from diabetic db/db mice compared to regular chow. The increased monocyte adhesion to aorta was normalized in db/db mice treated with ≥5% of SB powder for 4 weeks. Increased contents of Nicotinamide adenine dinucleotide phosphate oxidase (NADPH) oxidase-4, heat shock factor-1, monocyte chemotactic protein (MCP)-1, intracellular adhesion molecule (ICAM)-1, P-selectin, tumor necrosis factor-α, plasminogen activator inhibitor (PAI)-1 and urokinase plasminogen activator in aorta or heart apex, elevated plasma PAI-1 and MCP-1 were detected in db/db mice on chow compared to wild-type mice on the same diet; 5% SB powder inhibited the increases of inflammatory, fibrinolytic or stress regulators in aorta or heart apex of db/db mice. Monocyte adhesion positively correlated with blood glucose, cholesterol, body weight, heart MCP-1, PAI-1 or ICAM-1. CONCLUSION: The findings suggest that SB powder attenuated monocyte adhesion to aorta of db/db mice, which was potentially mediated through inhibiting the inflammatory, stress and/or fibrinolyic regulators.