Ascorbic acid prevents high glucose-induced apoptosis in human brain pericytes

High glucose concentrations due to diabetes increase apoptosis of vascular pericytes, impairing vascular regulation and weakening vessels, especially in brain and retina. We sought to determine whether vitamin C, or ascorbic acid, could prevent such high glucose-induced increases in pericyte apoptosis. Culture of human microvascular brain pericytes at 25 mM compared to 5 mM glucose increased apoptosis measured as the appearance of cleaved caspase 3. Loading the cells with ascorbate during culture decreased apoptosis, both at 5 and 25 mM glucose. High glucose-induced apoptosis was due largely to activation of the receptor for advanced glycation end products (RAGE), since it was prevented by specific RAGE inhibition. Culture of pericytes for 24 h with RAGE agonists also increased apoptosis, which was completely prevented by inclusion of 100 μM ascorbate. Ascorbate also prevented RAGE agonist-induced apoptosis measured as annexin V binding in human retinal pericytes, a cell type with relevance to diabetic retinopathy. RAGE agonists decreased intracellular ascorbate and GSH in brain pericytes. Despite this evidence of increased oxidative stress, ascorbate prevention of RAGE-induced apoptosis was not mimicked by several antioxidants. These results show that ascorbate prevents pericyte apoptosis due RAGE activation. Although RAGE activation decreases intracellular ascorbate and GSH, the prevention of apoptosis by ascorbate may involve effects beyond its function as an antioxidant.     

Astragaloside IV improved barrier dysfunction induced by acute high glucose in human umbilical vein endothelial cells

The purpose of the present study was to examine the effects of astragaloside IV, a saponin isolated from Astragalus membranaceus (Fisch) Bge, on the impairment of barrier function induced by acute high glucose in cultured human vein endothelial cells. High glucose (27.8 mM) induced a decrease in transendothelial electrical impedance and an increase in cell monolayer permeability in human umbilical vein endothelial cells. Endothelial barrier dysfunction stimulated by high glucose was accompanied by translocation and activation of protein kinase C (PKC), the redistribution of F-actin and formation of intercellular gaps, suggesting that increases in PKC activity and rearrangement of F-actin could be associated with endothelial barrier dysfunction induced by acute high glucose. Application of astragaloside IV inhibited high glucose-induced endothelial barrier dysfunction in a dose-dependent manner, which is compatible with inhibition of PKC translocation and improvement of F-actin rearrangements. Western blot analysis revealed that high glucose-induced PKC alpha and beta2 overexpression in the membrane fraction were significantly reduced by astragaloside IV. These findings indicate that astragaloside IV protected endothelial cells from high glucose-induced barrier impairment by inhibiting PKC activation, as well as improving cytoskeleton remodeling.     

Modulation of advanced glycation endproduct synthesis by kynurenines in human lens proteins

Human lens proteins (HLP) become chemically modified by kynurenines and advanced glycation end products (AGEs) during aging and cataractogenesis. We investigated the effects of kynurenines on AGE synthesis in HLP. We found that incubation with 5 mM ribose or 5 mM ascorbate produced significant quantities of pentosidine, and this was further enhanced in the presence of two different kynurenines (200–500 µM): N-formylkynurenine (Nfk) and kynurenine (Kyn). Another related compound, 3-hydroxykynurenine (3OH-Kyn), had disparate effects; low concentrations (10–200 µM) promoted pentosidine synthesis, but high concentrations (200–500 µM) inhibited it. 3OH-Kyn showed similar effects on pentosidine synthesis from Amadori-enriched HLP or ribated lysine. Chelex-100 treatment of phosphate buffer reduced pentosidine synthesis from Amadori-enriched HLP by ∼ 90%, but it did not inhibit the stimulating effect of 3OH-Kyn and EDTA. 3OH-Kyn (100–500 μM) spontaneously produced copious amounts of H₂O₂ (10–25 μM), but externally added H₂O₂ had only a mild stimulating effect on pentosidine but had no effect on N^ε-carboxymethyl lysine (CML) synthesis in HLP from ribose and ascorbate. Further, human lens epithelial cells incubated with ribose and 3OH-Kyn showed higher intracellular pentosidine than cells incubated with ribose alone. CML synthesis from glycating agents was inhibited 30 to 50% by 3OH-Kyn at concentrations of 100–500 μM. Argpyrimidine synthesis from 5 mM methylglyoxal was slightly inhibited by all kynurenines at concentrations of 100–500 μM. These results suggest that AGE synthesis in HLP is modulated by kynurenines, and such effects indicate a mode of interplay between kynurenines and carbohydrates important for AGE formation during lens aging and cataract formation.     

Ascorbic acid prevents oxidant-induced increases in endothelial permeability

Oxidative stress acutely increases the permeability of the vascular endothelium to large molecules that would not otherwise cross the barrier. Ascorbic acid is an antioxidant that tightens the endothelial permeability barrier, so we tested whether it might also prevent the increase in endothelial permeability due to cellular oxidative stress. Treatment of EA.hy926 endothelial cells cultured on filter inserts with H(2) O(2) , menadione, and buthionine sulfoximine increased endothelial permeability to radiolabeled inulin. Short-term ascorbate loading of the cells to what are likely physiologic concentrations of the vitamin by treating them with dehydroascorbate prevented the increase in endothelial permeability due to these agents. The nonphysiologic antioxidants dithiothreitol and tempol also prevented increases in endothelial barrier permeability induced by the agents. These results suggest that oxidative stress induced directly by oxidants or indirectly by glutathione depletion impairs endothelial barrier function and that intracellular ascorbate may serve to prevent this effect.     

Irreversibly glycated LDL induce oxidative and inflammatory state in human endothelial cells; added effect of high glucose

In diabetes, hyperglycemia and the associated formation of advanced glycation end-products (AGE) and AGE-modified low density lipoproteins (AGE-LDL) can directly affect the cells of the vascular wall. We hypothesize that AGE-LDL may act directly and induce oxidant and inflammatory alterations in human endothelial cells (HEC), this effect being amplified by high glucose. To test this assumption, the activity of NADPH oxidase (NADPHox) was evaluated and the expression of its subunits (p22^phox, NOX4, and p67^phox), of the AGE receptor (RAGE), and of the monocyte chemoattractant protein-1 (MCP-1) were assessed by real-time PCR and Western blot in confluent EA.hy926 cells incubated with AGE-LDL for 24 and 48 h, in normal and high glucose conditions. Exposure of HEC for 48 h to AGE-LDL in 5 mM glucose induced an increase of RAGE expression (50%), NADPHox activity (107%), p22^phox and NOX4 mRNA (50% and 188%, respectively) and MCP-1 expression (80%). AGE-LDL-stimulated p22^phox expression by activating p38 MAP kinase and NF-kB, and MCP-1 expression by activating NF-kB, as demonstrated by the use of specific inhibitors (SB203580 and Bay11-7085). The addition of 25 mM glucose in the culture medium enhanced the effect of AGE-LDL, but also of nLDL, on RAGE, p22^phox, NOX4, p67^phox, and MCP-1 gene expression. In conclusion, AGE-LDL induce an oxidative stress and a pro-inflammatory state in human endothelial cells. Both AGE-LDL and nLDL in the presence of high glucose amplify their effect, revealing a link between hyperlipidemia, diabetes, and endothelial cell dysfunction.     

Ascorbic acid prevents increased endothelial permeability caused by oxidized low density lipoprotein

Abstract

Mildly oxidized low density lipoprotein (mLDL) acutely increases the permeability of the vascular endothelium to molecules that would not otherwise cross the barrier. This study has shown that ascorbic acid tightens the permeability barrier in the endothelial barrier in cells, so this work tested whether it might prevent the increase in endothelial permeability due to mLDL. Treatment of EA.hy926 endothelial cells with mLDL decreased intracellular GSH and activated the cells to further oxidize the mLDL. mLDL also increased endothelial permeability over 2 h to both inulin and ascorbate in cells cultured on semi-permeable filters. This effect was blocked by microtubule and microfilament inhibitors, but not by chelation of intracellular calcium. Intracellular ascorbate both prevented and reversed the mLDL-induced increase in endothelial permeability, an effect mimicked by other cell-penetrant antioxidants. These results suggest a role for endothelial cell ascorbate in ameliorating an important facet of endothelial dysfunction caused by mLDL.     

Atorvastatin exerts its anti-atherosclerotic effects by targeting the receptor for advanced glycation end products

Recent studies demonstrated the beneficial role of atorvastatin in reducing the risk of cardiovascular morbidity and mortality in patients with diabetes mellitus and/or metabolic syndrome. To investigate the mechanisms underlying the anti-atheroscleroic action of atorvastatin, we examined the expression of the receptor for advanced glycation end products (RAGE) and its downstream target gene, monocyte chemoattractant protein-1 (MCP-1) using real-time PCR. In in vitro studies, exposure to high glucose or AGE induced oxidative stress and activation of the AGE/RAGE system in human umbilical vein endothelial cells. Treatment of the cells with atorvastatin significantly released the oxidative stress by restoring the levels of glutathione and inhibited the RAGE upregulation. In diabetic Goto Kakisaki (GK) rats fed with a high-fat diet for 12 weeks, RAGE and MCP-1 were upregulated in the aortas, and there was a significant correlation between RAGE and MCP-1 mRNA abundance (r = 0.482, P = 0.031). Treatment with atorvastatin (20 mg/kg qd) significantly downregulated the expression of RAGE and MCP-1. These data thus demonstrate a novel “pleiotropic” activity of atorvastatin in reducing the risk of cardiovascular diseases by targeting RAGE expression.     

Advanced glycation end-products (AGEs) induce concerted changes in the osteoblastic expression of their receptor RAGE and in the activation of extracellular signal-regulated kinases (ERK)

An increase in the interaction between advanced glycation end-products (AGEs) and their receptor RAGE is believed to contribute to the pathogenesis of chronic complications of Diabetes mellitus, which can include bone alterations such as osteopenia. We have recently found that extracellular AGEs can directly regulate the growth and development of rat osteosarcoma UMR106 cells, and of mouse calvaria-derived MC3T3E1 osteoblasts throughout their successive developmental stages (proliferation, differentiation and mineralisation), possibly by the recognition of AGEs moieties by specific osteoblastic receptors which are present in both cell lines. In the present study we examined the possible expression of RAGE by UMR106 and MC3T3E1 osteoblastic cells, by immunoblot analysis. We also investigated whether short-, medium- or long-term exposure of osteoblasts to extracellular AGEs, could modify their affinity constant and maximal binding for AGEs (by 125I-AGE-BSA binding experiments), their expression of RAGE (by immunoblot analysis) and the activation status of the osteoblastic ERK 1/2 signal transduction mechanism (by immunoblot analysis for ERK and P-ERK). Our results show that both osteoblastic cell lines express readily detectable levels of RAGE. Short-term exposure of phenotypically mature osteoblastic UMR106 cells to AGEs decrease the cellular density of AGE-binding sites while increasing the affinity of these sites for AGEs. This culture condition also dose-dependently increased the expression of RAGE and the activation of ERK. In proliferating MC3T3E1 pre-osteoblasts, 24–72 h exposure to AGEs did not modify expression of RAGE, ERK activation or the cellular density of AGE-binding sites. However, it did change the affinity of these binding sites for AGEs, with both higher- and lower-affinity sites now being apparent. Medium-term (1 week) incubation of differentiated MC3T3E1 osteoblasts with AGEs, induced a simultaneous increase in RAGE expression and in the relative amount of P-ERK. Mineralising MC3T3E1 cultures grown for 3 weeks in the presence of extracellular AGEs showed a decrease both in RAGE and P-ERK expression. These results indicate that, in phenotypically mature osteoblastic cells, changes in ERK activation closely follow the AGEs-induced regulation of RAGE expression. Thus, the AGEs-induced biological effects that we have observed previously in osteoblasts, could be mediated by RAGE in the later stages of development, and mediated by other AGE receptors in the earlier pre-osteoblastic stage.     

Higher glucose level can enhance the H. pylori adhesion and virulence related with type IV secretion system in AGS cells

Background

Hyperglycemia increases the risk of gastric cancer in H. pylori-infected patients. High glucose could increase endothelial permeability and cancer-associated signaling. These suggest high glucose may affect H. pylori or its infected status.We used two strains to investigate whether H. pylori growth, viability, adhesion and CagA-phosphorylation level in the infected-AGS cells were influenced by glucose concentration (100, 150, and 200 mg/dL).

Results

The growth curves of both strains in 200 mg/dL of glucose were maintained at the highest optimal density after 48 h and the best viability of both strains were retained in the same glucose condition at 72 h. Furthermore, adhesion enhancement of H. pylori was significantly higher in 200 mg/dL of glucose as compared to that in 100 and 150 mg/dL (p < 0.05). CagA protein also increased in higher glucose condition. The cell-associated CagA and phosphorylated-CagA was significantly increased in 150 and 200 mg/dL of glucose concentrations as compared to that of 100 mg/dL (p < 0.05), which were found to be dose-dependent.

Conclusion

Higher glucose could maintain H. pylori growth and viability after 48 h. H. pylori adhesion and CagA increased to further facilitate the enhancement of cell-associated CagA and phosphorylated CagA in higher glucose conditions.     

Green tea polysaccharide-conjugates protect human umbilical vein endothelial cells against impairments triggered by high glucose

Hot-water extracts of low-grade green tea were precipitated with ethanol, deproteinized with trichloroacetic acid, neutralized with NaOH and fractionated by DEAE-cellulose DE-52 column chromatography to yield three (3) of unexplored polysaccharide-conjugate fractions termed gTPC1, gTPC2 and gTPC3. Monosaccharide and amino acid composition, contents of total neutral sugars, proteins and moistures, HPGPC distribution and Zeta potentials of gTPC1-3 were investigated. Exposure of human umbilical vein endothelial (HUVE) cells to high glucose (33 mM) for 12 h significantly decreased cell viability relative to normal glucose control (p < 0.001). As compared with cell injury group, gTPC1-3 at all of three dose levels (50, 150 and 300 μg/mL) were found to possess remarkably protective effects on HUVE cells against impairments induced by high glucose in a dose-dependent manner (p < 0.05, p < 0.001). To contribute toward our understanding of the cell-based protection mechanism of gTPC1-3, the latter were subjected to self-oxidation of 1,2,3-phentriol assay, and their scavenging effects were observed as 55.1%, 47.6% and 47.9% at the concentration of 300 μg/mL, respectively. On the basis of the fact that high glucose-induced endothelial dysfunction involves in the overproduction of reactive oxygen species (ROS) and contributes to the vascular complications in patients with diabetes, inhibitory effects of gTPC1-3 on high glucose-mediated HUVE cell loss are, at least in part, correlated with their potential scavenging potency of ROS. Taken together, gTPC1-3 could be developed as non-cytotoxic candidates of therapeutic agent for diabetic vascular complications.     

Characterization of ecto-ATPase activity in the surface of LLC-PK1 cells and its modulation by ischemic conditions

Background

The concentration of extracellular nucleotides is regulated by enzymes that have their catalytic site facing the extracellular space, the so-called ecto-enzymes.

Methods

We used LLC-PK1 cells, a well-characterized porcine renal proximal tubule cell line, to biochemically characterize ecto-ATPase activity in the luminal surface. The [γ-³²P]Pi released after reaction was measured in aliquots of the supernatant by liquid scintillation.

Results

This activity was linear with time up to 20 min of reaction and stimulated by divalent metals. The ecto-ATPase activity measured in the presence of 5 mM MgCl₂ was (1) optimum at pH 8, (2) insensitive to different inhibitors of intracellular ATPases, (3) inhibited by 1 mM suramin, an inhibitor of ecto-ATPases, (4) sensitive to high concentrations of sodium azide (NaN₃) and (5) also able to hydrolyze ADP in the extracellular medium. The ATP:ADP hydrolysis ratio calculated was 4:1. The ecto-ADPase activity was also inhibited by suramin and NaN₃. The dose–response of ATP revealed a hyperbolic profile with maximal velocity of 25.2 ± 1.2 nmol Pi x mg^− 1 x min^− 1 and K_0.5 of 0.07 ± 0.01 mM. When cells were submitted to ischemia, the E-NTPDase activity was reduced with time, achieving 71% inhibition at 60 min of ischemia.

Conclusion

Our results suggest that the ecto-ATPase activity of LLC-PK1 cells has the characteristics of a type 3 E-NTPDase which is inhibited by ischemia.

General Significance

This could represent an important pathophysiologic mechanism that explains the increase in ATP concentration in the extracellular milieu in the proximal tubule during ischemia.     

American ginseng (Panax quinquefolius) prevents glucose-induced oxidative stress and associated endothelial abnormalities

Purpose

Ginseng (Araliaceae), demonstrates widespread biological effects because of its purported antioxidant and other properties. The present study was undertaken to investigate the effects of American ginseng root extract on glucose-induced oxidative stress and associated oxidative damage to human umbilical vein endothelial cells (HUVECs).

Methods

Following pretreatment with various concentrations of ginseng (alcoholic extract), HUVECs were incubated with various concentrations of d-glucose ranging from 5 to 25 mmol/l for 24 h. l-Glucose was used at a concentration of 25 mmol/l as a control.

Results

Glucose-induced oxidative stress detected by intracellular reactive oxygen species accumulation, superoxide anion generation and DNA damage in HUVECs were significantly prevented by ginseng. Treatment of HUVECs with ginseng further led to significant prevention of glucose-induced NF-κB activation. Glucose-induced increase in fibronectin (FN), EDB⁺FN (a splice variant of FN), endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF) mRNAs and protein levels were also prevented by ginseng treatment.

Conclusion

These data indicate that American ginseng prevented glucose-induced damage in the HUVECs through its antioxidant properties.     

Peptidylprolyl cis/trans isomerase,NIMA-interacting 1 (PIN1) regulates pulmonary effects of endotoxin and tumor necrosis factor-α in mice

Peptidylprolyl cis/trans isomerase, NIMA-interacting 1 (PIN1) modulates phospho-signaling by catalyzing rotation of the bond between a phosphorylated serine or threonine before proline in proteins. As depletion of PIN1 increased inflammatory protein expression in cultured endothelial cells treated with bacterial endotoxin (lipopolysaccharide, LPS) and interferon-γ, we hypothesized that PIN1 knockout would increase sensitivity to LPS-induced lung inflammation in mice. Mortality due to a high dose of LPS (30 mg/kg) was greater in knockout than wildtype mice. Lung myeloperoxidase activity, reflecting neutrophils, was increased to a 35% higher level in PIN1 knockout mouse lung, as compared with wildtype, after treatment with a sublethal dose of 3 mg LPS/kg, ip. Unexpectedly, plasma tumor necrosis factor-α (TNF) was approximately 50% less than in wildtype mice. Knockout mice, however, were more sensitive than wildtype to TNF-induced neutrophil accumulation. The neutrophil adhesion molecule, E-selectin, was also elevated in lungs of knockout mice treated with TNF, suggesting that PIN1 depletion increases endothelial sensitivity to TNF. Indeed, TNF induced more reactive oxygen species in cultured endothelial cells depleted of PIN1 with short hairpin RNA than in control cells. Collectively, the results indicate that while PIN1 normally facilitates TNF production in LPS-treated mice, it suppresses pulmonary and endothelial reactions to the cytokine. Tissue or cell-specific effects of PIN1 may affect the overall inflammatory response to LPS and other stimuli.     

Chelation of intracellular iron enhances endothelial barrier function: A role for vitamin C?

Ascorbic acid improves endothelial barrier function by decreasing the permeability of endothelial cells cultured on semi-porous membrane filters. This decrease was not due to enhanced collagen synthesis and was mimicked by the collagen synthesis inhibitor ethyl-3,4-dihydroxybenzoic acid (EDHB). Since EDHB is known to chelate intracellular free iron, the effects of two membrane-permeant iron chelators were tested on endothelial permeability. Both 2,2′-dipyridyl and desferrioxamine decreased trans-endothelial permeability in a concentration-dependent manner. Increasing intracellular iron with a chelate of 8-hydroxyquinoline and ferric iron prevented effects of both EDHB and intracellular ascorbate. That EDHB and ascorbate did in fact chelate intracellular iron was supported by finding that they both decreased the cellular fluorescence quenching of the iron-sensitive dye Phen green SK. These results show that chelation of intracellular iron decreases endothelial barrier permeability and implicate this mechanism in the ability of EDHB and possibly intracellular ascorbate to tighten the endothelial barrier.     

Mitochondria-targeted penetrating cations as carriers of hydrophobic anions through lipid membranes

High negative electric potential inside mitochondria provides a driving force for mitochondria-targeted delivery of cargo molecules linked to hydrophobic penetrating cations. This principle is utilized in construction of mitochondria-targeted antioxidants (MTA) carrying quinone moieties which produce a number of health benefitting effects by protecting cells and organisms from oxidative stress. Here, a series of penetrating cations including MTA were shown to induce the release of the liposome-entrapped carboxyfluorescein anion (CF), but not of glucose or ATP. The ability to induce the leakage of CF from liposomes strongly depended on the number of carbon atoms in alkyl chain (n) of alkyltriphenylphosphonium and alkylrhodamine derivatives. In particular, the leakage of CF was maximal at n about 10-12 and substantially decreased at n = 16. Organic anions (palmitate, oleate, laurylsulfate) competed with CF for the penetrating cation-induced efflux. The reduced activity of alkylrhodamines with n = 16 or n = 18 as compared to that with n = 12 was ascribed to a lower rate of partitioning of the former into liposomal membranes, because electrical current relaxation studies on planar bilayer lipid membranes showed rather close translocation rate constants for alkylrhodamines with n = 18 and n = 12. Changes in the alkylrhodamine absorption spectra upon anion addition confirmed direct interaction between alkylrhodamines and the anion. Thus, mitochondria-targeted penetrating cations can serve as carriers of hydrophobic anions across bilayer lipid membranes.     

Effect of ghrelin on human endothelial cells apoptosis induced by high glucose

Endothelial dysfunction is thought to be a major cause of vascular complications in diabetes. Our research shows that ghrelin attenuates high glucose-induced apoptosis in cultured human umbilical vein endothelial cells (ECV-304). Exposure to glucose (33.3mM) for 72 h caused a significant increase in apoptosis, as evaluated by TUNEL and flow cytometry, but pretreatment of ghrelin (10(-7)M) eliminated high glucose-induced apoptosis in ECV-304. Ghrelin also prevented the induction of caspase-3 activation, in cells incubated with glucose (33.3 mM). Exposure of cells to ghrelin (10(-7)M) caused rapid activation of Akt. PI3K inhibitor, LY294002 attenuated ghrelin's inhibitory effect on caspase-3 activity. Ghrelin protected endothelial cells from high glucose by inhibiting reactive oxygen species (ROS) generation. Results of our study indicate that ghrelin inhibits both high glucose-induced apoptosis via PI3K/Akt pathway and ROS production in ECV-304. This peptide may have potential in preventing diabetic complications, especially in obese patients.     

Concurrent regulation of AMP-activated protein kinase and SIRT1 in mammalian cells

We examined in HepG2 cells whether glucose-induced changes in AMP-activated protein kinase (AMPK) activity could be mediated by SIRT1, an NAD⁺-dependent histone/protein deacetylase that has been linked to the increase in longevity caused by caloric restriction. Incubation with 25 vs. 5 mM glucose for 6 h concurrently diminished the phosphorylation of AMPK (Thr 172) and ACC (Ser 79), increased lactate release, and decreased the abundance and activity of SIRT1. In contrast, incubation with pyruvate (0.1 and 1 mM) for 2 h increased AMPK phosphorylation and SIRT1 abundance and activity. The putative SIRT1 activators resveratrol and quercetin also increased AMPK phosphorylation. None of the tested compounds (low or high glucose, pyruvate, and resveratrol) significantly altered the AMP/ATP ratio. Collectively, these findings raise the possibility that glucose-induced changes in AMPK are linked to alterations in SIRT1 abundance and activity and possibly cellular redox state.