Glyceraldehyde-derived advanced glycation end-products having pyrrolopyridinium-based crosslinks

Reducing sugars and reactive aldehydes, such as glyceraldehyde, non-enzymatically react with amino or guanidino groups of proteins to form advanced glycation end-products (AGEs) by the Maillard reaction that involves Schiff base formation followed by Amadori rearrangement. AGEs are found relatively in abundance in the human eye and to accumulate at a higher rate in diseases that impair vision such as cataract, diabetic retinopathy or age-related macular degeneration. We identified two novel AGEs of pyrrolopyridinium lysine dimer derived from glyceraldehyde, PPG1 and PPG2, in the Maillard reaction of N^α-acetyl-l-lysine with glyceraldehyde under physiological conditions. Having fluorophores similar to that of vesperlysine A, which was isolated from the human lens, PPGs were found to act as photosensitizers producing singlet oxygen in response to blue light irradiation. Moreover, PPG2 interacts with receptor for AGE (RAGE) in vitro with a higher binding affinity than GLAP, a well-known ligand of the receptor. We also proposed a pathway to form PPGs and discussed how they would be formed in vitro. As glyceraldehyde-derived AGEs have been studied extensively in connection with various hyperglycemia-related diseases, further studies will be required to find PPGs in vivo such as in the lens or other tissues.     

Dihydrochalcones: Implication in resistance to oxidative stress and bioactivities against advanced glycation end-products and vasoconstriction

Flavonoids are a group of polyphenol compounds with known antioxidant activities. Among them, dihydrochalcones are mainly found in apple leaves (Malus domestica). Glycosylated dihydrochalcones were previously found in large amounts in leaves of two genotypes of Malus with contrasting resistance to fire blight, a bacterial disease caused by Erwinia amylovora. In the present study we demonstrate that soluble polyphenol patterns comprised phloridzin alone or in combination with two additional dihydrochalcones, identified as sieboldin and trilobatin. Presence of sieboldin in young leaves correlated well with a high 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity. Moreover, these leaves displayed enhanced tolerance to paraquat, a photooxidative-stress generating herbicide. Interestingly, phloridzin had a high activity in the oxygen radical absorbance capacity (ORAC) assay, but its presence alone in leaves did not correlate with tolerance to paraquat. In order to further characterise the activity of these compounds, we tested their ability to prevent oxidative-dependent formation of advanced glycation end-products (AGEs) and phenylephrine-induced contraction of isolated rat mesenteric arteries. The antioxidant capacity of sieboldin was clearly demonstrated by showing that this compound (i) prevented vasoconstriction and (ii) inhibited AGEs formation. Both assays provided interesting information concerning a potential use of sieboldin as a therapeutic. Hence, our results strongly argue for a bioactivity of dihydrochalcones as functional antioxidants in the resistance of Malus leaves to oxidative stress. In addition, we demonstrate for the first time that sieboldin is a powerful multipotent antioxidant, effective in preventing physiopathological processes. Further work should aim at demonstrating the potential use of this compound as a therapeutic in treating free radical-involving diseases.     

Mechanistic targeting of advanced glycation end-products in age-related diseases

Glycative stress, caused by the accumulation of cytotoxic and irreversibly-formed sugar-derived advanced glycation end-products (AGEs), contributes to morbidity associated with aging, age-related diseases, and metabolic diseases. In this review, we summarize pathways leading to formation of AGEs, largely from sugars and glycolytic intermediates, and discuss detoxification of AGE precursors, including the glyoxalase system and DJ-1/Park7 deglycase. Disease pathogenesis downstream of AGE accumulation can be cell autonomous due to aggregation of glycated proteins and impaired protein function, which occurs in ocular cataracts. Extracellular AGEs also activate RAGE signaling, leading to oxidative stress, inflammation, and leukostasis in diabetic complications such as diabetic retinopathy. Pharmaceutical agents have been tested in animal models and clinically to diminish glycative burden. We summarize existing strategies and point out several new directions to diminish glycative stress including: plant-derived polyphenols as AGE inhibitors and glyoxalase inducers; improved dietary patterns, particularly Mediterranean and low glycemic diets; and enhancing proteolytic capacities of the ubiquitin-proteasome and autophagy pathways that are involved in cellular clearing of AGEs.     

The neuroprotective role of rosiglitazone in advanced glycation end product treated human neural stem cells is PPARgamma-dependent

Hyperglycemia is accompanied by an accelerated formation rate of advanced glycation end products (AGEs), which is associated with the pathogenesis of diabetic neuronal deficits. Peroxisome proliferator-activated receptor gamma (PPARγ) belongs to a family of ligand-activated nuclear receptors and its ligands are known to control many physiological, pathological and inflammatory pathways. Weinvestigated the hypothesis that the PPARγ agonist (rosiglitazone) would abrogate AGEs-mediated neurotoxic effects on human neural stem cells (hNSCs), by whichAGEs may play a role in diabetic-related neuronal impairment. Here, we show that rosiglitazone treatment increases cell viability of hNSCs via downregulation of caspase 3 activity. These rescue effects were extended in our studies showingrosiglitazone-mediated activation of PPARγ reversed the expression levels of two neuroprotective factors (Bcl-2 and PGC1α) that were downregulated in hNSCs exposed to AGEs alone. The stimulation of mitochondrial function and anti-oxidative stress by rosiglitazone was associated with activation of the PGC1α pathway by up-regulation of mitochondrial (NRF-1 and Tfam) and oxidative defense (SOD1, SOD2 and Gpx1) genes. Moreover, rosiglitazone significantly normalized the inflammatory responses (TNF-α and IL-1β), NF-κB (p65), and inflammatory genes (iNOS and COX-2) in the hNSCs treated with AGEs. This neuroprotective effect of rosiglitazone was effectively blocked by PPARγ-specific antagonist (GW9662), demonstrating that the action of rosiglitazone was mediated by at PPARγ-dependent pathway. Collectively, these novel findings show AGEs induce neurotoxic effects in hNSCs, and provide important mechanistic insights that may explain the increased risk of neuronal impairment deficits in diabetic patients. More importantly, these data show rosiglitazone-mediated activation of PPARγ-dependent signaling is neuroprotective in AGE-treated hNSCs, and suggests PPARγ ligands may be useful in the therapeutic management of patients with neurodegenerative diseases     

Combined immunoelectron microscopic and computer-assisted image analyses to detect advanced glycation end-products in human myocardium

Advanced glycation end-products (AGEs) result from oxidation–reduction reactions that ensue when a sugar becomes adducted to a protein. AGEs cause various complications of diabetes mellitus (DM). Experimental and clinical evidence suggest that AGEs also contribute to the complications of hypertension (HTN). Little is known about the abundance and localization of AGEs in human myocardium. In a few light microscopic studies, the AGE carboxymethyl lysine (CML) has been immunolabeled and localized virtually exclusively to the walls of small arteries. To more precisely delineate the abundance and localization of CML, we developed an immunoelectron microscopic (IEM) detection method using anti-CML monoclonal antibody 6D12 in conjunction with computer-assisted image analysis. Antibody was pre-absorbed with purified AGE-bovine serum albumin to assure specificity. Antigen–antibody (ag–ab) complexes were individually identified with protein A-conjugated colloidal gold and counted with an automated system. We applied this method in 21 patients (pts) undergoing epicardial biopsy during coronary bypass grafting (CBG) [20 M, 1 F; mean age 65 ± 7.4 (± SEM) years]. Seven pts had neither DM nor HTN, seven had HTN, and seven had DM + HTN. In contrast to the prior light microscopic studies, we detected CML scattered throughout the cardiomyocyte in all pts, but in widely varying amounts. Ag–ab complexes were abundant in sections through myofilaments (mean count 23.6 ± 9.2 per μm², range 9.4–48) and even more so in mitochondria (mean count 34.4 ± 11.9 per μm², range 14.1–68.2, P < 0.001 vs. myofilaments). CML was also detected in vascular endothelial cells. There were no statistically significant differences based on presence or absence of HTN or DM. In conclusion, our IEM method is the first to provide detailed delineation of the localization and abundance of CML in myocardium. CML is very prevalent in CBG pts, suggesting that AGEs could play a role in abnormal cardiomyocyte function, including altered energy metabolism.     

Early and advanced glycation end-products are increased in dietary copper deficiency

The hypothesis that nonenzymatic glycosylation of proteins (glycation) contributes to damage associated with dietary copper deficiency has depended largely on indirect evidence. Thus far, the observation of an elevated percentage of glycated hemoglobin in copper-deficient rats has provided the only direct evidence of an increase in glycation. We sought further direct evidence of increased glycation in copper deficiency. Male weanling rats were fed a copper-adequate (CuA, 6.4 mg Cu/kg diet) or copper-deficient diet (CuD, 0.4 mg Cu/kg diet) for 5 weeks. Rats fed the CuD diet were copper deficient as judged by depressed organ copper concentrations and a variety of indirect indices. Measurements of hemoglobin A(1) and serum fructosamine (both early glycation end-products) as well as serum pentosidine (an advanced glycation end-product) indicated that all three compounds were elevated in CuD rats relative to CuA rats. This finding further supports the view that glycation is enhanced and thus may contribute to defects associated with dietary copper deficiency.     

Pioglitazone suppresses advanced glycation end product-induced expression of plasminogen activator inhibitor-1 in vascular smooth muscle cells

Advanced glycation end products (AGEs) play an important role in vascular complications of diabetes, including fibrinolytic abnormalities.Pioglitazone, a peroxisome proliferator-activated receptor gamma (PPARΥ) agonist, has recently been shown to reduce circulating plasminogen activator inhibitor-1 (PAI-1) levels in diabetes mellitus. In the present study, we investigated the effects of pioglitazone on the expression of local PAI-1 in rat vascular smooth muscle cells (VSMCs) induced by AGEs and the underlying mechanism. The result showed that AGEs could enhance the PAI-1 expression by 5.1-fold in mRNA and 2.7-fold in protein level, as evaluated by real-time RT-PCR and Western blotting,respectively. Pioglitazone was found to down-regulate the AGE-stimulated PAI-1 expression in VSMCs. However, these inhibitory effects were partially attenuated by the PPARΥ antagonist, GW9662. Furthermore, we found that AGEs induced a rapid increase in phosphorylation and activation of extracellular signal-regulated protein kinase 1/2 (ERK 1/2). The ERK kinase inhibitor, UO126, partially prevented the induction of PAI-1 by AGEs. Moreover, pioglitazone was also found to inhibit the phosphorylation of ERKi/2. Taken together, it was concluded that pioglitazone could inhibit AGE-induced PAI-1 expression, which was mediated by the ERK1/2 and PPARΥ pathways. Our findings suggestedpioglitazone had a therapeutic potential in improving fibrinolytic activity, and consequently preventing thromboembolic complications of diabetes and cardiovascular disease.     

Endothelin-1 stimulates glial cell line-derived neurotrophic factor expression in cultured rat astrocytes

Effects of endothelin-1 (ET-1) on glial cell line-derived neurotrophic factor (GDNF) production in cultured astrocytes were examined. Treatment of cultured astrocytes with ET-1 (100 nM) increased mRNA levels of GDNF in 1-6h. The effect of ET-1 was inhibited by BQ788, an ET(B) receptor antagonist, but not by FR139317, an ET(A) receptor antagonist. ET-1 stimulated release of GDNF into culture medium. Dexamethasone (1 microM) and pyrrolidine dithiocarbamate (PDTC, 100 microM), which inhibit activation of NFkappaB, prevented the increases in GDNF mRNA by H(2)O(2). In contrast, the effect of ET-1 was not affected by dexamethasone and PDTC. The increase of astrocytic GDNF mRNA by ET-1 was inhibited by BAPTA/AM (30 microM) and PD98059 (50 microM), but not by calphostin C, staurosporine, and cyclosporine A. These results suggest that ET-1 stimulated expression of astrocytic GDNF through ET(B) receptor-mediated increases in cytosolic Ca(2+) and ERK activation.     

Influence of +1245 A/G MT1A polymorphism on advanced glycation end-products (AGEs) in elderly: effect of zinc supplementation

Advanced glycation end-products (AGEs) stimulate reactive oxygen species (ROS) generation and represent a risk factor for atherosclerosis, while their formation seems to be prevented by zinc. Metallothioneins (MT), zinc-binding proteins exert an antioxidant function by regulating intracellular zinc availability and protecting cells from ROS damages. +1245 A/G MT1A polymorphism was implicated in type 2 diabetes and in cardiovascular disease development as well as in the modulation of antioxidant response. The purpose of this study was to investigate the influence of +1245 A/G MT1A polymorphism on AGEs and ROS production and to verify the effect of zinc supplementation on plasma AGEs, zinc status parameters and antioxidant enzyme activity in relation to this SNP. One hundred and ten healthy subjects (72 ± 6 years) from the ZincAge study were supplied with zinc aspartate (10 mg/day for 7 weeks) and screened for +1245 MT1A polymorphism. +1245 MT1A G+ (Arginine) genotype showed higher plasma AGEs and ROS production in peripheral blood mononuclear cells (PBMCs) than G− (Lysine) one at the baseline. No significant changes after zinc supplementation were observed for AGEs, ROS and MT levels as well as for enzyme antioxidant activity in relation to the genotype. Among zinc status parameters, major increases were observed for the intracellular labile zinc (iZnL) and the NO-induced release of zinc in PBMCs, in G+ genotype as compared to G− one. In summary, +1245 G+ carriers showed increased plasma AGEs and ROS production in PBMCs at baseline and a higher improvement in iZnL after zinc intervention with respect to G− individuals.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0426-2) contains supplementary material, which is available to authorized users.     

Stress-induced senescence in human and rodent astrocytes

There is an increasing awareness that astrocytes, the most abundant cell type in the central nervous system, are critical mediators of brain homeostasis, playing multifunctional roles including buffering potassium ions, maintaining the blood-brain barrier, releasing growth factors, and regulating neurotransmitter levels. Defects in astrocyte function have been implicated in a variety of diseases including age-related diseases such Alzheimer's disease and Parkinson's disease. However, little is known about the age-related changes that occur in astrocytes and if these cells are able to generate a senescent phenotype in response to stress. In this report we have examined whether astrocytes can initiate a senescence program similar to that described in other cell types in response to a variety of stresses. Our results indicate that after oxidative stress, proteasome inhibition, or exhausted replication, human and mouse astrocytes show changes in several established markers of cellular senescence. Astrocytes appear to be more sensitive to oxidative stress than fibroblasts, suggesting that stress-induced senescence may be more pronounced in the brain than in other tissues.     

Advanced glycation end-products and advanced oxidation protein products in patients with diabetes mellitus

Accelerated glycoxidation takes part in the development of diabetic complications. We determined advanced glycation end-products (AGEs) and advanced oxidation protein products (AOPP) in the sera of 52 patients with diabetes mellitus (DM) - 18 with DM Type 1 and 34 with DM Type 2 and examined their relationship to the compensation of the disease. AGEs were estimated spectrofluorimetrically (350 nm/440 nm) whereas AOPP were determined spectro-photometrically (340 nm). AGEs were elevated only in DM Type 2 (DM2 5.11+/-1.15 x 10(3) AU/g vs controls 4.08+/-0.71 x 10(3) AU/g, p<0.001, vs DM1 4.14+/-0.86 x 10(3) AU/g, p<0.005, DM1 vs controls were not significant). AOPP were elevated significantly in both types of DM with higher levels in DM Type 2 (DM2 157.50+/-75.15 micromol/l vs healthy subjects 79.80+/-23.72 micromol/l, p<0.001, vs DM1 97.50+/-30.91 micromol/l, p<0.005, DM1 vs controls p<0.05). There was a tight correlation between AGEs and AOPP in both types of DM (DM1 r=0.75, DM2 r=0.47 (p<0.05)) and both AGEs and AOPP correlated with triglycerides. In DM Type 1 only, AGEs correlated with HbA1c r=0.47 (p<0.05) and with blood glucose. Slight but not significant differences in AGEs and AOPP levels were observed in patients with or without diabetic complications. Oxidative stress is increased in both types of DM, more in Type 2 where it contributes to the formation of glycoxidation products.     

Protective effect of extractive and biotechnological chondroitin in insulin amyloid and advanced glycation end product-induced toxicity

Glycosaminoglycans are extracellular matrix components related to several biological functions and diseases. Chondroitin sulfate is a sulphated glycosaminoglycan synthesized as part of proteoglycan molecules. They are frequently associated with amyloid deposits and possess an active role in amyloid fibril formation. Recently, a neuroprotective effect of extracellular matrix components against amyloid toxicity and oxidative stress has been reported. Advanced glycation end products (AGEs), the end products of the glycation reaction, have been linked to amyloid-based neurodegenerative disease as associated with oxidative stress and inflammation. In this study we have analyzed the effect of chondroitin sulfate isolated from different species, in comparison with a new biotechnological unsulfated chondroitin, in the amyloid aggregation process of insulin, as well as the ability to prevent the formation of AGEs and related toxicity. The results have showed a determining role of chondroitin sulfate groups in modulating insulin amyloid aggregation. In addition, both sulfated and unsulfated chondroitins have shown protective properties against amyloid and AGEs-induced toxicity. These data are very relevant as a protective effect of these glycosaminoglycans in the AGE-induced toxicity was never observed before. Moreover, considering the issues related to the purity and safety of chondroitin from natural sources, this study suggests a new potential application for the biotechnological chondroitin.     

C-reactive protein down-regulates endothelial nitric oxide synthase expression and promotes apoptosis in endothelial progenitor cells through receptor for advanced glycation end-products

Objective

C-reactive protein (CRP), the prototypic marker of inflammation, has been shown to be an independent predictor of atherosclerosis. CRP can regulate receptor for advanced glycation end-products (RAGE) expression in endothelial progenitor cells (EPCs). Endothelial nitric oxide synthase (eNOS) deficiency is a pivotal event in atherogenesis. It is believed that decreased eNOS bioactivity occurs early in atherogenesis. Therefore, we tested the hypothesis that CRP can alter eNOS expression and promote apoptosis in EPCs through RAGE.

Methods and results

EPCs, isolated from bone marrow, were cultured in the presence or absence of LPS-free CRP (5, 10, 15, 20, and50 μg/ml). RAGE protein expression and siRNA were measured by flow cytometric analysis. PCR was used to detect eNOS mRNA expression. eNOS protein expression was measured by Western blot analysis. A spectrophotometer was used to assess eNOS activity. A modified Boyden's chamber was used to assess the migration of EPCs and the number of recultured EPCs was counted to measure adhesiveness. A MTT assay was used to determine proliferation. Apoptosis was evaluated by annexin V immunostaining and TUNEL staining. Co-culturing with CRP caused a significant down-regulation of eNOS expression, inhibited the proliferation, migration, and adhesion of EPCs, and induced EPC apoptosis. In addition, these effects were attenuated during RAGE protein expression blockade by siRNA.

Conclusions

CRP, at concentrations known to predict cardiovascular event, directly quenches the expression of eNOS and diminishes NO production, and may serve to impair EPC function and promote EPC apoptosis through RAGE. These data further support a direct role of CRP in the development and/or progression of atherosclerosis and indicate a new pathophysiologic mechanism of disturbed vascular adaptation in atherosclerosis.     

C-reactive protein decreases expression of VEGF receptors and neuropilins and inhibits VEGF165-induced cell proliferation in human endothelial cells

C-reactive protein (CRP) is associated with cardiovascular disease. However, its biological functions for the vascular system are largely unknown. The objective of this study was to determine whether CRP could affect endothelial cell proliferation and expression of VEGF receptors (VEGFRs) and/or neuropilins. Human coronary artery endothelial cells (HCAECs) treated with CRP showed a significant reduction of mRNA levels of VEGFR-2, VEGFR-3, NRP-1, and NRP-2 by 34%, 63%, 41%, and 43%, respectively, as compared to untreated control cells (p < 0.05) by real-time PCR analysis. In addition, VEGF165-induced cell proliferation was determined by [3H]thymidine incorporation and MTS assay as well as capillary-like tube formation on Matrigel. HCAECs pretreated with CRP significantly decreased VEGF165-induced [3H]thymidine incorporation by 73%, MTS absorbance by 44%, and capillary-like tube formation by 54% as compared to CRP-untreated cells (p < 0.05). These data demonstrate that CRP significantly attenuates VEGF165-induced HCAEC proliferation and capillary-like tube formation through downregulation of expression of VEGFRs and NRPs. This study suggests a new molecular mechanism underlying the adverse effect of CRP on the vascular system.     

Effects of advanced glycation end-products on the proliferation and differentiation of osteoblast-like cells

Dextran M20 was added to isolated rat liver mitochondria to mimic cytosolic macromolecules. Under these conditions, the morphological changes in the mitochondrial periphery that occur upon isolation of the organelle are restored, i.e. the volume of the intermembrane space decreases and the contact site frequency increases. The ADP routing from mitochondrial kinases at various locations was investigated by using the activities of oxidative phosphorylation and externally added pyruvate kinase as sensors for ADP transport into the matrix and extramitochondrial compartment, respectively. The studies reveal that a significant fraction of the ADP generated by either adenylate kinase in the intermembrane space or by outer membrane bound hexokinase isozyme I, is not accessible to extramitochondrial pyruvate kinase. Quantitative information on the ADP compartmentation in rat liver mitochondria was obtained by comparing the ADP supply from mitochondrial kinases to oxidative phosphorylation with that of non-bound, extramitochondrially located kinases. This approach allowed us to estimate the ADP diffusion gradients which were present across the outer membrane and between the compartment formed by bound hexokinase and the extramitochondrial compartment. In the presence of 10% dextran M20 these ADP gradients amounted to approximately 12 µM. The possible role of mitochondrial kinases in ADP transport into mitochondria in vivo is discussed. (Mol Cell Biochem 174: 43–51, 1997)