Reactive Dicarbonyl Research Articles

Pyridoxamine ameliorates methylglyoxal-induced macrophage dysfunction to facilitate tissue repair in diabetic wounds.

Methylglyoxal (MGO) is a highly reactive dicarbonyl compound formed during hyperglycaemia. MGO combines with proteins to form advanced glycation end products (AGEs), leading to cellular dysfunction and organ damage. In type 2 diabetes mellitus (T2DM), the higher the plasma MGO concentration, the higher the lower extremity amputation rate. Here, we aimed to identify the mechanisms of MGO‐induced dysfunction. We observed that the accumulation of MGO‐derived AGEs in human diabetic wounds increased, whereas the expression of glyoxalase 1 (GLO1), a key metabolic enzyme of MGO, decreased. We show for the first time that topical application of pyridoxamine (PM), a natural vitamin B6 analogue, reduced the accumulation of MGO‐derived AGEs in the wound tissue of type‐2 diabetic mice, promoted the influx of macrophages in the early stage of tissue repair, improved the dysfunctional inflammatory response, and accelerated wound healing. In vitro, MGO damaged the phagocytic functions of M1‐like macrophages induced by lipopolysaccharide (LPS), but not those of M0‐like macrophages induced by PMA or of M2‐like macrophages induced by interleukins 4 (IL‐4) and 13 (IL‐13); the impaired phagocytosis of M1‐like macrophages was rescued by PM administration. These findings suggest that the increase in MGO metabolism in vivo might contribute to macrophage dysfunction, thereby affecting wound healing. Our results indicate that PM may be a novel therapeutic approach for treating diabetic wounds. MGO forms protein adducts that cause macrophage dysfunction. These adducts cause cell and organ dysfunction that is common in diabetes. Pyridoxamine scavenges MGO to ameliorate this dysfunction, promoting wound healing. Pyridoxamine could be used therapeutically to treat non‐healing diabetic wounds.

The Dual-Role of Methylglyoxal in Tumor Progression - Novel Therapeutic Approaches.

One of the hallmarks of cancer cells is their metabolic reprogramming, which includes the preference for the use of anaerobic glycolysis to produce energy, even in presence of normal oxygen levels. This phenomenon, known as “Warburg effect”, leads to the increased production of reactive intermediates. Among these Methylglyoxal (MGO), a reactive dicarbonyl known as the major precursor of the advanced glycated end products (AGEs), is attracting great attention. It has been well established that endogenous MGO levels are increased in several types of cancer, however the MGO contribution in tumor progression is still debated. Although an anti-cancer role was initially attributed to MGO due to its cytotoxicity, emerging evidence has highlighted its pro-tumorigenic role in several types of cancer. These apparently conflicting results are explained by the hormetic potential of MGO, in which lower doses of MGO are able to establish an adaptive response in cancer cells while higher doses cause cellular apoptosis. Therefore, the extent of MGO accumulation and the tumor context are crucial to establish MGO contribution to cancer progression. Several therapeutic approaches have been proposed and are currently under investigation to inhibit the pro-tumorigenic action of MGO. In this review, we provide an overview of the early and latest evidence regarding the role of MGO in cancer, in order to define its contribution in tumor progression, and the therapeutic strategies aimed to counteract the tumor growth.

Protective role of Clitoria ternatea L. flower extract on methylglyoxal-induced protein glycation and oxidative damage to DNA

BackgroundMethylglyoxal (MG) is a highly reactive dicarbonyl precursor for the formation of advanced glycation end products (AGEs) associated with age-related diseases, including diabetes and its complications. Clitoria ternatea L. flower has been reported to possess antioxidant and antiglycating properties. Evidence indicates that the extract of Clitoria ternatea L. flower inhibits fructose-induced protein glycation and oxidative damage to bovine serum albumin (BSA). However, there is no evidence to support the inhibitory effect of CTE against MG-mediated protein glycation and oxidative damage to protein and DNA. Therefore, the aim of the present study was to investigate whether C. ternatea flower extract (CTE) prevents MG-induced protein glycation and oxidative DNA damage.MethodsThe formation of fluorescent AGEs in BSA was evaluated using spectrofluorometer. The protein carbonyl and thiol group content were used for detecting protein oxidation. DNA strand breakage in a glycation model comprising of MG, lysine and Cu2+ or a free radical generator 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH) systems was investigated using gel electrophoresis. Generation of superoxide anions and hydroxyl radicals in the MG/lysine system was assessed by the cytochrome c reduction assay and thiobarbituric acid reactive substances assay, respectively. High performance liquid chromatography (HPLC) was used to measure the MG-trapping ability.ResultsIn the BSA/MG system, CTE (0.25–1 mg/mL) significantly inhibited the formation of fluorescent AGEs and protein oxidation by reducing protein carbonyl content as well as preventing the protein thiol depletion. The concentration of CTE at 0.125–1 mg/mL prevented oxidative DNA cleavage in MG/lysine and AAPH systems associated with the inhibition of superoxide anion and hydroxyl radical formation. It also directly trapped MG in a concentration-dependent manner, ranging from 15 to 43%.ConclusionsThe study findings suggest that the direct carbonyl trapping ability and the free radical scavenging activity of CTE are the underlying mechanisms responsible for the prevention of protein glycation and oxidative DNA damage.

Protein glycation – biomarkers of metabolic dysfunction and early-stage decline in health in the era of precision medicine

Protein glycation provides a biomarker in widespread clinical use, glycated hemoglobin HbA1c (A1C). It is a biomarker for diagnosis of diabetes and prediabetes and of medium-term glycemic control in patients with established diabetes. A1C is an early-stage glycation adduct of hemoglobin with glucose; a fructosamine derivative. Glucose is an amino group-directed glycating agent, modifying N-terminal and lysine sidechain amino groups. A similar fructosamine derivative of serum albumin, glycated albumin (GA), finds use as a biomarker of glycemic control, particularly where there is interference in use of A1C. Later stage adducts, advanced glycation endproducts (AGEs), are formed by the degradation of fructosamines and by the reaction of reactive dicarbonyl metabolites, such as methylglyoxal. Dicarbonyls are arginine-directed glycating agents forming mainly hydroimidazolone AGEs. Glucosepane and pentosidine, an intense fluorophore, are AGE covalent crosslinks. Cellular proteolysis of glycated proteins forms glycated amino acids, which are released into plasma and excreted in urine. Development of diagnostic algorithms by artificial intelligence machine learning is enhancing the applications of glycation biomarkers. Investigational glycation biomarkers are in development for: (i) healthy aging; (ii) risk prediction of vascular complications of diabetes; (iii) diagnosis of autism; and (iv) diagnosis and classification of early-stage arthritis. Protein glycation biomarkers are influenced by heritability, aging, decline in metabolic, vascular, renal and skeletal health, and other factors. They are applicable to populations of differing ethnicities, bridging the gap between genotype and phenotype. They are thereby likely to find continued and expanding clinical use, including in the current era of developing precision medicine, reporting on multiple pathogenic processes and supporting a precision medicine approach.

The Isothiocyanate Sulforaphane Depends on the Nrf2/γ-GCL/GSH Axis to Prevent Mitochondrial Dysfunction in Cells Exposed to Methylglyoxal.

Methylglyoxal (MG) is a reactive dicarbonyl presenting both endogenous (e.g. glycolysis) and exogenous (e.g. food cooking) sources. MG induces neurotoxicity, at least in part, by affecting mitochondrial function, including a decline in the oxidative phosphorylation (OXPHOS) system activity, bioenergetics failure, and redox disturbances. Sulforaphane (SFN) is an isothiocyanate found mainly in cruciferous vegetables and exerts antioxidant and anti-inflammatory effects in mammalian cells. SFN also decreases mitochondrial vulnerability to several chemical stressors. SFN is a potent activator of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), which is a master regulator of the mammalian redox biology. Here, we have investigated whether and how SFN would be able to prevent the MG-induced mitochondrial collapse in the human neuroblastoma SH-SY5Y cells. The cells were exposed to SFN at 5µM for 24h prior to the administration of MG at 500µM for additional 24h. We found that SFN prevented the MG-induced OXPHOS dysfunction and mitochondrial redox impairment. SFN stimulated the activity of the enzyme γ-glutamylcysteine ligase (γ-GCL), leading to increased synthesis of glutathione (GSH). Inhibition of γ-GCL with buthionine sulfoximine (BSO) or silencing of Nrf2 using small interfering RNA (siRNA) against this transcription factor reduced the levels of GSH and abolished the mitochondrial protection promoted by SFN in the MG-treated cells. Thus, SFN protected mitochondria of the MG-challenged cells by a mechanism involving the Nrf2/γ-GCL/GSH axis.

Antioxidant and antiglycation effects of cucurbita argyrosperma seeds polysaccharide

Background: Diabetes is related with oxidative stress caused by free radicals and glycation of the proteins. Objectives: This study aimed to isolate a polysaccharide (PLY) from Cucurbita argyrosperma seeds and determine their antioxidant and antiglycation effects. Materials and Methods: The PLY fraction was isolated by hot water extraction and purified by ion-exchange chromatography (A103S). La antioxidant capacities were evaluated by using lipid peroxidation, superoxide dismutase, glutathione peroxidase (GSH-Px), catalase, and malondialdehyde activities, and intracellular reactive oxygen species (ROS) with stimulation H2O2 in pancreatic β INS-1 cells. In addition, scavenging activities of PLY were estimated in 2,2 diphenyl-1-picrylhydrazyl radical, 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid, β-carotene-linoleic acid, chelating activity, ferric reducing power, hydroxyl radical, nitric oxide radical, and superoxide anion radical assays. Therefore, the protective effect of PLY on reducing sugar-induced bovine serum albumin glycation was examined. Results: PLY consisted of mannose, galactose, and glucose in the molar ratio of 1.12:6.12:5.60 with an average molecular weight of 102326 Da. PLY had a pronounced radical scavenging potential as well as total antioxidant effect and prevented the cells ROS generation and increased the level of antioxidant enzymes. PLY inhibited the formation of carboxymethyllysine and fructosamine, prevented protein oxidation increasing the level of thiol, and decreasing protein carbonyl level, interacts with reactive dicarbonyl compounds (methylglyoxal), and decreased amyloid cross β-structure level. Conclusion: These results indicate that PLY enhanced antioxidant capacity and prevented oxidative stress and AGEs progression in its different stages. consequently, had promising effects on diabetes and associated disorders.

Expression dynamics of glyoxalase genes under high temperature stress in plants

Heat stress, owing to the recent climate change events, has emerged as one of the major environmental factors posing a grave threat to plant survival and crop productivity. Oxidative damage due to increased generation of reactive oxygen and dicarbonyl species in response to heat, is one of the serious consequences of high temperature stress. Methylglyoxal (MG), is one such cytotoxic dicarbonyl metabolite whose levels are known to increase in response to high temperature and various other stress conditions in plants. In fact, it is considered as a general consequence of stress in plants. The synergistic co-activation of reactive oxygen species scavenging and the MG detoxification pathways thus, plays a key role in regulation of stress responses for plant acclimation. Glyoxalase enzymes are the major MG detoxifying proteins in the living organisms. In plants, glyoxalases are encoded as multigenic families, known to be involved in plant growth and development, although, chiefly associated with multiple stress inducible behaviour and tolerance. While their role in salinity, drought and heavy metal stresses has been well-studied, their significance under high temperature stress has received limited coverage. In this review, we emphasize on the dynamic role of glyoxalases under high temperature stress for conferring thermotolerance. Through analysis of heat responsive transcriptomes and proteomes of various plants, we provide evidence for a hitherto, unexplored role of glyoxalases as a mechanism of mounting thermotolerance responses. We also, show that ‘priming’ induced stress/cross stress tolerance in plants involves the activation of glyoxalase pathway.

Glycation-mediated protein crosslinking and stiffening in mouse lenses are inhibited by carboxitin in vitro.

Proteins in the eye lens have negligible turnover and therefore progressively accumulate chemical modifications during aging. Carbonyls and oxidative stresses, which are intricately linked to one another, predominantly drive such modifications. Oxidative stress leads to the loss of glutathione (GSH) and ascorbate degradation; this in turn leads to the formation of highly reactive dicarbonyl compounds that react with proteins to form advanced glycation end products (AGEs). The formation of AGEs leads to the crosslinking and aggregation of proteins contributing to lens aging and cataract formation. To inhibit AGE formation, we developed a disulfide compound linking GSH diester and mercaptoethylguanidine, and we named it carboxitin. Bovine lens organ cultured with carboxitin showed higher levels of GSH and mercaptoethylguanidine in the lens nucleus. Carboxitin inhibited erythrulose-mediated mouse lens protein crosslinking, AGE formation and the formation of 3-deoxythreosone, a major ascorbate-derived AGE precursor in the human lens. Carboxitin inhibited the glycation-mediated increase in stiffness in organ-cultured mouse lenses measured using compressive mechanical strain. Delivery of carboxitin into the lens increases GSH levels, traps dicarbonyl compounds and inhibits AGE formation. These properties of carboxitin could be exploited to develop a therapy against the formation of AGEs and the increase in stiffness that causes presbyopia in aging lenses.

Abstract 13548: Dicarbonyl Stress, Mitochondrial Impairments, and Right Ventricular Dysfunction in Pulmonary Arterial Hypertension

Introduction: Right ventricular dysfunction (RVD) is the leading cause of mortality in pulmonary arterial hypertension (PAH), but the molecular mediators of RVD are incompletely understood. In PAH, RV glucose uptake is inversely proportional to RV function, which is a consequence of increased glycolytic flux due to mitochondrial dysfunction. Methylglyoxal (MG) is a highly reactive dicarbonyl that is generated during glycolysis. MG can deleteriously modulate protein function via post-translational modifications. MG is metabolized by two key enzymes: glyoxalase (Glo) 1 and 2. Finally, DJ-1 is a deglycase enzyme that reverses protein-glycation. The regulation of protein-glycation, the glyoxalase system, and DJ-1 in RVD due to PAH are unexplored. Methods: We analyzed protein-glycation, Glo-1/2, and DJ-1 expression in RV extracts from control, monocrotaline (MCT), and pulmonary artery banded (PAB) rats. The effects of protein-glycation on mitochondrial function in H9c2 cardiomyocytes were probed using Seahorse analysis. Finally, we examined the link between protein-glycation and RV function in PAH patients using hemoglobin-A1C as a surrogate measure of protein-glycation. Results: In MCT RVs, protein-glycation was increased (3.2±0.9-fold), Glo-1 (48±7%) was decreased, while Glo-2 (1.4±0.1-fold) and DJ-1 (1.5±0.4-fold) were increased. Conversely, in PAB rats, protein-glycation (1.4±0.3-fold) was not as drastically elevated. Moreover, Glo-1 (1.7±0.3-fold), Glo-2 (1.6±0.2-fold change), and DJ-1 (2.3±0.3-fold) levels were all increased. In H9c2 cardiomyocytes, MG treatment induced protein-glycation and impaired mitochondrial function. Finally, hemoglobin-A1C levels were inversely associated with RV contractility in PAH patients. Conclusions: Increased protein-glycation results in impaired mitochondrial function in vitro and is associated with more severe RV dysfunction in rodent and human PAH.

Abstract 16939: Metabolic and Electrophysiological Alterations Underlie Proarrhythmic Properties of Highly Reactive Isolevuglandins in Atrial Cardiomyocytes

Background: Hypertension is one of the most common risk factors for atrial fibrillation (AF), although the precise cellular and molecular mechanism(s) by which hypertension leads to AF are not well understood. Isolevuglandins (IsoLGs) are highly reactive dicarbonyl products of lipid peroxidation responsible for a major component of oxidative stress-related injury. In a mouse model of hypertension, we recently demonstrated that IsoLGs are elevated in hypertensive mouse atria and that an IsoLG scavenger reduced both IsoLG burden and AF susceptibility. Hypothesis: In this study, we hypothesized that IsoLGs can promote AF by inducing proarrhythmic metabolic and electrophysiologic (EP) changes in atrial cardiomyocytes. Methods and Results: Using standard patch clamp methods, we found significant changes in action potential properties of isolated mouse atrial cardiomyocytes exposed to IsoLGs (1μM, n=15 cells), including elevation of resting membrane potential, shortening of APD and reduction of V max . Acute IsoLG treatment led to a reduction of intracellular ATP production in atrial HL-1 cardiomyocytes, as measured by using a luminescence assay. Employing TMRM and Mitotracker Green staining for confocal and high-throughput screening (HTS) live-cell imaging assays, we also found that IsoLGs decreased mitochondrial membrane potential (compared to control, TMRM fluorescence decreased by 23%, 28%, 36% and 42%, respectively, when exposed to 0.01, 0.1, 0.5 and 1μM concentrations of IsoLG) accompanied by increased apoptosis (Cell Event Caspase-3/7 Green Detection Reagent) in a concentration-dependent manner, suggesting a prolonged mitochondrial transition pore opening. Moreover, cell metabolism assays performed using Agilent’s Seahorse XF96 extracellular flux analyzer revealed that IsoLGs exert a concentration dependent decrease in basal oxygen consumption rate and ATP production in HL-1 atrial cardiomyocytes. Conclusion: Together, these findings indicate that IsoLGs promote proarrhythmic EP and mitochondrial effects in atrial cells and thus may provide a novel therapeutic target for AF.

Plasma Methylglyoxal Levels Are Associated With Amputations and Mortality in Severe Limb Ischemia Patients With and Without Diabetes.

Diabetes is a risk factor for severe limb ischemia (SLI), a condition associated with high mortality, morbidity, and limb loss. The reactive glucose-derived dicarbonyl methylglyoxal (MGO) is a major precursor for advanced glycation end products (AGEs) and a potential driver of cardiovascular disease. We investigated whether plasma MGO levels are associated with poor outcomes in SLI. We measured plasma levels of MGO, free AGEs, and d-lactate, the detoxification end product of MGO, with ultraperformance liquid chromatography-tandem mass spectrometry at baseline in 160 patients (64.8 ± 13.3 years, 67.5% male, 37.5% with diabetes) with no-option SLI and recorded major adverse outcomes (n = 86, comprising n = 53 deaths and n = 49 amputations [first event counted]) over the 5-year follow-up. Data were analyzed with linear or Cox regression, after Ln-transformation of the independent variables, adjusted for sex, age, trial arm, diabetes, estimated glomerular filtration rate, systolic blood pressure, cholesterol levels, and BMI. Associations are reported per 1 SD plasma marker. Higher plasma MGO levels were associated with more adverse outcomes (relative risk 1.44; 95% CI 1.11-1.86) and amputations separately (1.55; 1.13-2.21). We observed a similar but weaker trend for mortality (1.28; 0.93-1.77). The MGO-derived AGE Nε-(carboxyethyl)lysine was also associated with more adverse outcomes (1.46; 1.00-2.15) and amputations (1.71; 1.04-2.79). d-Lactate was not associated with adverse incident outcomes. Higher plasma MGO levels were also associated with more inflammation and white blood cells and fewer progenitor cells. Plasma MGO levels are associated with adverse outcomes in SLI. Future studies should investigate whether MGO-targeting therapies improve outcomes in SLI.

Rabbit as an Aging Model in Reproduction: Advanced Maternal Age Alters GLO1 Expression in the Endometrium at the Time of Implantation

Advanced maternal age is associated with adverse pregnancy outcomes and the decline of female fertility in mammals. A potential reason for reduced fertility is metabolic changes due to protein modifications by advanced glycation end products. To elucidate the aging process in female reproduction, we analysed a key enzyme for detoxification of reactive dicarbonyls, the glyoxalase 1 (GLO1), in reproductive organs and blastocysts of young and old rabbits at the preimplantation stage. At day 6 post coitum, uterine, oviductal, ovarian tissue and blastocysts from young (16–20 weeks) and old rabbits (>108 weeks) were characterised for GLO1 expression. GLO1 amounts, enzymatic activity and localisation were quantified by qPCR, Simple Western, activity assay and immunohistochemistry. The GLO1 enzyme was present and active in all reproductive tract organs in a cell-type-specific pattern. Ovarian follicle and uterine epithelial cells expressed GLO1 to a high extent. In tertiary follicles, GLO1 expression increased, whereas it decreased in the endometrium of old rabbits at day 6 of pregnancy. In blastocysts of old animals, GLO1 expression remained unchanged. In early pregnancy, advanced maternal age leads to modified GLO1 expression in ovarian follicles and the endometrium, indicating an altered metabolic stress response at the preimplantation stage in older females.

Effect of glyoxal and 1-methylisatin on stress-induced fibrillation of Hen Egg White Lysozyme: Insight into the anti-amyloidogenic property of the compounds with possible therapeutic implications

Systemic amyloidosis is a hereditary disorder that mostly arises as a result of specific point mutations to the wild type gene of lysozyme, forming mutant lysozyme variants leading to aggregation of the protein. The small monomeric protein Hen Egg White Lysozyme (HEWL) is a structural homolog of Human Lysozyme and is widely used as a model protein to investigate protein aggregation. In the present study, we have investigated the effect of 1-methylisatin, an indole derivative and glyoxal, a reactive dicarbonyl compound, on stress-induced aggregation of HEWL. Interaction of the compounds with HEWL induced changes in structure and surface hydrophobicity of the protein as evident from CD spectroscopy, tryptophan fluorescence and ANS binding studies. Additional experiments (Thioflavin T fluorescence, AFM imaging and DLS studies) demonstrate that stress induces amyloid-like fibrillation of HEWL, however, prior modification of the protein with glyoxal or 1-methylisatin significantly reduces its susceptibility to aggregation. High resolution mass spectrometric analysis indicated that 1-methylisatin primarily complexes with the protein in the form of a dimer. On the other hand, glyoxal-mediated modification of the protein induces formation of glycated adducts (carboxymethyllysine, hydroimidazolone). The results highlight possible clinical implications of the compounds in treatment of systemic amyloidosis and protein conformational disorder.

Mitochondrial Isolevuglandins Contribute to Vascular Oxidative Stress and Mitochondria-Targeted Scavenger of Isolevuglandins Reduces Mitochondrial Dysfunction and Hypertension.

Hypertension remains a major health problem in Western Societies, and blood pressure is poorly controlled in a third of patients despite use of multiple drugs. Mitochondrial dysfunction contributes to hypertension, and mitochondria-targeted agents can potentially improve treatment of hypertension. We have proposed that mitochondrial oxidative stress produces reactive dicarbonyl lipid peroxidation products, isolevuglandins, and that scavenging of mitochondrial isolevuglandins improves vascular function and reduces hypertension. To test this hypothesis, we have studied the accumulation of mitochondrial isolevuglandins-protein adducts in patients with essential hypertension and Ang II (angiotensin II) model of hypertension using mass spectrometry and Western blot analysis. The therapeutic potential of targeting mitochondrial isolevuglandins was tested by the novel mitochondria-targeted isolevuglandin scavenger, mito2HOBA. Mitochondrial isolevuglandins in arterioles from hypertensive patients were 250% greater than in arterioles from normotensive subjects, and ex vivo mito2HOBA treatment of arterioles from hypertensive subjects increased deacetylation of a key mitochondrial antioxidant, SOD2 (superoxide dismutase 2). In human aortic endothelial cells stimulated with Ang II plus TNF (tumor necrosis factor)-α, mito2HOBA reduced mitochondrial superoxide and cardiolipin oxidation, a specific marker of mitochondrial oxidative stress. In Ang II-infused mice, mito2HOBA diminished mitochondrial isolevuglandins-protein adducts, raised Sirt3 (sirtuin 3) mitochondrial deacetylase activity, reduced vascular superoxide, increased endothelial nitric oxide, improved endothelium-dependent relaxation, and attenuated hypertension. Mito2HOBA preserved mitochondrial respiration, protected ATP production, and reduced mitochondrial permeability pore opening in Ang II-infused mice. These data support the role of mitochondrial isolevuglandins in endothelial dysfunction and hypertension. We conclude that scavenging of mitochondrial isolevuglandins may have therapeutic potential in treatment of vascular dysfunction and hypertension.

Scavenging Reactive Lipids to Prevent Oxidative Injury.

Oxidative injury due to elevated levels of reactive oxygen species is implicated in cardiovascular diseases, Alzheimer's disease, lung and liver diseases, and many cancers. Antioxidant therapies have generally been ineffective at treating these diseases, potentially due to ineffective doses but also due to interference with critical host defense and signaling processes. Therefore, alternative strategies to prevent oxidative injury are needed. Elevated levels of reactive oxygen species induce lipid peroxidation, generating reactive lipid dicarbonyls. These lipid oxidation products may be the most salient mediators of oxidative injury, as they cause cellular and organ dysfunction by adducting to proteins, lipids, and DNA. Small-molecule compounds have been developed in the past decade to selectively and effectively scavenge these reactive lipid dicarbonyls. This review outlines evidence supporting the role of lipid dicarbonyls in disease pathogenesis, as well as preclinical data supporting the efficacy of novel dicarbonyl scavengers in treating or preventing disease.

Deciphering the Nature of Caffeic Acid to Inhibit the HSA Aggregation Induced by Glyoxal.

Under certain circumstances, the path for protein folding deviates and attains an alternative path forming misfolded states, which are the key precursors for protein aggregation. Protein aggregation is associated with variety of diseases and leads to the cytotoxicity. These protein aggregate related diseases have been untreated so far. However, extensive attempts have been applied to develop anti-aggregating agents as possible approaches to overcome protein aggregation. Different types of substances have been reported to halt or decrease the formation of ordered protein aggregates both in vitro and in vivo, such as polyphenols and metal ions. In the present study the in vitro aggregation of human serum albumin (HSA) by using a reactive dicarbonyl glyoxal has been investigated, simultaneously an attempt has been done to inhibit the glyoxal (GO) induced aggregation of (HSA) by caffeic acid (CA). Different methods have been employed to investigate the process, fluorescence spectroscopy, circular dichroism, cango red binding assay, thioflavin T dye binding, turbidimetric analysis, docking study and transmission electron microscopy. Results have shown that elevated concentration of GO forms aggregates of HSA, and the activity of CA suggested the possibility of inhibiting the HSA aggregation at higher concentrations, and this compound was found to have an anti-aggregation property. The present study explained that micro molar concentrations of CA inhibits the aggregation of HSA and showed pronounced anti-aggregation effect at increasing concentrations in the presence of GO which is elevated in diabetic and hyperglycaemia conditions.

CD44, a Predominant Protein in Methylglyoxal-Induced Secretome of Muscle Cells, is Elevated in Diabetic Plasma.

Methylglyoxal (MG), a glycolytic intermediate and reactive dicarbonyl, is responsible for exacerbation of insulin resistance and diabetic complication. In this study, MG-induced secretome of rat muscle cells was identified and relatively quantified by SWATH-MS. A total of 643 proteins were identified in MG-induced secretome, of which 82 proteins were upregulated and 99 proteins were downregulated by more than 1.3-fold in SWATH analysis. Further, secretory proteins from the classical secretory pathway and nonclassical secretory pathway were identified using SignalP and SecretomeP, respectively. A total of 180 proteins were identified with SignalP, and 113 proteins were identified with SecretomeP. The differentially expressed proteins were functionally annotated by KEGG pathway analysis using Cytoscape software with plugin clusterMaker. The differentially expressed proteins were found to be involved in various pathways like extracellular matrix (ECM)–receptor interaction, leukocyte transendothelial migration, fluid shear stress and atherosclerosis, complement and coagulation cascades, and lysosomal pathway. Since the MG levels are high in diabetic conditions, the presence of MG-induced secreted proteins was inspected by profiling human plasma of healthy and diabetic subjects (n = 10 each). CD44, a predominant MG-induced secreted protein, was found to be elevated in the diabetic plasma and to have a role in the development of insulin resistance.

Promotion of Mitochondrial Protection by Emodin in Methylglyoxal-Treated Human Neuroblastoma SH-SY5Y Cells: Involvement of the AMPK/Nrf2/HO-1 Axis.

Mitochondrial dysfunction is part of the mechanism of several human diseases. This negative circumstance may be induced by certain toxicants, as methylglyoxal (MG). MG is a reactive dicarbonyl presenting both endogenous and exogenous sources and is also able to induce protein cross-linking and glycation. Emodin (EM; 1,3,8-trihydroxy-6-methylanthracene-9,10-dione; C15H10O5) is a cytoprotective agent. Nonetheless, it was not previously demonstrated whether EM would be able to promote mitochondrial protection in cells challenged with MG. Therefore, we investigated here whether and how EM would prevent the MG-induced mitochondrial collapse in the human neuroblastoma SH-SY5Y cells. We found that a pretreatment (for 4h) with EM at 40μM prevented the MG-induced mitochondrial dysfunction (i.e., decreased activity of the complexes I and V, reduced adenosine triphosphate levels, and loss of mitochondrial membrane potential) in the SH-SY5Y cells. EM also prevented the redox impairment induced by MG in mitochondrial membranes. Inhibiting the adenosine monophosphate-activated protein kinase (AMPK) or silencing of the nuclear factor erythroid 2-related factor 2 (Nrf2), transcription factor abolished the EM-induced protection. Inhibition of heme oxygenase-1 (HO-1) also blocked the EM-induced mitochondrial protection. Therefore, EM protected the mitochondria by a mechanism dependent on the AMPK/Nrf2/HO-1 signaling pathway in MG-challenged SH-SY5Y cells.

Abstract P074: Mitochondrial Isolevuglandins Contribute To Vascular Oxidative Stress And Mitochondria-targeted Scavenger Of Isolevuglandins Reduces Mitochondrial Dysfunction And Hypertension

Hypertension remains a major health problem in Western Societies, and blood pressure is poorly controlled in a third of patients despite use of multiple drugs. Mitochondrial dysfunction contributes to hypertension and mitochondria-targeted agents can potentially improve treatment of hypertension. We have proposed that mitochondrial oxidative stress produces reactive dicarbonyl lipid peroxidation products isolevuglandins (isoLGs) and that scavenging of mitochondrial isoLG improves vascular function and reduces hypertension. To test this hypothesis, we have studied the accumulation of mitochondrial isoLG-protein adducts in human patients with essential hypertension and angiotensin II mouse model of hypertension using mass spectrometry and Western blot analysis. The therapeutic potential of targeting mitochondrial isoLG was tested by the novel mitochondria-targeted isoLG scavenger, mito2HOBA. Mitochondrial isoLG in arterioles isolated from hypertensive patients were 250% greater than in arterioles from normotensive subjects, and ex vivo mito2HOBA treatment of arterioles from hypertensive subjects improved deacetylation of a key mitochondrial antioxidant, superoxide dismutase 2 (SOD2). In human aortic endothelial cells, mito2HOBA diminished mitochondrial superoxide and inhibited cardiolipin oxidation, a specific marker of mitochondrial oxidative stress. In angiotensin II-infused mice, mito2HOBA prevented accumulation of mitochondrial isoLG-protein adducts, improved Sirt3 mitochondrial deacetylase activity, reduced vascular superoxide, increased endothelial nitric oxide, improved endothelium-dependent relaxation, and attenuated hypertension. Mito2HOBA preserved mitochondrial respiration, protected ATP production, and reduced mitochondrial permeability pore opening in angiotensin II-infused mice. These data support the role of mitochondrial isoLGs in endothelial dysfunction and hypertension. We conclude that scavenging of mitochondrial isoLGs may have therapeutic potential in treatment of vascular dysfunction and hypertension.

Scavenging of reactive dicarbonyls with 2-hydroxybenzylamine reduces atherosclerosis in hypercholesterolemic Ldlr\u2212/\u2212 mice

Lipid peroxidation generates reactive dicarbonyls including isolevuglandins (IsoLGs) and malondialdehyde (MDA) that covalently modify proteins. Humans with familial hypercholesterolemia (FH) have increased lipoprotein dicarbonyl adducts and dysfunctional HDL. We investigate the impact of the dicarbonyl scavenger, 2-hydroxybenzylamine (2-HOBA) on HDL function and atherosclerosis in Ldlr−/− mice, a model of FH. Compared to hypercholesterolemic Ldlr−/− mice treated with vehicle or 4-HOBA, a nonreactive analogue, 2-HOBA decreases atherosclerosis by 60% in en face aortas, without changing plasma cholesterol. Ldlr−/− mice treated with 2-HOBA have reduced MDA-LDL and MDA-HDL levels, and their HDL display increased capacity to reduce macrophage cholesterol. Importantly, 2-HOBA reduces the MDA- and IsoLG-lysyl content in atherosclerotic aortas versus 4-HOBA. Furthermore, 2-HOBA reduces inflammation and plaque apoptotic cells and promotes efferocytosis and features of stable plaques. Dicarbonyl scavenging with 2-HOBA has multiple atheroprotective effects in a murine FH model, supporting its potential as a therapeutic approach for atherosclerotic cardiovascular disease.