Genistein Prevents Apoptosis and Oxidative Stress Induced by Methylglyoxal in Endothelial Cells.

Glycolytic overload promotes accumulation of the highly reactive dicarbonyl compounds, resulting in harmful conditions called dicarbonyl stress. Methylglyoxal (MG) is a highly reactive dicarbonyl species and its accumulation plays a crucial pathophysiological role in diabetes and its vascular complications. MG cytotoxicity is mediated by reactive oxygen species (ROS) generation, a key event underlying the intracellular signaling pathways leading to inflammation and apoptosis. The identification of compounds able to inhibit ROS signaling pathways and counteract the MG-induced toxicity is a crucial step for developing new therapeutic strategies in the treatment of diabetic vascular complications. In this study, the effect of genistein, a natural soybean isoflavone, has been evaluated on MG-induced cytotoxicity in human endothelial cells. Our results show that genistein is able to counteract the MG-induced apoptosis by restraining ROS production, thus inhibiting the MAPK signaling pathways and caspase-3 activation. These findings identify a beneficial role for genistein, providing new insights for its potential clinical applications in preserving endothelial function in diabetic vascular complications.

Association between Urinary AGEs and Circulating miRNAs in Children and Adolescents with Overweight and Obesity from the Italian I.Family Cohort: A Pilot Study.

Modern dietary habits are linked to high exposure to Advanced Glycation End products (AGEs) mainly due to the dramatic increase in the consumption of highly processed foods in recent years. Body levels of these compounds vary with food intake and are almost interconnected with age and health status, formally embodying indicators of oxidative stress and inflammation in adults. However, the relationship between AGEs and health issues has not been definitively understood in children, and several pediatric investigations have produced conflicting evidence. Besides, despite extensive research, there are no universally accepted analytical techniques for measuring AGE levels in the human body, with several approaches available, each with its advantages and disadvantages. This pilot study aimed to investigate the association between urinary AGEs, measured using spectrofluorimetry-based assays, and circulating microRNAs (c-miRNAs) in a subsample (n = 22) of Italian children participating in the I.Family Study. Anthropometric measurements, biochemical markers, and miRNA profiles were assessed. The first indication of a relationship between urinary AGEs and c-miRNAs in the context of obesity was found. Specifically, four miRNAs, hsa-miR-10b-5p, hsa-miR-501-5p, hsa-miR-874-3p, and hsa-miR-2355-5p were significantly associated with levels of urinary AGEs. The association between AGEs, obesity, inflammation markers, and specific miRNAs highlights the complex interplay between these factors and their potential impact on cellular and tissue homeostasis. The discovery of altered c-miRNAs profiling has the potential to offer innovative methods for assessing early changes in the body's AGE pool and allow recognition of an increased risk of disease susceptibility, routinely undetected until metabolic complications are identified.

Overview of the Role of Vanillin in Neurodegenerative Diseases and Neuropathophysiological Conditions.

Nowadays, bioactive natural products play key roles in drug development due to their safety profile and strong antioxidant power. Vanillin is a natural phenolic compound found in several vanilla beans and widely used for food, cosmetic, and pharmaceutical products. Besides its industrial applications, vanillin possesses several beneficial effects for human health, such as antioxidant activity in addition to anti-inflammatory, anti-mutagenic, anti-metastatic, and anti-depressant properties. Moreover, vanillin exhibits neuroprotective effects on multiple neurological disorders and neuropathophysiological conditions. This study reviews the mechanisms of action by which vanillin prevents neuroinflammation and neurodegeneration in vitro and in vivo systems, in order to provide the latest views on the beneficial properties of this molecule in chronic neurodegenerative diseases and neuropathophysiological conditions.

Association between Urinary Advanced Glycation End Products and Subclinical Inflammation in Children and Adolescents: Results from the Italian I.Family Cohort.

Advanced Glycation End Products (AGEs) have been positively correlated with inflammation in adults, while inconsistent evidence is available in children. We evaluated the association between urinary AGEs, measured by fluorescence spectroscopy, and biomarkers of subclinical inflammation in 676 healthy children/adolescents (age 11.8 ± 1.6 years, M ± SD) from the Italian cohort of the I.Family project. Urinary fluorescent AGEs were used as independent variable and high-sensitivity C-reactive protein (hs-CRP) was the primary outcome, while other biomarkers of inflammation were investigated as secondary outcomes. Participants with urinary AGEs above the median of the study population showed statistically significantly higher hs-CRP levels as compared to those below the median (hs-CRP 0.44 ± 1.1 vs. 0.24 ± 0.6 mg/dL, M ± SD p = 0.002). We found significant positive correlations between urinary AGEs and hs-CRP (p = 0.0001), IL-15 (p = 0.001), IP-10 (p = 0.006), and IL-1Ra (p = 0.001). At multiple regression analysis, urinary AGEs, age, and BMI Z-score were independent variables predicting hs-CRP levels. We demonstrated for the first time, in a large cohort of children and adolescents, that the measurement of fluorescent urinary AGEs may represent a simple, noninvasive, and rapid technique to evaluate the association between AGEs and biomarkers of inflammation. Our data support a role of AGEs as biomarkers of subclinical inflammation in otherwise healthy children and adolescents.

Hydroxytyrosol Prevents Doxorubicin-Induced Oxidative Stress and Apoptosis in Cardiomyocytes.

Doxorubicin (Dox) is a highly effective chemotherapeutic agent employed in the handling of hematological and solid tumors. The effective use of Dox in cancer therapy has been seriously limited due to its well-known cardiotoxic side effects, mainly mediated by oxidative damage. Therefore, the identification of an effective and safe antagonist against Dox-induced cardiotoxicity remains a challenge. In this respect, as plant polyphenols have attracted considerable interest due to their antioxidant properties and good safety profile, hydroxytyrosol (HT), the major phenolic compound in olive oil, could be a potential candidate due to its remarkable antioxidant and anticancer powers. In this study, the effect of HT was tested on Dox-induced cardiotoxicity by using a combination of biochemical and cellular biology techniques. Interestingly, HT was able to counteract Dox-induced cytotoxicity in cardiomyocytes by acting on the SOD2 level and the oxidative response, as well as on apoptotic mechanisms mediated by Bcl-2/Bax. At the same time, HT did not to interfere with the antitumorigenic properties of Dox in osteosarcoma cells. This study identifies new, beneficial properties for HT and suggests that it might be a promising molecule for the development of additional therapeutic approaches aimed at preventing anthracycline-related cardiotoxicity and improving long-term outcomes in antineoplastic treatments.

Hydroxytyrosol Selectively Affects Non-Enzymatic Glycation in Human Insulin and Protects by AGEs Cytotoxicity.

Hydroxytyrosol (HT), the major phenolic compound in olive oil, is attracting increasing interest for its beneficial properties including a notable antioxidant and anti-inflammatory power. In this study, using a combination of biophysical and cell biology techniques, we have tested the role of HT in the formation of advanced glycation end-products (AGEs). AGEs have a key role in clinical sciences as they have been associated to diabetes, neurodegenerative and cardiovascular diseases. In addition, as the incidence of Alzheimer's disease (AD) is strongly increased in diabetic patients, AGE formation is supposed to be involved in the development of the pathological hallmarks of AD. Our data show that HT selectively inhibits protein glycation reaction in human insulin, and it is able to counteract the AGE-induced cytotoxicity in human neurotypical cells by acting on SIRT1 level and oxidative stress, as well as on inflammatory response. This study identifies new beneficial properties for HT and suggests it might be a promising molecule in protecting against the AGE-induced toxicity, a key mechanism underlying the development and progression of neurodegenerative disorders.

Understanding the Role of Protein Glycation in the Amyloid Aggregation Process.

Protein function and flexibility is directly related to the native distribution of its structural elements and any alteration in protein architecture leads to several abnormalities and accumulation of misfolded proteins. This phenomenon is associated with a range of increasingly common human disorders, including Alzheimer and Parkinson diseases, type II diabetes, and a number of systemic amyloidosis characterized by the accumulation of amyloid aggregates both in the extracellular space of tissues and as intracellular deposits. Post-translational modifications are known to have an active role in the in vivo amyloid aggregation as able to affect protein structure and dynamics. Among them, a key role seems to be played by non-enzymatic glycation, the most unwanted irreversible modification of the protein structure, which strongly affects long-living proteins throughout the body. This study provided an overview of the molecular effects induced by glycation on the amyloid aggregation process of several protein models associated with misfolding diseases. In particular, we analyzed the role of glycation on protein folding, kinetics of amyloid formation, and amyloid cytotoxicity in order to shed light on the role of this post-translational modification in the in vivo amyloid aggregation process.

Vanillin Prevents Doxorubicin-Induced Apoptosis and Oxidative Stress in Rat H9c2 Cardiomyocytes.

Doxorubicin (doxo) is an effective anticancer compound in several tumor types. However, as a consequence of oxidative stress induction and ROS overproduction, its high cardiotoxicity demands urgent attention. Vanillin possesses antioxidant, antiproliferative, antidepressant and anti-glycating properties. Therefore, we investigated the potential vanillin protective effects against doxo-induced cardiotoxicity in H9c2 cells. Using multiparametric approach, we demonstrated that vanillin restored both cell viability and damage in response to doxo exposure. Contextually, vanillin decreased sub-G1 appearance and caspase-3 and PARP1 activation, reducing the doxo-related apoptosis induction. From a mechanistic point of view, vanillin hindered doxo-induced ROS accumulation and impaired the ERK phosphorylation. Notably, besides the cardioprotective effects, vanillin did not counteract the doxo effectiveness in osteosarcoma cells. Taken together, our results suggest that vanillin ameliorates doxo-induced toxicity in H9c2 cells, opening new avenues for developing alternative therapeutic approaches to prevent the anthracycline-related cardiotoxicity and to improve the long-term outcome of antineoplastic treatment.

Hydroxytyrosol Inhibits Protein Oligomerization and Amyloid Aggregation in Human Insulin.

Hydroxytyrosol (HT), one of the main phenolic components of olive oil, has attracted considerable interest for its biological properties, including a remarkable antioxidant and anti-inflammatory power and, recently, for its ability to interfere with the amyloid aggregation underlying several human diseases. We report here a broad biophysical approach and cell biology techniques that allowed us to characterize the molecular mechanisms by which HT affects insulin amyloid aggregation and the related cytotoxicity. Our data show that HT is able to fully inhibit insulin amyloid aggregation and this property seems to be ascribed to the stabilization of the insulin monomeric state. Moreover, HT completely reverses the toxic effect produced by amyloid insulin aggregates in neuroblastoma cell lines by fully inhibiting the production of toxic amyloid species. These findings suggest that the beneficial effects of olive oil polyphenols, including HT, may arise from multifunctional activities and suggest possible a application of this natural compound in the prevention or treatment of amyloid-associated diseases.

Pinocembrin Protects from AGE-Induced Cytotoxicity and Inhibits Non-Enzymatic Glycation in Human Insulin.

Advanced glycation end products (AGEs) are the end products of the glycation reaction and have a great importance in clinical science for their association with oxidative stress and inflammation, which play a major role in most chronic diseases, such as cardiovascular disease, neurodegenerative diseases, and diabetes. Their pathogenic effects are generally induced by the interaction between AGEs and the receptor for advanced glycation end product (RAGE) on the cell surface, which triggers reactive oxygen species production, nuclear factor kB (NF-kB) activation, and inflammation. Pinocembrin, the most abundant flavonoid in propolis, has been recently proven to interfere with RAGE activation in Aß-RAGE-induced toxicity. In the present study, we investigated the ability of pinocembrin to interfere with RAGE signaling pathways activated by AGEs. Interestingly, pinocembrin was able to inhibit oxidative stress and NF-kB activation in cells exposed to AGEs. In addition, it was able to block caspase 3/7 and 9 activation, thus suggesting an active role of this molecule in counteracting AGE-RAGE-induced toxicity mediated by NF-kB signaling pathways. The ability of pinocembrin to affect the glycation reaction has been also tested. Our data suggest that pinocembrin might be a promising molecule in protecting from AGE-mediated pathogenesis.

Urinary sucrose and fructose to validate self-reported sugar intake in children and adolescents: results from the I.Family study.

PURPOSE: Excessive consumption of free sugar increases the risk for non-communicable diseases where a proper assessment of this intake is necessary to correctly estimate its association with certain diseases. Urinary sugars have been suggested as objective biomarkers for total and free sugar intake in adults but less is known about this marker in children and adolescents. Therefore, the aim of this exploratory study is to evaluate the relative validity of self-reported intake using urinary sugars in children and adolescents. METHODS: The study was conducted in a convenience subsample of 228 participants aged 5-18 years of the I.Family study that investigates the determinants of food choices, lifestyle and health in European families. Total, free and intrinsic sugar intake (g/day) and sugar density (g/1000 kcal) were assessed using 24-h dietary recalls (24HDRs). Urinary sucrose (USUC) and urinary fructose (UFRU) were measured in morning urine samples and corrected for creatinine excretion (USUC/Cr, UFRU/Cr). Correlation coefficients, the method of triads and linear regression models were used to investigate the relationship between intake of different types of sugar and urinary sugars. RESULTS: The correlation between usual sugar density calculated from multiple 24HDRs and the sum of USUC/Cr and UFRU/Cr (USUC/Cr + UFRU/Cr) was 0.38 (p < 0.001). The method of triads revealed validity coefficients for the 24HDR from 0.64 to 0.87. Linear regression models showed statistically significant positive associations between USUC/Cr + UFRU/Cr and the intake of total and free sugar. CONCLUSIONS: These findings support the relative validity of total and free sugar intake assessed by self-reported 24HDRs in children and adolescents.

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.

Insights into Insulin Fibril Assembly at Physiological and Acidic pH and Related Amyloid Intrinsic Fluorescence.

Human insulin is a widely used model protein for the study of amyloid formation as both associated to insulin injection amyloidosis in type II diabetes and highly prone to form amyloid fibrils in vitro. In this study, we aim to gain new structural insights into insulin fibril formation under two different aggregating conditions at neutral and acidic pH, using a combination of fluorescence, circular dichroism, Fourier-transform infrared spectroscopy, and transmission electron miscroscopy. We reveal that fibrils formed at neutral pH are morphologically different from those obtained at lower pH. Moreover, differences in FTIR spectra were also detected. In addition, only insulin fibrils formed at neutral pH showed the characteristic blue-green fluorescence generally associated to amyloid fibrils. So far, the molecular origin of this fluorescence phenomenon has not been clarified and different hypotheses have been proposed. In this respect, our data provide experimental evidence that allow identifying the molecular origin of such intrinsic property.

Vanillin Affects Amyloid Aggregation and Non-Enzymatic Glycation in Human Insulin.

Curcumin is known for its anti-inflammatory, antioxidant and anticancer activity, as well as for its ability to interfere with amyloid aggregation and non-enzymatic glycation reaction, that makes it an attractive potential drug. However, curcumin therapeutic use is limited because of its low systemic bioavailability and chemical stability as it undergoes rapid hydrolysis in physiological conditions. Recently, much attention has been paid to the biological properties of curcumin degradation products as potential bioactive molecules. Between them, vanillin, a natural vanilla extract, is a stable degradation product of curcumin that could be responsible for mediating its beneficial effects. We have analyzed the effect of vanillin, in comparison with curcumin, in the amyloid aggregation process of insulin as well as its ability to prevent the formation of the advanced glycation end products (AGEs). Employing biophysical, biochemical and cell based assays, we show that vanillin and curcumin similarly affect insulin amyloid aggregation promoting the formation of harmless fibrils. Moreover, vanillin restrains AGE formation and protects from AGE-induced cytotoxicity. Our novel findings not only suggest that the main health benefits observed for curcumin can be ascribed to its degradation product vanillin, but also open new avenues for developing therapeutic applications of curcumin degradation products.

The Role of Metal Binding in the Amyotrophic Lateral Sclerosis-Related Aggregation of Copper-Zinc Superoxide Dismutase.

Protein misfolding and conformational changes are common hallmarks in many neurodegenerative diseases involving formation and deposition of toxic protein aggregates. Although many players are involved in the in vivo protein aggregation, physiological factors such as labile metal ions within the cellular environment are likely to play a key role. In this review, we elucidate the role of metal binding in the aggregation process of copper-zinc superoxide dismutase (SOD1) associated to amyotrophic lateral sclerosis (ALS). SOD1 is an extremely stable Cu-Zn metalloprotein in which metal binding is crucial for folding, enzymatic activity and maintenance of the native conformation. Indeed, demetalation in SOD1 is known to induce misfolding and aggregation in physiological conditions in vitro suggesting that metal binding could play a key role in the pathological aggregation of SOD1. In addition, this study includes recent advances on the role of aberrant metal coordination in promoting SOD1 aggregation, highlighting the influence of metal ion homeostasis in pathologic aggregation processes.

Glycation in Demetalated Superoxide Dismutase 1 Prevents Amyloid Aggregation and Produces Cytotoxic Ages Adducts.

Superoxide dismutase 1 (SOD1) has been implicated with familial amyotrophic lateral sclerosis (fALS) through accumulation of protein amyloid aggregates in motor neurons of patients. Amyloid aggregates and protein inclusions are a common pathological feature of many neurological disorders in which protein aggregation seems to be directly related to neurotoxicity. Although, extensive studies performed on the aggregation process of several amyloidogenic proteins in vitro allowed the identification of many physiological factors involved, the molecular mechanisms underlying the formation of amyloid aggregates in vivo and in pathological conditions are still poorly understood. Post-translational modifications are known to affect protein structure and function and, recently, much attention has been devoted to the role played by non-enzymatic glycation in stimulating amyloid aggregation and cellular toxicity. In particular, glycation seems to have a determining role both in sporadic and familial forms of ALS and SOD1 has been shown to be glycated in vivo The aim of this study was to investigate the role of glycation on the amyloid aggregation process of both wild-type SOD1 and its ALS-related mutant G93A. To this aim, the glycation kinetics of both native and demetalated SOD have been followed using two different glycating agents, i.e., D-ribose and methylglyoxal. The effect of glycation on the structure and the amyloid aggregation propensity of native and ApoSOD has been also investigated using a combination of biophysical and biochemical techniques. In addition, the effect of SOD glycated species on cellular toxicity and reactive oxygen species (ROS) production has been evaluated in different cellular models. The results provided by this study contribute to clarify the role of glycation in amyloid aggregation and suggest a direct implication of glycation in the pathology of fALS.

Glycation of Wild-Type Apomyoglobin Induces Formation of Highly Cytotoxic Oligomeric Species.

Protein glycation is a non-enzymatic, irreversible modification of protein amino groups by reactive carbonyl species leading to the formation of advanced glycation end products (AGEs). Several proteins implicated in neurodegenerative diseases have been found to be glycated in vivo and the extent of glycation is related to the pathologies of the patients. Although it is now accepted that there is a direct correlation between AGEs formation and the development of neurodegenerative diseases related to protein misfolding and amyloid aggregation, several questions still remain unanswered: whether glycation is the triggering event or just an additional factor acting on the aggregation pathway. We have recently shown that glycation of the amyloidogenic W7FW14F apomyoglobin mutant significantly accelerates the amyloid fibrils formation providing evidence that glycation actively participates to the process. In the present study, to test if glycation can be considered also a triggering factor in amyloidosis, we evaluated the ability of different glycation agents to induce amyloid aggregation in the soluble wild-type apomyoglobin. Our results show that glycation covalently modifies apomyoglobin and induces conformational changes that lead to the formation of oligomeric species that are not implicated in amyloid aggregation. Thus, AGEs formation does not trigger amyloid aggregation in the wild-type apomyoglobin but only induce the formation of soluble oligomeric species able to affect cell viability. The molecular bases of cell toxicity induced by AGEs formed upon glycation of wild-type apomyoglobin have been also investigated.

The effect of glycosaminoglycans (GAGs) on amyloid aggregation and toxicity.

Amyloidosis is a protein folding disorder in which normally soluble proteins are deposited extracellularly as insoluble fibrils, impairing tissue structure and function. Charged polyelectrolytes such as glycosaminoglycans (GAGs) are frequently found associated with the proteinaceous deposits in tissues of patients affected by amyloid diseases. Experimental evidence indicate that they can play an active role in favoring amyloid fibril formation and stabilization. Binding of GAGs to amyloid fibrils occurs mainly through electrostatic interactions involving the negative polyelectrolyte charges and positively charged side chains residues of aggregating protein. Similarly to catalyst for reactions, GAGs favor aggregation, nucleation and amyloid fibril formation functioning as a structural templates for the self-assembly of highly cytotoxic oligomeric precursors, rich in ß-sheets, into harmless amyloid fibrils. Moreover, the GAGs amyloid promoting activity can be facilitated through specific interactions via consensus binding sites between amyloid polypeptide and GAGs molecules. We review the effect of GAGs on amyloid deposition as well as proteins not strictly related to diseases. In addition, we consider the potential of the GAGs therapy in amyloidosis.

Platelet-activating factor mediates the cytotoxicity induced by W7FW14F apomyoglobin amyloid aggregates in neuroblastoma cells.

W7FW14F apomyoglobin (W7FW14F ApoMb) amyloid aggregates induce cytotoxicity in SH-SY5Y human neuroblastoma cells through a mechanism not fully elucidated. Amyloid neurotoxicity process involves calcium dyshomeostasis and reactive oxygen species (ROS) production. Another key mediator of the amyloid neurotoxicity is Platelet-Activating Factor (PAF), an inflammatory phospholipid implicated in neurodegenerative diseases. Here, with the aim at evaluating the possible involvement of PAF signaling in the W7FW14F ApoMb-induced cytotoxicity, we show that the presence of CV3899, a PAF receptor (PAF-R) antagonist, prevented the detrimental effect of W7FW14F ApoMb aggregates on SH-SY5Y cell viability. Noticeably, we found that the activation of PAF signaling, following treatment with W7FW14F ApoMb, involves a decreased expression of the PAF acetylhydroase II (PAF-AH II). Interestingly, the reduced PAF-AH II expression was associated with a decreased acetylhydrolase (AH) activity and to an increased sphingosine-transacetylase activity (TA(S)) with production of N-acetylsphingosine (C2-ceramide), a well known mediator of neuronal caspase-dependent apoptosis. These findings suggest that an altered PAF catabolism takes part to the molecular events leading to W7FW14F ApoMb amyloid aggregates-induced cell death.