Lipidome Investigation of Carnosine Effect on Nude Mice Skin to Prevent UV-A Damage.

The lipid profile of skin is fundamental in the maintenance of the protective barrier against the external environment. Signaling and constitutive lipids of this large organ are involved in inflammation, metabolism, aging, and wound healing, such as phospholipids, triglycerides, FFA, and sphingomyelin. Skin exposure to ultraviolet (UV) radiation results in a photoaging process that is an accelerated form of aging. UV-A radiation deeply penetrates the dermis and promotes damage to DNA, lipids, and proteins by increasing the generation of reactive oxygen species (ROS). Carnosine, an endogenous ß-alanyl-L-histidine dipeptide, demonstrated antioxidant properties that prevent photoaging and modification of skin protein profiling, making carnosine a compelling ingredient to consider for use in dermatology. The aim of this research was to investigate the modification of skin lipidome after UV-A treatment in presence or not of topic administration of carnosine. Quantitative analyses based on high-resolution mass spectrometry of nude mice skin-extracted lipids resulted in several modifications of barrier composition after UV-A radiation, with or without carnosine treatment. In total, 328 out of 683 molecules showed significant alteration-262 after UV-A radiation and 126 after UV-A and carnosine treatment versus controls. Importantly, the increased oxidized TGs after UV-A radiation, responsible of dermis photoaging, were completely reverted by carnosine application to prevent the UV-A damage. Network analyses also showed that the production of ROS and the calcium and TNF signaling were modulated by UV-A and carnosine. In conclusion, lipidome analyses attested the carnosine activity to prevent the UV-A damage, reducing the lipid oxidation, the inflammation, and the dysregulation of lipid skin barrier.

Targeting Nrf2 and NF-κB Signaling Pathways in Cancer Prevention: The Role of Apple Phytochemicals.

Plant secondary metabolites, known as phytochemicals, have recently gained much attention in light of the "circular economy", to reutilize waste products deriving from agriculture and food industry. Phytochemicals are known for their onco-preventive and chemoprotective effects, among several other beneficial properties. Apple phytochemicals have been extensively studied for their effectiveness in a wide range of diseases, cancer included. This review aims to provide a thorough overview of the main studies reported in the literature concerning apple phytochemicals, mostly polyphenols, in cancer prevention. Although there are many different mechanisms targeted by phytochemicals, the Nrf2 and NF-κB signaling pathways are the ones this review will be focused on, highlighting also the existing crosstalk between these two systems.

The Effect of Carnosine on UVA-Induced Changes in Intracellular Signaling of Human Skin Fibroblast Spheroids.

Dermis fibroblasts are very sensitive to penetrating UVA radiation and induce photo-damage. To protect skin cells against this environmental damage, there is an urgent need for effective compounds, specifically targeting UVA-induced mitochondrial injury. This study aimed to analyze the effect of carnosine on the proteome of UVA-irradiated human skin fibroblast, cultured in a three-dimensional (3D) biological system recapitulating dermal compartment as a test system to investigate the altered cellular pathways after 48 h and 7 days of culture with or without carnosine treatment. The obtained results indicate that UVA dysregulates Oxidative Phosphorylation, the Fibrosis Signaling Pathway, Glycolysis I and Nrf2-mediated Oxidative Stress Response. Carnosine exercises provide a protective function against the harmful effects of UVA radiation by activating the Nrf2 pathway with the upregulations of some ROS-detoxifying enzymes such as the glutathione S-transferase (GST) protein family. Additionally, carnosine regulates the activation of the Epithelial Adherens Junction and Wound Healing Signaling Pathway by mediating the activation of structural proteins such as vinculin and zyxin as well as fibronectin 1 and collagen type XVIII alpha 1 chain against UVA-induced changes.

Novel insights about albumin in cardiovascular diseases: Focus on heart failure.

The Human Plasma Proteome has always been the most investigated compartment in proteomics-based biomarker discovery, and is considered the largest and deepest version of the human proteome, reflecting the state of the body in health and disease. Even if efforts have been always dedicated to the refinement of proteomic approaches to investigate more deeply the plasma proteome, it should not be forgotten that also highly abundant plasma proteins, like human serum albumin (HSA), often neglected in these studies, might provide fundamental physiological functions in plasma, and should be better considered. This review summarizes the important roles of HSA in the context of cardiovascular diseases (CVD), and in particular in heart failure. Notwithstanding much attention has been historically directed toward the association of HSA levels and CVD risk, the advances in the field of mass spectrometry research allow also a better characterization of the effects of oxidative modifications that could alter not only the structure but also the function of HSA.

Proteomic studies on apoB-containing lipoprotein in cardiovascular research: A comprehensive review.

The complexity of cardiovascular diseases (CVDs), which remains the leading cause of death worldwide, makes the current clinical pathway for cardiovascular risk assessment unsatisfactory, as there remains a substantial unexplained residual risk. Simultaneous assessment of a large number of plasma proteins may be a promising tool to further refine risk assessment, and lipoprotein-associated proteins have the potential to fill this gap. Technical advances now allow for high-throughput proteomic analysis in a reproducible and cost-effective manner. Proteomics has great potential to identify and quantify hundreds of candidate marker proteins in a sample and allows the translation from isolated lipoproteins to whole plasma, thus providing an individual multiplexed proteomic fingerprint. This narrative review describes the pathophysiological roles of atherogenic apoB-containing lipoproteins and the recent advances in their mass spectrometry-based proteomic characterization and quantitation for better refinement of CVD risk assessment.

N-Acetylcysteine Regenerates In Vivo Mercaptoalbumin.

Human serum albumin (HSA) represents the most abundant plasma protein, with relevant antioxidant activity due to the presence of the sulfhydryl group on cysteine at position 34 (Cys34), the latter being one of the major target sites for redox-dependent modifications leading to the formation of mixed disulfide linkages with low molecular weight thiols. Thiolated forms of HSA (Thio-HSA) may be useful as markers of an unbalanced redox state and as a potential therapeutic target. Indeed, we have previously reported that albumin Cys34 can be regenerated in vitro by N-Acetylcysteine (NAC) through a thiol-disulfide breaking mechanism, with a full recovery of the HSA antioxidant and antiplatelet activities. With this case study, we aimed to assess the ability of NAC to regenerate native mercaptoalbumin (HSA-SH) and the plasma antioxidant capacity in subjects with redox unbalance, after oral and intravenous administration. A placebo-controlled crossover study, single-blinded, was performed on six hypertensive subjects, randomized into two groups, on a one-to-one basis with NAC (600 mg/die) or a placebo, orally and intravenously administered. Albumin isoforms, HSA-SH, Thio-HSA, and glutathione levels were evaluated by means of mass spectrometry. The plasma antioxidant activity was assessed by a fluorimetric assay. NAC, orally administered, significantly decreased the Thio-HSA levels in comparison with the pre-treatment conditions (T0), reaching the maximal effect after 60 min (-24.7 ± 8%). The Thio-HSA reduction was accompanied by a concomitant increase in the native HSA-SH levels (+6.4 ± 2%). After intravenous administration of NAC, a significant decrease of the Thio-HSA with respect to the pre-treatment conditions (T0) was observed, with a maximal effect after 30 min (-68.9 ± 10.6%) and remaining significant even after 6 h. Conversely, no effect on the albumin isoforms was detected with either the orally or the intravenously administered placebo treatments. Furthermore, the total antioxidant activity of the plasma significantly increased after NAC infusion with respect to the placebo (p = 0.0089). Interestingly, we did not observe any difference in terms of total glutathione corrected for hemoglobin, ruling out any effect of NAC on the intracellular glutathione and supporting its role as a disulfide-breaking agent. This case study confirms the in vitro experiments and demonstrates for the first time that NAC is able to regenerate mercaptoalbumin in vivo, allowing us to hypothesize that the recovery of Cys34 content can modulate in vivo oxidative stress and, hopefully, have an effect in oxidative-based diseases.

Protein Profiling of a Cellular Model of NAFLD by Advanced Bioanalytical Approaches.

Advanced quantitative bioanalytical approaches in combination with network analyses allow us to answer complex biological questions, such as the description of changes in protein profiles under disease conditions or upon treatment with drugs. In the present work, three quantitative proteomic approaches-either based on labelling or not-in combination with network analyses were applied to a new in vitro cellular model of nonalcoholic fatty liver disease (NAFLD) for the first time. This disease is characterized by the accumulation of lipids, inflammation, fibrosis, and insulin resistance. Hepatic G2 cells were used as model, and NAFLD was induced by a complex of oleic acid and bovine albumin. The development of the disease was verified by lipid vesicle staining and by the increase in the expression of perilipin-2-a protein constitutively present in the vesicles during NAFLD. The nLC-MS/MS analyses of peptide samples obtained from three different proteomic approaches resulted in accurate and reproducible quantitative data of protein fold-change expressed in NAFLD versus control cells. The differentially regulated proteins were used to evaluate the involved and statistically enriched pathways. Network analyses highlighted several functional and disease modules affected by NAFLD, such as inflammation, oxidative stress defense, cell proliferation, and ferroptosis. Each quantitative approach allowed the identification of similar modulated pathways. The combination of the three approaches improved the power of statistical network analyses by increasing the number of involved proteins and their fold-change. In conclusion, the application of advanced bioanalytical approaches in combination with pathway analyses allows the in-depth and accurate description of the protein profile of an in vitro cellular model of NAFLD by using high-resolution quantitative mass spectrometry data. This model could be extremely useful in the discovery of new drugs to modulate the equilibrium NAFLD health state.

Erratum: Altomare et al. In-Depth AGE and ALE Profiling of Human Albumin in Heart Failure: Ex Vivo Studies. Antioxidants 2021, 10, 358.

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Immature Circulating SP-B, Bound to HDL, Represents an Early Sign of Smoke-Induced Pathophysiological Alterations.

Cigarette smoking is a major independent risk factor for cardiovascular diseases (CVD). The underlying mechanisms, however, are not clearly understood. Lungs are the primary route of exposure to smoke, with pulmonary cells and surfactant being the first structures directly exposed, resulting in the leakage of the immature proteoform of surfactant protein B (proSP-B). Herein, we evaluated whether proSP-B joined the cargo of high-density lipoprotein (HDL) proteins in healthy young subjects (n = 106) without any CVD risk factor other than smoking, and if HDL-associated proSP-B (HDL-SPB) correlated with pulmonary function parameters, systemic inflammation, and oxidative stress. At univariable analysis, HDL-SPB resulted significantly higher in smokers (2.2-fold, p < 0.001) than in non-smokers. No significant differences have been detected between smokers and non-smokers for inflammation, oxidation variables, and alveolar-capillary diffusion markers. In a multivariable model, HDL-SPB was independently associated with smoking. In conclusion, HDL-SPB is not only a precocious and sensitive index of the acute effects of smoke, but it might be also a potential causal factor in the onset of the vascular damage induced by modified HDL. These findings contribute to the emerging concept that the quality of the HDL proteome, rather than the quantity of particles, plays a central role in CVD risk protection.

In-Depth AGE and ALE Profiling of Human Albumin in Heart Failure: Ex Vivo Studies.

Advanced glycation end-products (AGEs) and advanced lipoxidation end-products (ALEs), particularly carboxymethyl-lysine (CML), have been largely proposed as factors involved in the establishment and progression of heart failure (HF). Despite this evidence, the current literature lacks the comprehensive identification and characterization of the plasma AGEs/ALEs involved in HF (untargeted approach). This work provides the first ex vivo high-resolution mass spectrometry (HR-MS) profiling of AGEs/ALEs occurring in human serum albumin (HSA), the most abundant protein in plasma, characterized by several nucleophilic sites and thus representing the main protein substrate for AGE/ALE formation. A set of AGE/ALE adducts in pooled HF-HSA samples was defined, and a semi-quantitative analysis was carried out in order to finally select those presenting in increased amounts in the HF samples with respect to the control condition. These adducts were statistically confirmed by monitoring their content in individual HF samples by applying a targeted approach. Selected AGEs/ALEs proved to be mostly CML derivatives on Lys residues (i.e., CML-Lys12, CML-Lys378, CML-Lys402), and one deoxy-fructosyl derivative on the Lys 389 (DFK-Lys 389). The nature of CML adducts was finally confirmed using immunological methods and in vitro production of such adducts further confirmed by mass spectrometry.

Multiplexed MRM-Based Proteomics Identified Multiple Biomarkers of Disease Severity in Human Heart Failure.

Heart failure (HF) is a complex disease due to the intricate interplay of several mechanisms, which therefore implies the need for a multimarker strategy to better personalize the care of patients with HF. In this study, we developed a targeted mass spectrometry approach based on multiple reaction monitoring (MRM) to measure multiple circulating protein biomarkers, involved in cardiovascular disease, to address their relevance in the human HF, intending to assess the feasibility of the workflow in the disease monitoring and risk stratification. In this study, we analyzed a total of 60 plasma proteins in 30 plasma samples from eight control subjects and 22 age- and gender- matched HF patients. We identified a panel of four plasma proteins, namely Neuropilin-2, Beta 2 microglobulin, alpha-1-antichymotrypsin, and complement component C9, that were more abundant in HF patients in relation to disease severity and pulmonary dysfunction. Moreover, we showed the ability of the combination of these candidate proteins to discriminate, with sufficient accuracy, HF patients from healthy subjects. In conclusion, we demonstrated the feasibility and potential of a proteomic workflow based on MRM mass spectrometry for the evaluation of multiple proteins in human plasma and the identification of a panel of biomarkers of HF severity.

Proteomics of Extracellular Vesicles: Update on Their Composition, Biological Roles and Potential Use as Diagnostic Tools in Atherosclerotic Cardiovascular Diseases.

Extracellular vesicles (EVs) are lipid-bound vesicles released from cells under physiological and pathological conditions. Basing on biogenesis, dimension, content and route of secretion, they can be classified into exosomes, microvesicles (MVs) and apoptotic bodies. EVs have a key role as bioactive mediators in intercellular communication, but they are also involved in other physiological processes like immune response, blood coagulation, and tissue repair. The interest in studying EVs has increased over the years due to their involvement in several diseases, such as cardiovascular diseases (CVDs), and their potential role as biomarkers in diagnosis, therapy, and in drug delivery system development. Nowadays, the improvement of mass spectrometry (MS)-based techniques allows the characterization of the EV protein composition to deeply understand their role in several diseases. In this review, a critical overview is provided on the EV's origin and physical properties, as well as their emerging functional role in both physiological and disease conditions, focusing attention on the role of exosomes in CVDs. The most important cardiac exosome proteomic studies will be discussed giving a qualitative and quantitative characterization of the exosomal proteins that could be used in future as new potential diagnostic markers or targets for specific therapies.

S-Thiolation Targets Albumin in Heart Failure.

Human serum albumin (HSA) is associated with several physiological functions, such as maintaining oncotic pressure and microvascular integrity, among others. It also represents the major and predominant antioxidant in plasma due to the presence of the Cys34 sulfhydryl group. In this study, we assessed qualitative and quantitative changes in HSA in patients with heart failure (HF) and their relationship with the severity of the disease. We detected by means of mass spectrometry a global decrease of the HSA content in the plasma of HF patients in respect to control subjects, a significant increase of thio-HSA with a concomitant decrease in the reduced form of albumin. Cysteine and, at a lesser extent, homocysteine represent the most abundant thiol bound to HSA. A strong inverse correlation was also observed between cysteine-HSA and peak VO2/kg, an index of oxygen consumption associated with HF severity. Moreover, in HL-1 cardiomyocytes incubated with H2O2, we showed a significant decrease of cell viability in cells treated with thio-HSA in respect to restored native-HSA. In conclusion, we found for the first time that S-thiolation of albumin is increased in the plasma of HF patients and induced changes in the structure and antioxidant function of HSA, likely contributing to HF progression.