Bile Acids and Bilirubin Role in Oxidative Stress and Inflammation in Cardiovascular Diseases.

Bile acids (BAs) and bilirubin, primarily known for their role in lipid metabolism and as heme catabolite, respectively, have been found to have diverse effects on various physiological processes, including oxidative stress and inflammation. Indeed, accumulating evidence showed that the interplay between BAs and bilirubin in these processes involves intricate regulatory mechanisms mediated by specific receptors and signaling pathways under certain conditions and in specific contexts. Oxidative stress plays a significant role in the development and progression of cardiovascular diseases (CVDs) due to its role in inflammation, endothelial dysfunction, hypertension, and other risk factors. In the cardiovascular (CV) system, recent studies have suggested that BAs and bilirubin have some opposite effects related to oxidative and inflammatory mechanisms, but this area of research is still under investigation. This review aims to introduce BAs and bilirubin from a biochemical and physiological point of view, emphasizing their potential protective or detrimental effects on CVDs. Moreover, clinical studies that have assessed the association between BAs/bilirubin and CVD were examined in depth to better interpret the possible link between them.

The Use of Microbial Modifying Therapies to Prevent Psoriasis Exacerbation and Associated Cardiovascular Comorbidity.

Psoriasis has emerged as a systemic disease characterized by skin and joint manifestations as well as systemic inflammation and cardiovascular comorbidities. Many progresses have been made in the comprehension of the immunological mechanisms involved in the exacerbation of psoriatic plaques, and initial studies have investigated the mechanisms that lead to extracutaneous disease manifestations, including endothelial disfunction and cardiovascular disease. In the past decade, the involvement of gut dysbiosis in the development of pathologies with inflammatory and autoimmune basis has clearly emerged. More recently, a major role for the skin microbiota in establishing the immunological tolerance in early life and as a source of antigens leading to cross-reactive responses towards self-antigens in adult life has also been evidenced. Gut microbiota can indeed be involved in shaping the immune and inflammatory response at systemic level and in fueling inflammation in the cutaneous and vascular compartments. Here, we summarized the microbiota-mediated mechanisms that, in the skin and gut, may promote and modulate local or systemic inflammation involved in psoriatic disease and endothelial dysfunction. We also analyze the emerging strategies for correcting dysbiosis or modulating skin and gut microbiota composition to integrate systemically existing pharmacological therapies for psoriatic disease. The possibility of merging systemic treatment and tailored microbial modifying therapies could increase the efficacy of the current treatments and potentially lower the effect on patient's life quality.

Emerging Microfluidic Tools for Simultaneous Exosomes and Cargo Biosensing in Liquid Biopsy: New Integrated Miniaturized FFF-Assisted Approach for Colon Cancer Diagnosis.

The early-stage diagnosis of cancer is a crucial clinical need. The inadequacies of surgery tissue biopsy have prompted a transition to a less invasive profiling of molecular biomarkers from biofluids, known as liquid biopsy. Exosomes are phospholipid bilayer vesicles present in many biofluids with a biologically active cargo, being responsible for cell-to-cell communication in biological systems. An increase in their excretion and changes in their cargo are potential diagnostic biomarkers for an array of diseases, including cancer, and they constitute a promising analyte for liquid biopsy. The number of exosomes released, the morphological properties, the membrane composition, and their content are highly related to the physiological and pathological states. The main analytical challenge to establishing liquid biopsy in clinical practice is the development of biosensors able to detect intact exosomes concentration and simultaneously analyze specific membrane biomarkers and those contained in their cargo. Before analysis, exosomes also need to be isolated from biological fluids. Microfluidic systems can address several issues present in conventional methods (i.e., ultracentrifugation, size-exclusion chromatography, ultrafiltration, and immunoaffinity capture), which are time-consuming and require a relatively high amount of sample; in addition, they can be easily integrated with biosensing systems. A critical review of emerging microfluidic-based devices for integrated biosensing approaches and following the major analytical need for accurate diagnostics is presented here. The design of a new miniaturized biosensing system is also reported. A device based on hollow-fiber flow field-flow fractionation followed by luminescence-based immunoassay is applied to isolate intact exosomes and characterize their cargo as a proof of concept for colon cancer diagnosis.

Peto's paradox: Nature has used multiple strategies to keep cancer at bay while evolving long lifespans and large body masses. A systematic mini-review.

Comparative oncology is an understudied field of science. We are far from understanding the key mechanisms behind Peto's paradox, i.e., understanding how long-lived and large animals are not subject to a higher cancer burden despite the longer exposure time to mutations and the larger number of cells exposed. In this work, we investigated the scientific evidence on such mechanisms through a systematic mini-review of the literature about the relation of longevity and/or large body mass with physiological, genetic, or environmental traits among mammalian species. More than forty thousand articles were retrieved from three repositories, and 383 of them were screened using an active-learning-based tool. Of those, 36 articles on longevity and 37 on body mass were selected for the review. Such articles were examined focusing on: number and type of species considered, statistical methods used, traits investigated, and observed relationship with longevity and/or body mass. Where applicable, the traits investigated were matched with one or more hallmarks of cancer. We obtained a list of potential candidate traits to explain Peto's paradox related to replicative immortality, cell senescence, genome instability and mutations, proliferative signaling, growth suppression evasion, and cell resistance to death. Our investigation suggests that different strategies have been followed to prevent cancer in large and long-lived species. The large number of papers retrieved emphasizes that more studies can be launched in the future, using more efficient analytical approaches to comprehensively evaluate the convergent biological mechanisms essential for acquiring longevity and large body mass without increasing cancer risk.

The spontaneous immortalization probability of mammalian cell culture strains, as their proliferative capacity, correlates with species body mass, not longevity.

BACKGROUND: The Peto's paradox consists in the observation that individuals from long-lived and large animal species do not experience a higher cancer incidence, despite being exposed for longer time to the possibility of accumulating mutations and having more target cells exposed to the phenomenon. The existence of this paradox has been recently confirmed (Vincze et al., 2022). Concurrently, robust evidence has been published that longevity involves a convergent evolution of cellular mechanisms that prevent the accumulation of mutations (Cagan et al., 2022). It remains unclear which cellular mechanisms are critical to allow the evolution of a large body mass while keeping cancer at bay. METHODS: Adding to existing data linking cellular replicative potential and species body mass (Lorenzini et al., 2005), we have grown a total of 84 skin fibroblast cell strains from 40 donors of 17 mammalian species and analyzed their Hayflick's limit, i.e., their senescent plateau, and eventual spontaneous immortalization escape. The correlation of immortalization and replicative capacity of the species with their longevity, body mass and metabolism has been assessed through phylogenetic multiple linear regression (MLR). RESULTS: The immortalization probability is negatively related to species body mass. The new evaluation and additional data about replicative potential strengthen our previous observation, confirming that stable and extended proliferation is strongly correlated with the evolution of a large body mass rather than lifespan. CONCLUSION: The relation between immortalization and body mass suggests a need to evolve stringent mechanisms that control genetic stability during the evolution of a large body mass.

Treatment with PCSK9 Inhibitor Evolocumab Improves Vascular Oxidative Stress and Arterial Stiffness in Hypercholesterolemic Patients with High Cardiovascular Risk.

Atherosclerosis and atherosclerotic-related cardiovascular diseases (ASCVD) are characterized by high serum levels of low-density lipoprotein cholesterol (LDL-C) that can promote the generation of reactive oxygen species (ROS). To answer the need for better LDL-C control in individuals at high and very high risk for CVD, a new injectable innovative family of lipid-lowering (LL) monoclonal antibodies against the protein convertase subtilisin/kexin type 9 (PCSK9) has been approved. However, the effect of these drugs on vascular function, such as ROS generation and arterial stiffness, has not already been extensively described. In this report, we present data from 18 males with high to very high CV risk undergoing LL treatment (LLT) with either statin and ezetimibe or ezetimibe monotherapy, who experienced, after a 2-month treatment with Evolocumab, a significant improvement in blood pressure (BP)-adjusted carotid-femoral pulse wave velocity (cfPWV) (p-value = 0.0005 in the whole cohort, p-value = 0.0046 in the sub-cohort undergoing background LLT with statin and ezetimibe, p-value = 0.015 in the sub-cohort undergoing background LLT with ezetimibe monotherapy), which was significantly associated with a decrease in freshly isolated leukocytes (PBMCS)-derived H2O2 production (p-value = 0.004, p-value = 0.02 and p-value = 0.05, respectively, in the whole cohort, in the statin + ezetimibe sub-cohort, and the ezetimibe sub-cohort). Our observations support the role of systemic oxidative stress in atherosclerosis and give a further rationale for using Evolocumab also for its effect in vascular disorders linked to oxidative processes.

Agri-Food Waste from Apple, Pear, and Sugar Beet as a Source of Protective Bioactive Molecules for Endothelial Dysfunction and Its Major Complications.

Endothelial damage is recognized as the initial step that precedes several cardiovascular diseases (CVD), such as atherosclerosis, hypertension, and coronary artery disease. It has been demonstrated that the best treatment for CVD is prevention, and, in the frame of a healthy lifestyle, the consumption of vegetables, rich in bioactive molecules, appears effective at reducing the risk of CVD. In this context, the large amount of agri-food industry waste, considered a global problem due to its environmental and economic impact, represents an unexplored source of bioactive compounds. This review provides a summary regarding the possible exploitation of waste or by-products derived by the processing of three traditional Italian crops-apple, pear, and sugar beet-as a source of bioactive molecules to protect endothelial function. Particular attention has been given to the bioactive chemical profile of these pomaces and their efficacy in various pathological conditions related to endothelial dysfunction. The waste matrices of apple, pear, and sugar beet crops can represent promising starting material for producing "upcycled" products with functional applications, such as the prevention of endothelial dysfunction linked to cardiovascular diseases.

New Insights into Bile Acids Related Signaling Pathways in the Onset of Colorectal Cancer.

Colorectal cancer (CRC) ranks as the second among the causes of tumor death worldwide, with an estimation of 1.9 million new cases in 2020 and more than 900,000 deaths. This rate might increase by 60% over the next 10 years. These data are unacceptable considering that CRC could be successfully treated if diagnosed in the early stages. A high-fat diet promotes the hepatic synthesis of bile acids (BAs) increasing their delivery to the colonic lumen and numerous scientific reports correlate BAs, especially secondary BAs, with CRC incidence. We reviewed the physicochemical and biological characteristics of BAs, focusing on the major pathways involved in CRC risk and progression. We specifically pointed out the role of BAs as signaling molecules and the tangled relationships among their nuclear and membrane receptors with the big bang of molecular and cellular events that trigger CRC occurrence.

Fermentation of Vaccinium floribundum Berries with Lactiplantibacillus plantarum Reduces Oxidative Stress in Endothelial Cells and Modulates Macrophages Function.

Accumulating evidence suggests that high consumption of natural antioxidants promotes health by reducing oxidative stress and, thus, the risk of developing cardiovascular diseases. Similarly, fermentation of natural compounds with lactic acid bacteria (LAB), such as Lactiplantibacillus plantarum, enhances their beneficial properties as regulators of the immune, digestive, and cardiovascular system. We investigated the effects of fermentation with Lactiplantibacillus plantarum on the antioxidant and immunomodulatory effects of Pushgay berries (Vaccinium floribundum, Ericaceae family) in human umbilical vein endothelial cells (HUVECs) and macrophage cell line RAW264.7. Polyphenol content was assayed by Folin-Ciocalteu and HPLC-MS/MS analysis. The effects of berries solutions on cell viability or proliferation were assessed by WST8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt and Lactate dehydrogenase (LDH) release, Trypan blue exclusion test, and Alamar blue assay. Antioxidant activity was evaluated by a cell-based chemiluminescent probe for the detection of intracellular H2O2 production in HUVECs. Heme oxygenase-1 (HO-1) expression levels were investigated by RT-qPCR. Glutathione reductase (GR), glutathione peroxidase (Gpx), superoxide dismutase (SOD), and catalase (CAT) activities, as markers of intracellular antioxidant defense, were evaluated by spectrophotometric analysis. The immunomodulatory activity was examined in RAW 264.7 by quantification of inducible nitric oxide synthase (iNOS) and Tumor Necrosis Factor-alpha (TNFα) by RT-qPCR. Data showed that fermentation of Pushgay berries (i) enhances the content of quercetin aglycone, and (ii) increases their intracellular antioxidant activity, as indicated by the reduction in H2O2-induced cell death and the decrease in H2O2-induced HO-1 gene expression in HUVECs treated for 24 h with fermented berries solution (10 µg/mL). Moreover, treatment with Pushgay berries for 72 h (10 µg/mL) promotes cells growth in RAW 264.7, and only fermented Pushgay berries increase the expression of iNOS in the same cell line. Taken together, our results show that LAB fermentation of Pushgay berries enhances their antioxidant and immunomodulatory properties.

Grape Pomace for Topical Application: Green NaDES Sustainable Extraction, Skin Permeation Studies, Antioxidant and Anti-Inflammatory Activities Characterization in 3D Human Keratinocytes.

Food waste is a global problem due to its environmental and economic impact, so there is great demand for the exploitation of new functional applications. The winemaking process leads to an incomplete extraction of high-value compounds, leaving the pomace still rich in polyphenols. This study was aimed at optimising and validating sustainable routes toward the extraction and further valorisation of these polyphenols, particularly for cosmeceutical applications. New formulations based on red grape pomace polyphenols and natural deep eutectic solvents (NaDESs) were here investigated, namely betaine combined with citric acid (BET-CA), urea (BET-U) and ethylene glycol (BET-EG), in which DESs were used both as extracting and carrying agents for polyphenols. The flavonoid profile determined by HPLC-MS/MS analysis showed similar malvidin content (51-56 µg mL-1) in the DES combinations, while BET-CA gave the best permeation performance in Franz cells, so it was further investigated in 3D human keratinocytes (HaCat spheroids) injured with the pro-oxidant agent menadione. BET-CA treatment showed good intracellular antioxidant activity (IC50 0.15 ± 0.02 µg mL-1 in malvidin content) and significantly decreased (p < 0.001) the release of the pro-inflammatory cytokine IL-8, improving cell viability. Thus, BET-CA formulation is worthy of investigation for potential use as a cosmetic ingredient to reduce oxidative stress and inflammation, which are causes of skin aging.

Effect of Lactobacillus acidophilus Fermented Broths Enriched with Eruca sativa Seed Extracts on Intestinal Barrier and Inflammation in a Co-Culture System of an Enterohemorrhagic Escherichia coli and Human Intestinal Cells.

Lactic acid bacteria (LAB) "fermentates" confer a beneficial effect on intestinal function. However, the ability of new fermentations to improve LAB broth activity in preventing pathogen-induced intestinal inflammation and barrier dysfunction has not yet been studied. The objective of this study was to determine if broths of LAB fermented with Eruca sativa or Barbarea verna seed extracts prevent gut barrier dysfunction and interleukin-8 (CXCL8) release in vitro in human intestinal Caco-2 cells infected with enterohemorrhagic Escherichia coli (EHEC) O157:H7. LAB broths were assayed for their effects on EHEC growth and on Caco-2 viability; thereafter, their biological properties were analysed in a co-culture system consisting of EHEC and Caco-2 cells. Caco-2 cells infected with EHEC significantly increased CXCL8 release, and decreased Trans-Epithelial Electrical Resistance (TEER), a barrier-integrity marker. Notably, when Caco-2 cells were treated with LAB broth enriched with E. sativa seed extract and thereafter infected, both CXCL8 expression and epithelial dysfunction reduced compared to in untreated cells. These results underline the beneficial effect of broths from LAB fermented with E. sativa seed extracts in gut barrier and inflammation after EHEC infection and reveal that these LAB broths can be used as functional bioactive compounds to regulate intestinal function.

Estrogen-mediated protection against coronary heart disease: The role of the Notch pathway.

Estrogen regulates a plethora of biological processes, under physiological and pathological conditions, by affecting key pathways involved in the regulation of cell proliferation, fate, survival and metabolism. The Notch receptors are mediators of communication between adjacent cells and are key determinants of cell fate during development and in postnatal life. Crosstalk between estrogen and the Notch pathway intervenes in many processes underlying the development and maintenance of the cardiovascular system. The identification of molecular mechanisms underlying the interaction between these types of endocrine and juxtacrine signaling are leading to a deeper understanding of physiological conditions regulated by these steroid hormones and, potentially, to novel therapeutic approaches to prevent pathologies linked to reduced levels of estrogen, such as coronary heart disease, and cardiotoxicity caused by hormone therapy for estrogen-receptor-positive breast cancer.

The Use of Nutraceuticals to Counteract Atherosclerosis: The Role of the Notch Pathway.

Despite the currently available pharmacotherapies, today, thirty percent of worldwide deaths are due to cardiovascular diseases (CVDs), whose primary cause is atherosclerosis, an inflammatory disorder characterized by the buildup of lipid deposits on the inside of arteries. Multiple cellular signaling pathways have been shown to be involved in the processes underlying atherosclerosis, and evidence has been accumulating for the crucial role of Notch receptors in regulating the functions of the diverse cell types involved in atherosclerosis onset and progression. Several classes of nutraceuticals have potential benefits for the prevention and treatment of atherosclerosis and CVDs, some of which could in part be due to their ability to modulate the Notch pathway. In this review, we summarize the current state of knowledge on the role of Notch in vascular health and its modulation by nutraceuticals for the prevention of atherosclerosis and/or treatment of related CVDs.

Advanced biosensors for monitoring astronauts' health during long-duration space missions.

Long-duration space missions pose important health concerns for astronauts, especially regarding the adverse effects of microgravity and exposure to high-energy cosmic rays. The long-term maintenance of crew health and performance mainly relies on prevention, early diagnoses, condition management, and medical interventions in situ. In-flight biosensor diagnostic devices and medical procedures must use few resources and operate in a microgravity environment, which complicates the collection and management of biological samples. Moreover, the biosensors must be certified for in-flight operation according to strict design and safety regulations. Herein, we report on the state of the art and recent advances in biosensing diagnostic instrumentation for monitoring astronauts' health during long-duration space missions, including portable and wearable biosensors. We discuss perspectives on new-format biosensors in autonomous space clinics. We also describe our own work in developing biosensing devices for non-invasively diagnosing space-related diseases, and how they are used in long-duration missions. Finally, we discuss the benefits of space exploration for Earth-based medicine.

Combined analytical approaches to define biodistribution and biological activity of semi-synthetic berberrubine, the active metabolite of natural berberine.

Berberine (BBR) is a natural alkaloid obtained from Berberis species plants, known for its protective effects against several diseases. Among the primary BBR metabolites, berberrubine (M1) showed the highest plasma concentration but few and conflicting data are available regarding its concentration in biological fluids related to its new potential activity on vascular cells. A combined analytical approach was applied to study biodistribution of M1 in comparison with BBR. The optimization of sample clean-up combined with a fully validated HPLC-ESI-MS/MS tailored for M1 allows sufficient detectability and accuracy to be reached in the different studied organs even when administered at low dose, comparable to that assumed by human. A predictive human vascular endothelial cell-based assay to measure intracellular xanthine oxidase has been developed and applied to study unexplored activities of M1 alongside other common activities. Results showed that oral M1 treatment exhibits higher plasma levels than BBR, reaching maximum concentration 400-fold higher than BBR (204 vs 0.5 ng/mL); moreover, M1 exhibits higher concentrations than BBR also in all the biological compartments analyzed. Noteworthy, the two compounds follow two different excretion routes: M1 through urine, while BBR through feces. In vitro studies demonstrated that M1 inhibited intracellular xanthine oxidase activity, one of the major sources of reactive oxygen species in vasculature, with an IC50 = 9.90 ± 0.01 µg/mL and reduced the expression of the inflammatory marker ICAM-1. These peculiar characteristics allow new perspectives to be opened up for the direct use of M1 instead of BBR in endothelial dysfunction treatment.

Advanced bioanalytics for precision medicine.

Precision medicine is a new paradigm that combines diagnostic, imaging, and analytical tools to produce accurate diagnoses and therapeutic interventions tailored to the individual patient. This approach stands in contrast to the traditional "one size fits all" concept, according to which researchers develop disease treatments and preventions for an "average" patient without considering individual differences. The "one size fits all" concept has led to many ineffective or inappropriate treatments, especially for pathologies such as Alzheimer's disease and cancer. Now, precision medicine is receiving massive funding in many countries, thanks to its social and economic potential in terms of improved disease prevention, diagnosis, and therapy. Bioanalytical chemistry is critical to precision medicine. This is because identifying an appropriate tailored therapy requires researchers to collect and analyze information on each patient's specific molecular biomarkers (e.g., proteins, nucleic acids, and metabolites). In other words, precision diagnostics is not possible without precise bioanalytical chemistry. This Trend article highlights some of the most recent advances, including massive analysis of multilayer omics, and new imaging technique applications suitable for implementing precision medicine. Graphical abstract Precision medicine combines bioanalytical chemistry, molecular diagnostics, and imaging tools for performing accurate diagnoses and selecting optimal therapies for each patient.

Estrogen receptor ß-dependent Notch1 activation protects vascular endothelium against tumor necrosis factor α (TNFα)-induced apoptosis.

Unlike age-matched men, premenopausal women benefit from cardiovascular protection. Estrogens protect against apoptosis of endothelial cells (ECs), one of the hallmarks of endothelial dysfunction leading to cardiovascular disorders, but the underlying molecular mechanisms remain poorly understood. The inflammatory cytokine TNFα causes EC apoptosis while dysregulating the Notch pathway, a major contributor to EC survival. We have previously reported that 17ß-estradiol (E2) treatment activates Notch signaling in ECs. Here, we sought to assess whether in TNFα-induced inflammation Notch is involved in E2-mediated protection of the endothelium. We treated human umbilical vein endothelial cells (HUVECs) with E2, TNFα, or both and found that E2 counteracts TNFα-induced apoptosis. When Notch1 was inhibited, this E2-mediated protection was not observed, whereas ectopic overexpression of Notch1 diminished TNFα-induced apoptosis. Moreover, TNFα reduced the levels of active Notch1 protein, which were partially restored by E2 treatment. Moreover, siRNA-mediated knockdown of estrogen receptor ß (ERß), but not ERα, abolished the effect of E2 on apoptosis. Additionally, the E2-mediated regulation of the levels of active Notch1 was abrogated after silencing ERß. In summary, our results indicate that E2 requires active Notch1 through a mechanism involving ERß to protect the endothelium in TNFα-induced inflammation. These findings could be relevant for assessing the efficacy and applicability of menopausal hormone treatment, because they may indicate that in women with impaired Notch signaling, hormone therapy might not effectively protect the endothelium.

Effect of apple polyphenols on vascular oxidative stress and endothelium function: a translational study.

SCOPE: We aimed examining apple polyphenols' effect on uricemia and endothelial function in a sample of overweight subjects. METHODS AND RESULTS: This was a two-phased study. In vitro experiment aimed to evaluate apple polyphenols' ability to lower uric acid in comparison with allopurinol. In vivo study consisted in a randomized, double-blind, parallel placebo-controlled clinical trial involving 62 overweight volunteers with suboptimal values of fasting plasma glucose (100 mg/dL≤FPG≤125 mg/dL), randomized to 300 mg apple polyphenols or placebo for 8 weeks. Apple polyphenols extract inhibited xanthine oxidase activity, with an IC50 = 130 ± 30 ng/mL; reducing uric acid production with an IC50 = 154 ± 28 ng/mL. During the trial, after the first 4 weeks of treatment, FPG decreased in the active treated group (-6.1%, p < 0.05), while no significant changes were observed regarding the other hematochemistry parameters. After 4 more weeks of treatment, active-treated patients had an improvement in FPG compared to baseline (-10.3%, p < 0,001) and the placebo group (p < 0,001). Uric acid (-14.0%, p < 0.05 versus baseline; p < 0.05 versus placebo) and endothelial reactivity (0.24±0.09, p = 0.009 versus baseline; p < 0.05 versus placebo) significantly improved too. CONCLUSION: In vivo, apple polyphenols extract has a positive effect on vascular oxidative stress and endothelium function and reduce FPG and uric acid by inhibiting xanthine oxidase, as our In vitro experiment attests.