Selective chemiluminescent TURN-ON quantitative bioassay and imaging of intracellular hydrogen peroxide in human living cells.

Hydrogen peroxide is an unavoidable by-product of cell metabolism, but when it is not properly managed by the body it can lead to several pathologies (e.g., premature aging, cardiovascular and neurodegenerative diseases, cancer). Several methods have been proposed for the measurement of intracellular H2O2 but none of them has proven to be selective. We developed a rapid all-in-one chemiluminescent bioassay for the quantification of H2O2 in living cells with a low limit of detection (0.15 µM). The method relies on an adamantylidene-1,2-dioxetane lipophilic probe containing an arylboronate moiety; upon reaction with H2O2 the arylboronate moiety is converted to the correspondent phenol and the molecule decomposes leading to an excited-state fragment that emits light. The probe has been successfully employed for quantifying intracellular H2O2 in living human endothelial, colon and keratinocyte cells exposed to different pro-oxidant stimuli (i.e., menadione, phorbol myristate acetate and lipopolysaccharide). Imaging experiments clearly localize the chemiluminescence emission inside the cells. Treatment of cells with antioxidant molecules leads to a dose-dependent decrease of intracellular H2O2 levels. As a proof of concept, the bioassay has been used to measure the antioxidant activity of extracts from Brassica juncea wastes, which contain glucosinolates, isothiocyanates and other antioxidant molecules.

Peptides from Cauliflower By-Products, Obtained by an Efficient, Ecosustainable, and Semi-Industrial Method, Exert Protective Effects on Endothelial Function.

The large amount of cauliflower industry waste represents an unexplored source of bioactive compounds. In this work, peptide hydrolysates from cauliflower leaves were characterized by combined bioanalytical approaches. Twelve peptide fractions were studied to evaluate unexplored biological activities by effect-based cellular bioassays. A potent inhibition of intracellular xanthine oxidase activity was observed in human vascular endothelial cells treated with one fraction, with an IC50 = 8.3 ± 0.6 µg/ml. A different fraction significantly induced the antioxidant enzyme superoxide dismutase 1 and decreased the tumor necrosis factor α-induced VCAM-1 expression, thus leading to a significant improvement in the viability of human vascular endothelial cells. Shotgun peptidomics and bioinformatics were used to retrieve the most probable bioactive peptide sequences. Our study shows that peptides from cauliflower waste should be recycled for producing valuable products useful for the prevention of endothelial dysfunction linked to atherogenesis progression.

Identification and quantification of oxo-bile acids in human faeces with liquid chromatography-mass spectrometry: A potent tool for human gut acidic sterolbiome studies.

Bile acids (BAs) are endogenous steroids involved in the transport of lipids in bile, acting also as molecular signaling hormones. Primary BAs synthesized in the liver undergo several metabolic pathways in the intestine by gut microbiota to produce secondary BAs. Together with secondary BAs, other metabolites have been recovered from human faeces, including many oxo-BA analogues produced in the colon through oxidation of BA hydroxy groups. However, the complete oxo-BA characterization in biospecimens (particularly intestinal content and faeces) has not been reported yet, hampering the assessment of their potential physiological role. Herein, we have developed and validated a new RP-HPLC-ESI-MS/MS method in negative ionization mode for the simultaneous analysis of 21 oxo-BAs and their 7 metabolic BAs precursors in human faeces. The elution was performed in gradient mode and 28 compounds, including primary, secondary BAs, and their oxo-derivatives, were separated within 50 min at 40 °C column temperature. The method is accurate (bias% <13%), precise (CV% <10%), with limits of quantification (LOQ <30 ng/mLextract samples), similar for all the studied compounds. The matrix effect does not significantly affect the analysis accuracy, allowing the use of standard solutions for the quantifications, without matrix-matched protocols. Thanks to the high detectability and the relatively high concentration of oxo-BAs (about µg/gwet faeces), the method does not require a pre-analytical clean-up step. This method was used to identify and quantify oxo-BAs in human faecal samples from healthy subjects, serving as a proof of concept for application in patients with hepatobiliary disease and bacteria overgrowth.

Heart rate reduction with ivabradine in the early phase of atherosclerosis is protective in the endothelium of ApoE-deficient mice.

Ivabradine, a heart rate reducing agent, protects the vascular system by unidentified mechanisms. We sought to determine the effects of the treatment with ivabradine, started before plaque formation, on early transcriptional changes and endothelium lesions in regions of aorta subjected to disturbed blood flow. Six week-old apolipoprotein E-deficient (ApoE-/-) mice, fed a low-fat diet, were treated with ivabradine to determine the effect on transcriptional changes (2-and 4-week treatment) and on lesions formation (19-week treatment) in the endothelium of the aortic arch. Microarrays analysis (60k probes) of endothelium-enriched RNA was carried out to detect changes in gene expression induced by treatment. Endothelium damage was assessed by en-face immunofluorescence staining for vascular endothelial (VE) cadherin. According to microarray analysis, 930 transcripts were affected by the treatment. We found downregulation of pro-apoptotic and pro-inflammatory genes, the majority of which are nuclear factor-κB (NF-κB)-and/or angiotensin II-regulated genes, and upregulation of anti-inflammatory genes. Many shear stress-responsive genes were affected by the treatment and the MAPK, Notch signalling and sterol metabolic processes were among the most significantly affected pathways. Consistently, we observed increased levels of Hes5, a Notch target gene, together with a reduction of endothelium damage, in the lower aortic arch of treated- compared with untreated- mice. We concluded that an early treatment with ivabradine protected the endothelium of the aortic arch of ApoE-/- mice. Activation of the Notch signalling could be part of the mechanism underlying this protection.

Novel role of the nutraceutical bioactive compound berberine in lectin-like OxLDL receptor 1-mediated endothelial dysfunction in comparison to lovastatin.

BACKGROUND AND AIMS: Oxidized LDL (oxLDL) or pro-inflammatory stimuli lead to increased oxidative stress linked to endothelial dysfunction and atherosclerosis. The oxLDL receptor-1 (LOX1) is elevated within atheromas and cholesterol-lowering statins inhibit LOX1 expression. Berberine (BBR), an alkaloid extracted from plants of gender Berberis, has lipid-lowering and anti-inflammatory activity. However, its role in regulating LOX1-mediated signaling is still unknown. The aim of this study was to investigate the effect of BBR on oxLDL- and TNFα-induced endothelial dysfunction in human umbilical vein endothelial cells (HUVECs) and to compare it with that of lovastatin (LOVA). METHODS AND RESULTS: Cytotoxicity was determined by lactate dehydrogenase assay. Antioxidant capacity was measured with chemiluminescent and fluorescent method and intracellular ROS levels through a fluorescent dye. Gene and protein expression levels were assayed by qRT-PCR and western blot, respectively. HUVECs exposure to oxLDL (30 µg/ml) or TNFα (10 ng/ml) for 24 h led to a significant increase in LOX1 expression, effect abrogated by BBR (5 µM) and LOVA (5 µM). BBR but not LOVA treatment abolished the TNFα-induced cytotoxicity and restored the activation of Akt signaling. In spite of a low direct antioxidant capacity, both compounds reduced intracellular ROS levels generated by treatment of TNFα but only BBR inhibited NOX2 expression, MAPK/Erk1/2 signaling and subsequent NF-κB target genes VCAM and ICAM expression, induced by TNFα. CONCLUSIONS: These findings demonstrated for the first time that BBR could prevent the oxLDL and TNFα - induced LOX1 expression and oxidative stress, key events that lead to NOX, MAPK/Erk1/2 and NF-κB activation linked to endothelial dysfunction. CHEMICAL COMPOUNDS STUDIED IN THIS ARTICLE: Berberine (PubChem CID: 2353); Lovastatin (PubChem CID: 53232).

A new sensitive and quantitative chemiluminescent assay to monitor intracellular xanthine oxidase activity for rapid screening of inhibitors in living endothelial cells.

Xanthine oxidase (XO) is an important enzyme, expressed at high levels in the vasculature in endothelial cells, that catalyzes the hydroxylation of hypoxanthine to xanthine and xanthine to uric acid. Excessive production of uric acid results in hyperuricemia linked to gout and cardiovascular diseases. Testing inhibition of XO is important for detection of potentially effective drugs or natural products that could be used to treat diseases caused by increased XO activity. In the present study, for the first time, we developed an in vitro chemiluminescent bioassay to determine XO activity in living endothelial cells and the IC50 value of oxypurinol, the active metabolite of the inhibitor drug allopurinol. Intracellular XO activity was measured in less than 20 min with a luminol/catalyst-based chemiluminescence assay able to measure XO with a limit of 0.4 µU/mL. Oxypurinol addition to 5 × 103 cells (ranging from 5.0 to 0.0 µM) caused a linear decrease in XO activity, with an IC50 of 1.0 ± 0.5 µM. The detection system developed was low-cost, rapid, reproducible, and easily miniaturizable so suitable to be used on small quantities of cells.

ROS, Notch, and Wnt signaling pathways: crosstalk between three major regulators of cardiovascular biology.

Reactive oxygen species (ROS), traditionally viewed as toxic by-products that cause damage to biomolecules, now are clearly recognized as key modulators in a variety of biological processes and pathological states. The development and regulation of the cardiovascular system require orchestrated activities; Notch and Wnt/ß -catenin signaling pathways are implicated in many aspects of them, including cardiomyocytes and smooth muscle cells survival, angiogenesis, progenitor cells recruitment and differentiation, arteriovenous specification, vascular cell migration, and cardiac remodelling. Several novel findings regarding the role of ROS in Notch and Wnt/ß-catenin modulation prompted us to review their emerging function in the cardiovascular system during embryogenesis and postnatally.