The endocrine disruptor vinclozolin causes endothelial injury via eNOS/Nox4/IRE1α signaling.

Vinclozolin (VCZ) is a common dicarboximide fungicide used to protect crops from diseases. It is also an endocrine disruptor, and its effects on various organs have been described but its influence on vasculature has not yet been addressed. This study focuses on the potential mechanism of VCZ-induced vascular injury. The effect of VCZ on vascular function in terms of relaxing and contracting response was evaluated in mice aorta. A short exposure to VCZ affected the endothelial but not the smooth muscle component. Specifically, it caused a disruption of the eNOS/NO signaling. In line, a short exposure to VCZ in bovine aortic endothelial cells promoted eNOS uncoupling resulting in a reduction of NO bioavailability and eNOS dimer/monomer ratio, and in turn an increase of nitro-tyrosine levels and ROS formation. Prolonging the exposure to VCZ (3 and 6h) an up-regulation of Nox4, enzyme-generating ROS constitutively expressed in endothelial cells, and an increase in ROS and malondialdehyde content coupled with a reduction in NO levels were found. These events were strictly linked to endoplasmic reticulum stress as demonstrated by the phosphorylation of inositol-requiring transmembrane kinase endoribonuclease 1α (IRE1α), a stress sensor and its reversion by using a selective inhibitor. Collectively, these results demonstrated that VCZ provokes endothelial dysfunction by oxidative stress involving eNOS/Nox4/IRE1α axis. The rapid exposure affected the endothelial function promoting eNOS uncoupling while a post-transcriptional modification, involving Nox4/IRE1α signaling, occurred following prolonged exposure. Thus, exposure to VCZ could contribute to the onset and/or progression of cardiovascular diseases associated with endothelial dysfunction.

Rewiring Endothelial Sphingolipid Metabolism to Favor S1P Over Ceramide Protects From Coronary Atherosclerosis.

BACKGROUND: Growing evidence correlated changes in bioactive sphingolipids, particularly S1P (sphingosine-1-phosphate) and ceramides, with coronary artery diseases. Furthermore, specific plasma ceramide species can predict major cardiovascular events. Dysfunction of the endothelium lining lesion-prone areas plays a pivotal role in atherosclerosis. Yet, how sphingolipid metabolism and signaling change and contribute to endothelial dysfunction and atherosclerosis remain poorly understood. METHODS: We used an established model of coronary atherosclerosis in mice, combined with sphingolipidomics, RNA-sequencing, flow cytometry, and immunostaining to investigate the contribution of sphingolipid metabolism and signaling to endothelial cell (EC) activation and dysfunction. RESULTS: We demonstrated that hemodynamic stress induced an early metabolic rewiring towards endothelial sphingolipid de novo biosynthesis, favoring S1P signaling over ceramides as a protective response. This finding is a paradigm shift from the current belief that ceramide accrual contributes to endothelial dysfunction. The enzyme SPT (serine palmitoyltransferase) commences de novo biosynthesis of sphingolipids and is inhibited by NOGO-B (reticulon-4B), an ER membrane protein. Here, we showed that NOGO-B is upregulated by hemodynamic stress in myocardial EC of ApoE-/- mice and is expressed in the endothelium lining coronary lesions in mice and humans. We demonstrated that mice lacking NOGO-B specifically in EC (Nogo-A/BECKOApoE-/-) were resistant to coronary atherosclerosis development and progression, and mortality. Fibrous cap thickness was significantly increased in Nogo-A/BECKOApoE-/- mice and correlated with reduced necrotic core and macrophage infiltration. Mechanistically, the deletion of NOGO-B in EC sustained the rewiring of sphingolipid metabolism towards S1P, imparting an atheroprotective endothelial transcriptional signature. CONCLUSIONS: These data demonstrated that hemodynamic stress induced a protective rewiring of sphingolipid metabolism, favoring S1P over ceramide. NOGO-B deletion sustained the rewiring of sphingolipid metabolism toward S1P protecting EC from activation under hemodynamic stress and refraining coronary atherosclerosis. These findings also set forth the foundation for sphingolipid-based therapeutics to limit atheroprogression.

A reverse translational approach reveals the protective roles of Mangifera indica in inflammatory bowel disease.

Inflammatory bowel diseases (IBDs) are chronic intestinal disorders often characterized by a dysregulation of T cells, specifically T helper (Th) 1, 17 and T regulatory (Treg) repertoire. Increasing evidence demonstrates that dietary polyphenols from Mangifera indica L. extract (MIE, commonly known as mango) mitigate intestinal inflammation and splenic Th17/Treg ratio. In this study, we aimed to dissect the immunomodulatory and anti-inflammatory properties of MIE using a reverse translational approach, by initially using blood from an adult IBD inception cohort and then investigating the mechanism of action in a preclinical model of T cell-driven colitis. Of clinical relevance, MIE modulates TNF-α and IL-17 levels in LPS spiked sera from IBD patients as an ex vivo model of intestinal barrier breakdown. Preclinically, therapeutic administration of MIE significantly reduced colitis severity, pathogenic T-cell intestinal infiltrate and intestinal pro-inflammatory mediators (IL-6, IL-17A, TNF-α, IL-2, IL-22). Moreover, MIE reversed colitis-induced gut permeability and restored tight junction functionality and intestinal metabolites. Mechanistic insights revealed MIE had direct effects on blood vascular endothelial cells, blocking TNF-α/IFN-γ-induced up-regulation of COX-2 and the DP2 receptors. Collectively, we demonstrate the therapeutic potential of MIE to reverse the immunological perturbance during the onset of colitis and dampen the systemic inflammatory response, paving the way for its clinical use as nutraceutical and/or functional food.

Erucin, an H2S-Releasing Isothiocyanate, Exerts Anticancer Effects in Human Triple-Negative Breast Cancer Cells Triggering Autophagy-Dependent Apoptotic Cell Death.

Breast cancer is the most frequent form of cancer occurring in women of any age. Among the different types, the triple-negative breast cancer (TNBC) subtype is recognized as the most severe form, being associated with the highest mortality rate. Currently, there are no effective treatments for TNBC. For this reason, the research of novel therapeutics is urgently needed. Natural products and their analogs have historically made a major contribution to pharmacotherapy and the treatment of various human diseases, including cancer. In this study, we explored the potential anti-cancer effects of erucin, the most abundant H2S-releasing isothiocyanate present in arugula (Eruca sativa) in MDA-MB-231 cells, a validated in vitro model of TNBC. We found that erucin, in a concentration-dependent manner, significantly inhibited MDA-MB-231 cell proliferation by inducing apoptosis and autophagy. Additionally, erucin prevented intracellular ROS generation promoting the expression of key antioxidant genes and halted MDA-MB-231 cell migration, invasion, and colony formation. In conclusion, using a cellular and molecular biology approach, we show that the consumption of erucin could represent a novel and promising strategy for intervention against TNBC.

Hydrogen sulfide donor AP123 restores endothelial nitric oxide-dependent vascular function in hyperglycemia via a CREB-dependent pathway.

Diabetes is associated with severe vascular complications involving the impairment of endothelial nitric oxide synthase (eNOS) as well as cystathionine γ-lyase (CSE) activity. eNOS function is suppressed in hyperglycaemic conditions, resulting in reduced NO bioavailability, which is paralleled by reduced levels of hydrogen sulfide (H2S). Here we have addressed the molecular basis of the interplay between the eNOS and CSE pathways. We tested the impact of H2S replacement by using the mitochondrial-targeted H2S donor AP123 in isolated vessels and cultured endothelial cells in high glucose (HG) environment, at concentrations not causing any vasoactive effect per se. Aorta exposed to HG displayed a marked reduction of acetylcholine (Ach)-induced vasorelaxation that was restored by the addition of AP123 (10 nM). In HG condition, bovine aortic endothelial cells (BAEC) showed reduced NO levels, downregulation of eNOS expression, and suppression of CREB activation (p-CREB). Similar results were obtained by treating BAEC with propargylglycine (PAG), an inhibitor of CSE. AP123 treatment rescued eNOS expression, as well as NO levels, and restored p-CREB expression in both the HG environment and the presence of PAG. This effect was mediated by a PI3K-dependent activity since wortmannin (PI3K inhibitor) blunted the rescuing effects operated by the H2S donor. Experiments performed in the aorta of CSE-/- mice confirmed that reduced levels of H2S not only negatively affect the CREB pathway but also impair Ach-induced vasodilation, significantly ameliorated by AP123. We have demonstrated that the endothelial dysfunction due to HG involves H2S/PI3K/CREB/eNOS route, thus highlighting a novel aspect of the H2S/NO interplay in the vasoactive response.

Anti-inflammatory and immunomodulatory activity of Mangifera indica L. reveals the modulation of COX-2/mPGES-1 axis and Th17/Treg ratio.

In the context of inflammation and immunity, there are fragmented and observational studies relating to the pharmacological activity of Mangifera indica L. and its main active component, mangiferin. Therefore, we aimed to analyze the potential beneficial effects of this plant extract (MIE, 90 % in mangiferin) in a mouse model of gouty arthritis, to allow the evaluation of cellular immune phenotypes and the biochemical mechanism/s beyond MIE activity. Gouty arthritis was induced by the intra-articular administration of MSU crystals (200 µg 20 µl-1), whereas MIE (0.1-10 mg kg-1) or corresponding vehicle (DMSO/saline 1:3) were orally administrated concomitantly with MSU (time 0), 6 and 12 h after the stimulus. Thereafter, knee joint score and oedema were evaluated in addition to western blot analysis for COX-2/mPGES-1 axis. Moreover, the analysis of pro/anti-inflammatory cyto-chemokines coupled with the phenotyping of the cellular infiltrate was performed. Treatment with MIE revealed a dose-dependent reduction in joint inflammatory scores with maximal inhibition observed at 10 mg kg-1. MIE significantly reduced leukocyte infiltration and activation and the expression of different pro-inflammatory cyto-chemokines in inflamed tissues. Furthermore, biochemical analysis revealed that MIE modulated COX-2/mPGES-1 and mPGDS-1/PPARγ pathways. Flow cytometry analysis also highlighted a prominent modulation of inflammatory monocytes (CD11b+/CD115+/LY6Chi), and Treg cells (CD4+/CD25+/FOXP3+) after MIE treatment. Collectively, the results of this study demonstrate a novel function of MIE to positively affect the local and systemic inflammatory/immunological perturbance in the onset and progression of gouty arthritis.

Endogenous and exogenous hydrogen sulfide modulates urothelial bladder carcinoma development in human cell lines.

The role of H2S in urothelial carcinoma (UC) is still unclear. Here we have evaluated the expression of H2S producing enzymes as well as the effect of endogenous and exogenous H2S on human bladder UC cells. In human UC cells the expression of cystathionine ß-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST); is significantly lower as compared to healthy cells. A modulatory role for the H2S pathway is supported by the finding that, the overexpression of CSE or CBS, but not 3-MST, inhibits cell proliferation and promotes apoptosis. A similar effect is obtained by using exogenous H2S. Diallyl trisulfide (DATS), which is a fully characterized H2S donor, inhibits the proliferation of UC cells in a time and concentration-dependent manner as well as promotes apoptosis. Moreover, DATS also induces autophagy, as determined by transcriptomic and western blot analysis. Finally, DATS inhibits mRNA expression levels of canonical markers of epithelial-mesenchymal transition by limiting migration and clonogenic ability of human UC cells in vitro. In conclusion, in urothelial carcinoma, there is an impairment of H2S pathway that involves CSE and CBS- derived hydrogen sulfide. Thus, targeting H2S signaling pathway in urothelial carcinoma could represent a novel therapeutic strategy.

Phosphodiesterases S-sulfhydration contributes to human skeletal muscle function.

The increase in intracellular calcium is influenced by cyclic nucleotides (cAMP and cGMP) content, which rating is governed by phosphodiesterases (PDEs) activity.Despite it has been demonstrated a beneficial effect of PDEs inhibitors in different pathological conditions involving SKM, not much is known on the role exerted by cAMP-cGMP/PDEs axis in human SKM contractility. Here, we show that Ssulfhydration of PDEs modulates human SKM contractility in physiological and pathological conditions. Having previously demonstrated that, in the rare human syndrome Malignant Hyperthermia (MH), there is an overproduction of hydrogen sulfide (H2S) within SKM contributing to hyper-contractility, here we have used MH negative diagnosed biopsies (MHN) as healthy SKM, and MH susceptible diagnosed biopsies (MHS) as a pathological model of SKM hypercontractility. The study has been performed on MHS and MHN human biopsies after diagnosis has been made and on primary SKM cells derived from both MHN and MHS biopsies. Our data demonstrate that in normal conditions PDEs are S-sulfhydrated in both quadriceps' biopsies and primary SKM cells. This post translational modification (PTM) negatively regulates PDEs activity with consequent increase of both cAMP and cGMP levels. In hypercontractile biopsies, due to an excessive H2S content, there is an enhanced Ssulfhydration of PDEs that further increases cyclic nucleotides levels contributing to SKM hyper-contractility. Thus, the identification of a new endogenous PTM modulating PDEs activity represents an advancement in SKM physiopathology understanding.