Francesco Cimino

Cyanidin-3-O-glucoside Protection against TNF-α-Induced Endothelial Dysfunction: Involvement of Nuclear Factor-κB Signaling

Oxidative stress and inflammation are considered to play a pivotal role in vascular endothelial dysfunction by triggering activation of transcription factors, such as NF-κB, functionally dependent on cellular redox status. The anthocyanin cyanidin-3-O-glucoside (C3G), as well as other phytochemicals recognized as potent antioxidants and free radical scavengers, may act as modulators of gene regulation and signal transduction pathways. This study demonstrates that C3G is able to protect human endothelial cells against alterations induced by TNF-α, including the activation of NF-κB, increased gene expression of adhesion molecules, leukocyte adhesion to endothelium, and intracellular accumulation of H2O2 and lipid peroxidation byproducts. These observations contribute to provide a conceptual background for the understanding of the mechanisms underlying the role of C3G, as well as other dietary plant polyphenols, in the prevention of diseases associated with inflammation and oxidative stress, including atherosclerosis.

Phytocomplexes from liquorice (Glycyrrhiza glabra L.) leaves--chemical characterization and evaluation of their antioxidant, anti-genotoxic and anti-inflammatory activity.

Three extracts of different polarities of Glycyrrhiza glabra L. leaves were characterized and evaluated for their antioxidant, anti-genotoxic and anti-inflammatory activity. In total, thirty components have been identified and quantified through the use of liquid chromatography (LC) with ultraviolet-visible diode-array-detector (UV-vis-DAD) and mass spectrometry (MS). The main components belong to the polyphenols family, being flavonoid and dihydrostilbene derivatives. The extracts have been investigated for their antioxidant, anti-genotoxic and anti-inflammatory activities, which are fundamental requirements of efficacious chemo-preventive agents. The ethyl acetate extract proved to be the most valuable, evidently for the conspicuous presence of several polyphenols, namely flavonoids and dihydrostilbenes.

Bioavailability and molecular activities of anthocyanins as modulators of endothelial function

Anthocyanins (AC) are water-soluble natural pigments found in various parts of higher plants. Despite their limited oral bioavailability and very low post-absorption plasma concentrations, the dietary consumption of these pigments has been proposed to be associated with a significant protection against several human pathological conditions, including cardiovascular diseases. Many studies highlighted that some health benefits of AC localize in particular at endothelium level, contributing to vascular homeostasis and also to the control of angiogenesis, inflammation, and platelet aggregation. This review reports and comments on the large existing literature addressing the molecular mechanisms that, beyond the antioxidant properties, may have a significant role in the effects of AC and AC-rich foods on vessel endothelium. Among these, AC have been reported to prevent peroxynitrite-mediated endothelial dysfunction in endothelial cells (ECs), thanks to their capability to modulate the expression and activity of several enzymes involved in NO metabolism. Furthermore, evidence indicates that AC can prevent the expression of adhesion molecules and the adhesion of monocytes to ECs challenged by pro-inflammatory agents. Overall, the activity of AC could be associated with the ability to elicit cell adaptive responses involving the transcription factor Nrf2 by affecting the “nucleophilic tone” of the organism. This review confirms the importance of specific nutritional molecules for human health and suggests new avenues for nutrition-based interventions to reduce the risk of cardiovascular disease in the population.

Palmitate-induced endothelial dysfunction is attenuated by cyanidin-3-O-glucoside through modulation of Nrf2/Bach1 and NF-κB pathways.

Free fatty acids (FFA), commonly elevated in diabetes and obesity, have been shown to impair endothelial functions and cause oxidative stress, inflammation, and insulin resistance. Anthocyanins represent one of the most important and interesting classes of flavonoids and seem to play a role in preventing cardiovascular diseases. Herein, we investigated the in vitro protective effects of cyanidin-3-O-glucoside (C3G) on cell signaling pathways in human umbilical vein endothelial cells (HUVECs) exposed to palmitic acid (PA), the most prevalent saturated FFA in circulation. Our data reported a significant augmentation of free radicals and oxidative stress in HUVECs exposed to PA for 3h, while C3G pretreatment improved intracellular redox status altered by FFA. Moreover, C3G significantly inhibited NF-κB proinflammatory pathway and adhesion molecules induced by PA, and these effects were attributed to the activation of Nrf2/EpRE pathway. In fact, C3G induced Nrf2 nuclear localization and activation of cellular antioxidant and cytoprotective genes at baseline and after PA exposure in endothelial cells. Our data confirm the hypothesis that natural Nrf2 inducers, such as C3G, might be a potential therapeutic strategy to protect vascular system against various stressors preventing several pathological conditions.

Synthesis and “double-faced” antioxidant activity of polyhydroxylated 4-thiaflavans

A simple synthetic methodology, based on the inverse electron demand hetero Diels-Alder reaction of electron-poor dienic o-thioquinones with electron-rich styrenes used as dienophiles, allowed the preparation of several polyhydroxylated 4-thiaflavans. Such compounds, as a function of the nature and position of the substituents on the aromatic rings, as well as of the oxidation state of the sulfur atom, are able to behave in vitro as efficient antioxidants mimicking the action of catechol containing flavonoids or/and tocopherols. The possibility of joining together the potentialities of two relevant families of natural polyphenolic antioxidants appears particularly appealing since an efficient protection against free radicals and other reactive oxygen species (ROS) depends in vivo upon the synergic action of different antioxidant derivatives.

Cyanidin‐3‐O‐glucoside counters the response to TNF‐alpha of endothelial cells by activating Nrf2 pathway

SCOPE Many dietary phytochemicals have been shown able to prevent a large spectrum of diseases, including cardiovascular disorders, with a mechanism commonly ascribed to an antioxidant effect. However, these in vivo beneficial effects are unlikely to be explained on the base of this mechanism. The discovery of specific genes regulated by the antioxidant responsive element (ARE) affected by antioxidants/electrophiles, led to the hypothesis that some phytochemicals may act as modulators of signal transduction pathways. The aim of the study was to investigate if in vitro pharmacological activation of Nrf2 pathway by cyanidin-3-O-glucoside (C3G) may be involved in its antiatherogenic effects. METHODS AND RESULTS Herein, we investigated the in vitro effects of C3G on cell signaling pathways in human umbilical vein endothelial cells (HUVECs) challenged with tumor necrosis factor-α (TNF-α). Pretreatment with C3G prevented oxidative stress, improved antioxidant systems, and activated Nrf2/ARE pathway, at baseline and after TNF-α treatment. Furthermore, we demonstrated the involvement of specific mitogen-activated protein kinases (MAPKs) (ERK1/2) in C3G induction of Nrf2/ARE pathway. Finally, the inactivation of ERK1/2 activity by the inhibitor PD98059 abolished the increase of Nrf2 nuclear accumulation induced by C3G, and also increased NF-κB p65 nuclear translocation in TNF-α challenged cells. CONCLUSION Our data confirm the hypothesis that natural Nrf2 and HO-1 inducers, such as C3G and other dietary phytochemicals, might be a potential therapeutic strategy to protect vascular system against various stressors preventing several pathological conditions.

In vitro protective effects of two extracts from bergamot peels on human endothelial cells exposed to tumor necrosis factor-α (TNF-α).

Bergamot ( Citrus bergamia Risso) is a less commercialized Citrus fruit, mainly used for its essential oil extracted from the peel. Bergamot peel (BP) represents about 60% of the processed fruits and is regarded as primary waste. However, it contains good amounts of useful compounds, such as pectins and flavonoids. Many of the bioactivities of Citrus flavonoids appear to impact vascular endothelial cells. Herein, we report the protective effect of two flavonoid-rich extracts from BP (endowed with radical-scavenging properties and lacking genotoxic activity) against alterations in cell modifications induced by the pleiotropic inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) on human umbilical vein endothelial cells (HUVECs), as demonstrated by monitoring intracellular levels of malondialdehyde/4-hydroxynonenal, reduced and oxidized glutathione and superoxide dismutase activity, and the activation status of nuclear factor-kappaB (NF-kappaB). Thus, BP appears to be a potential source of natural antioxidant/anti-inflammatory phytocomplexes to be employed as ingredients of nutraceutical products or functional foods.

Cyanidin-3-O-glucoside inhibits NF-kB signalling in intestinal epithelial cells exposed to TNF-α and exerts protective effects via Nrf2 pathway activation

Chronic intestinal inflammatory disorders, such as Inflammatory Bowel Diseases (IBDs), are characterized by excessive release of proinflammatory mediators, intestinal barrier dysfunction and excessive activation of NF-kB cascade. Previous studies shown that TNF-α plays a central role in intestinal inflammation of IBDs and supported beneficial effects of flavonoids against chronic inflammatory diseases. In this study, we employed an in vitro model of acute intestinal inflammation using intestinal Caco-2 cells exposed to TNF-α. The protective effects of cyanidin-3-glucoside (C3G), an anthocyanin widely distributed in mediterranean diet, were then evaluated. Caco-2 cells exposure to TNF-α activated NF-kB proinflammatory pathway and induced IL6 and COX-2 expression. Cells pretreatment for 24h with C3G (20-40μM) prevented TNF-α-induced changes, and improved intracellular redox status. Our results demonstrated that C3G, also without any kind of stimulus, increased the translocation of the transcription factor Nrf2 into the nucleus so activating antioxidant and detoxifying genes. In conclusion, C3G exhibited protective effects through the inhibition of NF-kB signalling in Caco-2 cells and these beneficial effects appear to be due to its ability to activate cellular protective responses modulated by Nrf2. These data suggest that anthocyanins could contribute, as complementary or preventive approaches, to the management of chronic inflammatory diseases.

Cyanidin-3-O-glucoside modulates intracellular redox status and prevents HIF-1 stabilization in endothelial cells in vitro exposed to chronic hypoxia

The term hypoxia refers to conditions characterized by a relative restriction of oxygen supply. It is usually associated to a paradoxical overproduction of reactive oxygen species (ROS) and to the activation of several transcription factors, including HIF-1α, which in turn trigger angiogenic and apoptotic response. In this study we have investigated the mechanisms by which the anthocyanin cyanidin-3-O-glucoside (C3G) modulates hypoxia induced response in human endothelial cells (HUVECs). In fact, hypoxia induces an increase of ROS generation in HUVECs paralleled by a loss of antioxidant cellular capacity. According to the observed increase of HO-1 mRNA expression, pretreatment of C3G to HUVEC reduces the entity of oxidative stress thanks to the activation of cellular antioxidant response. C3G also attenuates HIF-1α protein accumulation conditions supporting the hypothesis of a major role of oxidative stress in the presence of low oxygen. Furthermore, the increased expression of angiogenesis and apoptosis markers (MMP-2 and caspase-3) due to HIF-1α activation by hypoxia is reduced in C3G pretreated cells. Overall, our data suggest that the modulation of intracellular redox status induced by C3G may be an important protective mechanism against endothelial damage in hypoxic conditions.

Cellular adaptive response to glutathione depletion modulates endothelial dysfunction triggered by TNF-α

Several interrelated cellular signaling molecules are involved in modulating adaptive compensatory changes elicited by low exposures to toxins and other stressors. The most prominent example of signaling pathway typically involved in this adaptive stress response, is represented by the activation of a redox-sensitive gene regulatory network mediated by the NF-E2-related factor-2 (Nrf2) which is intimately involved in mediating the Antioxidant Responsive Element (ARE)-driven response to oxidative stress and xenobiotics. We investigated if Nrf2 pathway activation following intracellular glutathione depletion through buthionine sulfoximine (BSO) exposure, might be able to alter the response to TNF-α, a proinflammatory cytokine, in cultured human umbilical vein endothelial cells. Herein, we revealed that such a change in the cellular redox status is able to reduce TNF-α induced endothelial activation (as shown by a decreased gene expression of adhesion molecules) by activating an adaptive response mediated by an increased Nrf2 nuclear translocation and overexpression of the ARE genes HO-1 and NQO-1. Furthermore, we have demonstrated the involvement of ERK1/2 kinases in Nrf2 nuclear translocation activated by BSO-induced glutathione depletion. The coordinate induction of endogenous cytoprotective proteins through adaptive activation of Nrf2 pathway is a field of great interest for potential application in prevention and therapy of inflammatory diseases such as atherosclerosis.