Browsing by Author "Festa, Joseph"

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    Anthocyanins and Vascular Health: A Matter of Metabolites
    (MDPI, 2023-04-26) Festa, Joseph; Hussain, Aamir; Al-Hareth, Zakia; Singh, Harprit; Da Boit, Mariasole
    Anthocyanins are a subgroup of flavonoid polyphenols previously investigated for improv ing cardiovascular health and preventing the development of endothelial dysfunction. However, their poor bioavailability raises the question of whether the observed biological activity is due to their metabolites. Phenolic metabolites can reach higher plasma concentrations and can persist in the circulation for periods much longer than their original anthocyanin form; therefore, the biological activity and health promoting effects of anthocyanins may differ from their metabolites. To address this, recent studies have facilitated different cell models, in vivo studies and explored physiologically relevant concentrations to better understand their mechanisms of action. The criteria were chosen based on previous reports demonstrating that anthocyanins can improve endothelial function via modulation of the Akt-endothelial nitric oxide synthase pathway and transcription factors Nrf2 and NF-κB, which made it critical to assess the phenolic metabolites’ modes of action via these pathways. This review demonstrates how phenolic metabolites differ in bioactivity from their precursor an thocyanin, demonstrating improved endothelial function in response to inflammatory mediators at concentrations that are tolerated in vivo. The review highlights the crucial need for further studies to focus on improving the bioavailability of metabolites in isolation and explore the effect of metabolites in mixtures.
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    C2- linked alkynyl poly-ethylene glycol(PEG) adenosine conjugates as water-soluble adenosine receptor agonists
    (Wiley, 2022-08-22) Ferguson, Lindsay; Madieh, Nasrin Shokrzadeh; Brucoli, Federico; Vaideanu, Alexandra; Schatzlein, Andreas; Festa, Joseph; Singh, Harprit; Wells, Geoffrey; Bhakta, Sanjib
    A series of 12 novel polyethylene-glycol(PEG)-alkynyl C2-adenosine(ADN) conjugates were synthesized using a robust Sonogashira coupling protocol and characterized by NMR spectroscopy and mass spectrometry analysis. The ADN-PEG conjugates showed null to moderate toxicity in murine macrophages and 12c was active against Mycobacterium aurum growth (MIC = 62.5 mg/L). The conjugates were not active against Mycobacterium bovis BCG. Conjugates 10b and 11b exhibited high water solubility with solubility values of 1.22 and 1.18 mg/ml, respectively, in phosphate buffer solutions at pH 6.8. Further, 10b and 11b induced a significant increase in cAMP accumulation in RAW264.7 cells comparable with that induced by adenosine. Analogues 10c, 11c and 12c were docked to the A1, A2A, A2B and A3 adenosine receptors (ARs) using crystal-structures and homology models. ADN-PEG-conjugates bearing chains with up to five ethyleneoxy units could be well accommodated within the binding sites of A1, A2A and A3 ARs. Docking studies showed that compound 10b and 11b were the best A2A receptor binders of the series, whereas 12c was the best binder for A1 AR. In summary, introduction of hydrophilic PEG substituents at the C2 of adenine ring significantly improved water solubility and did not affect AR binding properties of the ADN-PEG conjugates.
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    Cyanidin-3-glucoside phenolic metabolites, protocatechuic acid and vanillic acid prevent TNF-α induced endothelial dysfunction in vitro: Implications for anthocyanins and vascular health
    (De Montfort University, 2023) Festa, Joseph
    Anthocyanins are a subgroup of flavonoid polyphenols, derived from many fruits and vegetables, previously reported to improve cardiovascular health. However, their poor bioavailability in vivo raises the question of whether the observed biological activity is due to their metabolites. It is known that elderberry (EB) has a high content of cyanidin-3-glucoside (C3G), which is known to induce cardiovascular protective properties by improving endothelial function. After the consumption of C3G, it is rapidly degraded to many metabolites although mainly in the form of protocatechuic acid (PCA), which can be methylated to form vanillic acid (VA). Both have been shown to reach higher plasma concentrations than its original C3G form. However, their potential positive effect on endothelial health is yet to be investigated in inflammatory conditions. An understanding of their action will help to better define the in vivo vascular health benefits of anthocyanin rich foods such as EB. Purpose: The main aim of this thesis was to investigate the effects of PCA and VA on endothelial function at physiological concentrations in the presence of TNF-α, a pro-inflammatory mediator. Moreover, it was aimed to determine whether metabolites could be identified as the bioactive compounds responsible for the observed effect of anthocyanins, specifically focusing on C3G as a representative anthocyanin compound. Methods: Primary human umbilical vein endothelial cells (HUVEC) were pre-treated with either EB (50 μg/ml) or C3G, PCA and VA at various concentrations including 1,5 or 10 μM at various timepoints stimulated with or without TNF-α (10 or 20 ng/ml) as stated. Cell viability, apoptosis, oxidative stress; Akt and eNOS at the mRNA level and protein level as well as NF-κB, Nrf2, ERK1/2, NOX-4 protein level was measured. Additionally, a co-culture model between endothelial cells and monocytes to mimic the initial stages of atherosclerosis were implemented within this thesis. For monocyte adherence protocol, fluorescently labelled monocytes (THP-1) were added to HUVECs for 1 h after TNF-α stimulation and measured using a fluorescence plate reader. Supernatant was removed pre- and post-monocyte adherence to measure the concentration of cytokine release including IL-6, MCP-1, and IL-1β. Adhesion molecules on the surface of endothelial cells including VCAM-1 and ICAM-1 were also measured. Results: HUVECs pre-treated with EB, C3G, PCA and VA all prevented TNF-α induced monocyte adherence to endothelial cells (p < 0.03). This effect may have been a result of the treatments showing a reduction in TNF-α induced oxidative stress and inflammation (p < 0.05), as well as improving endothelial cell viability (p <0.05). Phenolic metabolites PCA and VA only increased eNOS activity in response to TNF-α (p <0.05), moreover VA showed increased activity by reducing NOX-4 and increasing HO-1 expression at physiological relevant concentrations. Discussion: The current thesis provides evidence that pre-exposing endothelial cells to phenolic metabolites, such as PCA and VA, enhances endothelial function by regulating multiple molecular pathways associated with endothelial function/dysfunction. Moreover, phenolic metabolites reduce the adhesion of monocytes to endothelial cells, which is a crucial initial stage in the progression of atherosclerosis. Importantly, these effects are observed at concentrations that are physiologically relevant. This was also associated with all treatments displaying improvements in cell viability, reduction in TNF-α induced apoptosis and reduction in oxidative stress. It is likely that the induction of inflammation is a pre-requisite for phenolic metabolites to induce its protective properties. Further research should explore these findings as mixture of metabolites or in more in vivo like models. The findings of this thesis could help further research with understanding which compounds have cardio-protective properties in polyphenol rich foods.
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    Elderberries as a potential supplement to improve vascular function in a SARS-CoV-2 environment
    (Wiley, 2022-02-03) Festa, Joseph; Singh, Harprit; Hussain, Aamir; Da Boit, Mariasole
    Coronavirus disease 2019 (COVID-19) pandemic has been triggered by the severe acute respiratory syndrome coronavirus (SARS-CoV-2). Although recent studies demonstrate that SARS-CoV-2 possibly does not directly infect endothelial cells (EC), the endothelium may be affected as a secondary response due to the damage of neighboring cells, circulating pro-inflammatory cytokines, and/or other mechanisms. Long-term COVID-19 symptoms specifically nonrespiratory symptoms are due to the persistence of endothelial dysfunction (ED). Based on the literature, anthocyanins a major subgroup of flavonoid polyphenols found in berries, have been well researched for their vascular protective properties as well as the prevention of cardiovascular disease (CVD)-related deaths. Elderberries have been previously used as a natural remedy for treating influenza, cold, and consequently cardiovascular health due to a high content of cyanidin-3-glucoside (C3G) a major anthocyanin found in the human diet. The literature reported many studies demonstrating that EE has both antiviral and vascular protective properties that should be further investigated as a nutritional component used against the (in)direct effect of SARS-CoV-2 in vascular function.
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    Elderberry extract improves molecular markers of endothelial dysfunction linked to atherosclerosis
    (Wiley, 2023-05-10) Festa, Joseph; Hussain, Aamir; Hackney, Amon; Desai, Unmesh; Sahota, T. S.; Singh, Harprit; Da Boit, Mariasole
    Endothelial dysfunction (ED), secondary to diminished nitric oxide (NO) production and oxidative stress, is an early subclinical marker of atherosclerosis. Reduced NO bioavailability enhances the adhesion of monocytes to endothelial cells and promotes atherosclerosis. Elderberry extract (EB) is known to contain high levels of anthocyanins which could exert vascular protective effects. Specifically, we investigated the functional capacity of EB on various markers of ED. Human umbilical vein endothelial cells (HUVEC) were pretreated with EB 50 μg/mL and stimulated with TNF-α 10 ng/mL. Cell viability, apoptosis, oxidative stress; eNOS, Akt, Nrf2, NOX-4, and NF-κB at the protein level were measured. A co-culture model was used to determine whether EB could prevent the adhesion of monocytes (THP-1) to HUVECs. Moreover, the expression of adhesion molecules and pro-inflammatory cytokines were also measured. It was demonstrated that EB prevented TNF-α induced apoptosis and reactive oxygen species production in HUVECs. Additionally, EB upregulated Akt and eNOS activity, and Nrf2 expression in response to TNF-α, whereas it decreased NOX-4 expression and NF-κB activity. EB prevented the adhesion of monocytes to HUVECs, as well as reduced IL-6 and MCP-1 levels, which was associated with inhibition of VCAM-1 expression. Our results demonstrate that EB upregulates key cellular markers of endothelial function and ameliorates markers of ED. EB could be used as a potential nutritional aid for preventing atherosclerosis progression.
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    Elderberry extract inhibits tumour necrosis factor induced monocyte adhesion to endothelial cells via modulation of the NF-κB pathway
    (Oxford University Press, 2022-06-10) Festa, Joseph; Singh, Harprit; Hussain, Aamir; Da Boit, Mariasole
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    Phenolic Metabolites Protocatechuic Acid and Vanillic Acid Improve Nitric Oxide Bioavailability via the Akt-eNOS Pathway in Response to TNF-α Induced Oxidative Stress and Inflammation in Endothelial Cells
    (MDPI, 2024-11-11) Festa, Joseph; Hussain, Aamir; Al-Hareth, Zakia; Bailey, Stephen J.; Singh, Harprit; Da Boit, Mariasole
    Background/Objectives: Reduced nitric oxide (NO) bioavailability secondary to excess-superoxide-driven oxidative stress is central to endothelial dysfunction. Previous studies suggest that phenolic metabolites may improve NO bioavailability, yet limited research is available in response to an inflammatory mediator. Therefore, we assessed the effects of cyanidin-3-glucoside (C3G) and its phenolic metabolites protocatechuic acid (PCA) and vanillic acid (VA) on NO bioavailability in a TNF-α induced inflammatory environment. Methods: Primary human umbilical vein endothelial cells (HUVECs) were supplemented with either C3G, PCA, or VA at 1 μM for 24 h before being stimulated with TNF-α 20 ng/mL for an additional 24 h. Measurements included cell viability, apoptosis, reactive oxygen species (ROS), nitrite concentrations, and endothelial nitric oxide synthase (eNOS) and Akt at the mRNA and protein level. Results: Phenolic metabolites did not increase the eNOS expression or nitrite levels in the unstimulated environment; rather, the metabolites mediated NO bioavailability in response to TNF-α induced oxidative stress, with increased viability, eNOS mRNA, phosphorylation, and nitrite levels. Conclusions: Phenolic metabolites, in the presence of TNF-α, can improve NO bioavailability at physiologically relevant concentrations via the Akt-eNOS pathway. This demonstrates that the induction of inflammation is a prerequisite for phenolic metabolites to promote protective properties in endothelial cells by activating the Akt-eNOS pathway.
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    Potential Benefits of Berry Anthocyanins on Vascular Function
    (Wiley, 2021-08-03) Festa, Joseph; Hussain, Aamir; Da Boit, Mariasole; Singh, Harprit