Havovi Chichger

Pulmonary vascular remodelling, driven by dysregulated angiogenesis, is a hallmark of several cardiopulmonary diseases, yet the molecular pathways regulating endothelial responses remain incompletely understood. Bitter taste receptors (T2Rs), traditionally recognised as chemosensory receptors, have emerged as important modulators of vascular function. However, the contribution of T2Rs to pulmonary angiogenesis remains unexplored. Here, we investigated the role of T2R14 in vascular endothelial growth factor (VEGF)‐mediated angiogenic responses in human pulmonary microvascular endothelial cells (HPMECs). Using the selective T2R14 antagonist LW129, we examined the effects of T2R14 inhibition on key endothelial angiogenic processes, including VEGF‐induced proliferation, migration and capillary‐like tube formation in vitro. Pharmacological antagonism of T2R14 significantly attenuated VEGF‐driven endothelial proliferative, migratory and morphogenic responses, revealing a novel contribution of T2R14 signalling to pulmonary microvascular angiogenesis. Proteome Profiler analysis identified a range of molecular regulators of angiogenic processes which were dysregulated by LW129 and VEGF exposure, suggesting a potential role for CXCL16, endoglin, FGF‐7, and VEGF‐C. These findings identify T2R14 as a novel regulator of VEGF‐dependent endothelial activation and suggest that targeting T2R14 signalling may represent a therapeutic strategy to limit pathological pulmonary vascular remodelling associated with aberrant angiogenesis.

EVs released from LPS-stimulated human pulmonary microvascular endothelial cells compromise barrier integrity and enhance migration via TLR4-mediated signaling. Inhibition of ceramide-dependent EV biogenesis with GW4869 attenuated barrier dysfunction, implicating specific EV subpopulations as pathogenic mediators. EV-TLR4 activation upregulates MyD88 and IL-6, suggesting an autocrine loop amplifying vascular injury in sepsis-induced ARDS, and identifying EV biogenesis and EV-mediated TLR4 signaling as novel therapeutic targets.

The development of acute lung injury in COVID‐19 patients is a major cause of lethality. Activation of the pulmonary vasculature, with inflammation, oxidative stress and barrier disruption, is initiated by the SARS‐CoV‐2 spike protein (Sp1) at the endothelial cell surface. We previously identified the presence of a novel G‐Protein Coupled Receptor (GPCR) in the lung microvasculature, T1R3. The T1R3 agonist (T1R3‐A) protects the endothelium from endotoxin‐induced lung injury therefore we sought to investigate the effect of T1R3‐A on Sp1‐induced damage to the pulmonary endothelium. Sp1 induced significant disruption to the endothelium with elevated gap formation, monolayer leak and ROS accumulation. This damage was attenuated following exposure of the lung microvasculature to T1R3‐A. We further demonstrated that this agonist promotes ACE2 expression at the endothelial cell surface. Using immunoprecipitation studies, we demonstrated that T1R3‐A increases ACE2:T1R3 binding which may be a mechanism for protecting against Sp1‐induced endothelial cell injury. Our findings highlight the protective effect of T1R3 activation in the pulmonary endothelium and that there is a close link between T1R3 and ACE2. We propose that T1R3 activation protects the endothelium by preventing Sp1‐induced internalisation of ACE2, thereby blocking downstream ACE2 signalling leading to pulmonary vascular injury.

Background: Pulmonary drug delivery represents a promising approach for the potential localised treatment of respiratory of non-small-cell lung cancer (NSCLC). However, the efficient delivery of poorly water-soluble drugs remains challenging due to limited solubility and inadequate aerodynamic performance. This study aimed to develop and characterise inhalable dry powder formulations of zinc diethyldithiocarbamate (Zn(DDC)₂) complexes with hydroxypropyl-β-cyclodextrin (HP-β-CD) and sulfobutylether-β-cyclodextrin (SBE-β-CD) for potential pulmonary administration. Methods: Formulations were prepared by freeze-drying and spray-drying, with leucine incorporated at 0%, 5%, and 10% w/w. Formulations were prepared via freeze-drying and spray-drying with leucine incorporation (0%, 5% and 10% w/w) to evaluate their physicochemical properties, flowability and aerodynamic performance. Results: Spray-dried formulations exhibited significantly lower densities (as low as 1.03 ± 0.71 g/cm³), enhanced flowability, improved aerosolisation and higher fine particle fraction (FPF) values (up to 40.12 ± 0.60%) compared to freeze-dried powders (20.03 ± 2.79%). The incorporation of leucine further reduced powder density down to 0.72 ± 0.34 g/cm³ and increased surface corrugation as shown in SEM images, improving aerosolisation performance, with FPF values up to 76.77 ± 1.18%. Next Generation Impactor (NGI) analysis confirmed that leucine-containing formulations exhibited a greater proportion of particles within the respirable aerodynamic diameter range (1–5 μm), suggesting suitability for deep lung deposition. Conclusions: These results demonstrate that spray-dried Zn(DDC)₂–cyclodextrin powders, particularly those modified with 10% leucine, offer excellent potential for pulmonary delivery in NSCLC therapy. Further in vivo studies are warranted to evaluate therapeutic efficacy and safety.

Introduction: The increase in e-cigarette use in the population has led to substantial interest in the health impacts associated with e-cigarette smoking. E-cigarette smoking represents a key external environmental cell stressor. Whilst there have been several studies to investigate the effect of nicotine-containing e-cigarette fluid, there is still a significant lack of understanding of how nicotine-free e-cigarette smoking can impact individuals. However, preliminary studies indicate that nicotine-free e-cigarette smoking can cause impaired endothelial function in humans. Materials and Methods: In the present study, we therefore used a common brand of nicotine-free e-cigarette and human umbilical vein endothelial cells to assess angiogenic processes in vitro . Results: We observed a significant upregulation in endothelial cell adhesion, migration and new tube formation with exposure to nicotine-free e-cigarette condensate (eVape) which was abrogated with exposure to the antioxidant, N-acetyl cysteine. Proteome analysis demonstrated that eVape exposure increased expression of the pro-angiogenic factors, angiogpoeitin-2, endoglin (CD105), PIGF and VEGF, as well as the ADP ribosylation factor, ARF6, and ARF6-GEF, ARNO. Chemical inhibition of ARNO reduced eVape-induced oxidative stress, angiogenic processes, and release of angiogpoeitin-2, endoglin (CD105) and VEGF. Discussion: These findings demonstrate that nicotine-free eVape causes aberrant upregulated angiogenesis in an in vitro model of the human endothelium through ARNO-dependent signalling. This study is the first to demonstrate the molecular mechanisms in response to the cellular stressor, nicotine-free eVape which underlie impaired vascular function.

Impaired intestinal barrier function is a precursor to various metabolic diseases which can occur when the intestinal epithelium is directly exposed to certain dietary components. Previous studies have demonstrated the barrier disruptive effect of artificial sweeteners such as sucralose and saccharin, in the intestinal epithelium. In the present study, we aimed to evaluate the impact of 3 non-synthetic sweeteners, stevia, miraculin, and mogroside V, on Caco-2 monolayer barrier integrity, reactive oxygen species (ROS) production, and tight junction protein expression as compared to the artificial sweetener saccharin. Miraculin and mogroside V exerted a significant impact on cell numbers with decreased cell viability at both 24 and 48 h. ROS production was also significantly increased by miraculin and mogroside V and epithelial barrier function, assessed by transepithelial electrical resistance and FITC-dextran leak, was significantly disrupted with both miraculin and mogroside V. Stevia exhibited no effect on epithelial cell viability, ROS accumulation or barrier function, despite a range of concentrations investigated. Knockdown of the sweet taste receptor, T1R3, significantly attenuated miraculin- and mogroside V-induced loss of cell viability, ROS accumulation and barrier disruption suggesting a T1R3-dependent mechanism. Gene expression analysis revealed differential regulation of cell junction-related genes by mogroside V and miraculin with upregulated expression of genes such as CAV1 , CLDN2 and CLDN10 , and downregulated expression of genes such as CAV3 and CDH2 . These findings demonstrate that miraculin and mogroside V, but not stevia, negatively regulate ROS formation and intestinal epithelial barrier function in a T1R3-dependent manner and potentially through modulation of tight junction proteins. This highlights the need for further investigation into the long-term dietary implications of natural sweeteners, particularly miraculin and mogroside V, on gut health.

Background/Objectives: There is current scientific interest pertaining to the therapeutic effects of olive-derived polyphenols (ODPs), in particular their associated anti-inflammatory properties, following the wealth of research surrounding the physiological impact of the Mediterranean Diet (MD). Despite this association, the majority of the current literature investigates ODPs in conjunction with metabolic diseases. There is limited research focusing on ODPs and acute inflammation following exercise, regardless of the knowledge surrounding the elevated inflammatory response during this time. Therefore, the aim of this scoping review is to understand the impact ODPs may have on exercise-induced inflammation. Methods: This scoping review was undertaken in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScRs). The literature searches were conducted in PubMed and EBSCOhost and considered for review if records reported original data, examined olives, olive-derived nutrients, food sources, or ODPs in conjunction with exercise-induced inflammation (including known causes, associations, and proxy measures). Results: Seven studies investigated ODPs and exercise-induced inflammation, providing commentary on reduced oxidative stress, inflammatory biomarkers, and immune biomarkers, enhanced antioxidant defenses and modulations in mitochondrial function, albeit in low numbers. An average of 100.9 mg∙d⁻¹ ODPs were supplemented for an average of 40 days, with hydroxytyrosol (HT) being the primary ODP investigated. Six studies employed individual aerobic exercise as their stimulus, whilst one study investigated the impact of an acute dose of ODP. Conclusions: There is a limited consensus on the direction of isolated HT in human models, whereas animal models suggest a reduced inflammatory response following ≥2 weeks HT supplementation in conjunction with chronic exercise. Future research should initially investigate the inflammatory response of ODP, with particular focus on HT, and aim to identify an optimum dose and time course for supplementation surrounding exercise to support acute recovery and exercise adaptations.

Introduction: Recent studies have indicated considerable health risks associated with the consumption of artificial sweeteners. Neotame is a relatively new sweetener in the global market however there is still limited data on the impact of neotame on the intestinal epithelium or the commensal microbiota.Methods: In the present study, we use a model of the intestinal epithelium (Caco-2) and microbiota (Escherichia coli and Enterococcus faecalis) to investigate how physiologically-relevant exposure of neotame impacts intestinal epithelial cell function, gut bacterial metabolism and pathogenicity, and gut epithelium-microbiota interactions.Results: Our findings show that neotame causes intestinal epithelial cell apoptosis and death with siRNA knockdown of T1R3 expression significantly attenuating the neotame-induced loss to cell viability. Similarly, neotame exposure results in barrier disruption with enhanced monolayer leak and reduced claudin-3 cell surface expression through a T1R3-dependent pathway. Using the gut bacteria models, E. coli and E. faecalis, neotame significantly increased biofilm formation and metabolites of E. coli, but not E. faecalis, reduced Caco-2 cell viability. In co-culture studies, neotame exposure increased adhesion capacity of E. coli and E. faecalis onto Caco-2 cells and invasion capacity of E. coli. Neotame-induced biofilm formation, E.coli-specific Caco-2 cell death, adhesion and invasion was identified to be meditated through a taste-dependent pathway.Discussion: Our study identifies novel pathogenic effects of neotame on the intestinal epithelium or bacteria alone, and in co-cultures to mimic the gut microbiome. These findings demonstrate the need to better understand food additives common in the global market and the molecular mechanisms underlying potential negative health impacts.

Zinc diethyldithiocarbamate (Zn (DDC)₂), a disulfiram metabolite (anti-alcoholism drug), has shown a strong anti-cancer activity in vitro. However, its application was limited by its low aqueous solubility and rapid metabolism. In this study, the solubility enhancement of Zn (DDC)₂ is investigated by forming inclusion complexes with cyclodextrins. The inclusion complexes were prepared using two different types of beta-cyclodextrins, SBE-CD and HP-CD. Phase solubility diagrams for the resulting solutions were assessed; subsequently, the solutions were freeze-dried for further characterisation studies using DSC, TGA, XRD, and FTIR. The cytotoxic activity of the produced inclusion complexes was evaluated on human lung carcinoma cells using the MTT assay. The solubility of Zn (DDC)₂ increased significantly upon adding beta-cyclodextrins, reaching approximately 4 mg/mL for 20% w/w CD solutions. The phase solubility diagram of Zn (DDC)₂ was of the Ap-type according to the Higuchi and Connors model. Characterisation studies confirmed the inclusion of the amorphous drug in the CD-Zn (DDC)₂ complexes. The cytotoxicity of Zn (DDC)₂ was enhanced 10-fold by the inclusion complexes compared to the free drug. Overall, the resulting CD-Zn (DDC)₂ inclusion complexes have a potential for treatment against lung cancer.