Electronic vape fluid activates the pulmonary endothelium and disrupts vascular integrity in vitro through an ARF6-dependent pathway.

The use of e-cigarettes or vapes is increasingly popular amongst a range of different demographics however the research in this area is surprisingly sparse. Clinical reports of e-cigarette- or vaping use-associated lung injury (EVALI) and vascular disruption, in both nicotine-containing and nicotine-free e-cigarette smokers, prompts the need for further research with a focus on the pulmonary endothelium. Using a common brand of e-cigarette (eVape) and an in vitro model of the human lung microvasculature, we investigated the effect of nicotine-free eVape fluid on pulmonary endothelial barrier integrity, oxidative stress and inflammation profile. Findings demonstrate reactive oxygen species-dependent breakdown of the pulmonary endothelium and release of inflammatory cytokines. These phenotypic changes, following exposure to nicotine-free eVape fluid, were accompanied by dysregulation of a number of adheren junctions-related genes of which ARF6 was most abundantly overexpressed. Further investigation of ARF6 identified it as a key regulator in eVape-induced barrier disruption and ROS accumulation. This study demonstrates, for the first time, the barrier disruptive effect of nicotine-free e-cigarette fluid on the pulmonary microvasculature and the ARF6 and ROS-dependent molecular mechanisms underlying this damage. Whilst these studies focus on a human in vitro model of the pulmonary microvasculature, the results support clinical case studies on EVALI and demonstrate a need for further investigation of the impact of nicotine-free e-cigarettes on the lung.

The Effect of a Hydroxytyrosol-Rich, Olive-Derived Phytocomplex on Aerobic Exercise and Acute Recovery.

There is current scientific interest in naturally sourced phenolic compounds and their potential benefits to health, as well as the effective role polyphenols may provide in an exercise setting. This study investigated the chronic effects of supplementation with a biodynamic and organic olive fruit water phytocomplex (OliPhenolia® [OliP]), rich in hydroxytyrosol (HT), on submaximal and exhaustive exercise performance and respiratory markers of recovery. Twenty-nine recreationally active participants (42 ± 2 yrs; 71.1 ± 2.1 kg; 1.76 ± 0.02 m) consumed 2 × 28 mL∙d−1 of OliP or a taste- and appearance-matched placebo (PL) over 16 consecutive days. Participants completed a demanding, aerobic exercise protocol at ~75% maximal oxygen uptake (V˙O2max) for 65 min 24 h before sub- and maximal performance exercise tests prior to and following the 16-day consumption period. OliP reduced the time constant (τ) (p = 0.005) at the onset of exercise, running economy (p = 0.015) at lactate threshold 1 (LT1), as well as the rating of perceived exertion (p = 0.003) at lactate turnpoint (LT2). Additionally, OliP led to modest improvements in acute recovery based upon a shorter time to achieve 50% of the end of exercise V˙O2 value (p = 0.02). Whilst OliP increased time to exhaustion (+4.1 ± 1.8%), this was not significantly different to PL (p > 0.05). Phenolic compounds present in OliP, including HT and related metabolites, may provide benefits for aerobic exercise and acute recovery in recreationally active individuals. Further research is needed to determine whether dose-response or adjunct use of OliP alongside longer-term training programs can further modulate exercise-associated adaptations in recreationally active individuals, or indeed support athletic performance.

Preparation and Characterisation of Zinc Diethyldithiocarbamate-Cyclodextrin Inclusion Complexes for Potential Lung Cancer Treatment.

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.

The Impact of a Natural Olive-Derived Phytocomplex (OliPhenolia®) on Exercise-Induced Oxidative Stress in Healthy Adults.

The role of natural polyphenols in reducing oxidative stress and/or supporting antioxidant mechanisms, particularly relating to exercise, is of high interest. The aim of this study was to investigate OliPhenolia® (OliP), a biodynamic and organic olive fruit water phytocomplex, rich in hydroxytyrosol (HT), for the first time within an exercise domain. HT bioavailability from OliP was assessed in fifteen healthy volunteers in a randomized, double-blind, placebo controlled cross-over design (age: 30 ± 2 yrs; body mass: 76.7 ± 3.9 kg; height: 1.77 ± 0.02 m), followed by a separate randomized, double-blinded, cohort trial investigating the short-term impact of OliP consumption (2 × 28 mL∙d−1 of OliP or placebo (PL) for 16-days) on markers of oxidative stress in twenty-nine recreationally active participants (42 ± 2 yrs; 71.1 ± 2.1 kg; 1.76 ± 0.02 m). In response to a single 28 mL OliP bolus, plasma HT peaked at 1 h (38.31 ± 4.76 ng∙mL−1), remaining significantly elevated (p < 0.001) until 4 h. Plasma malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH) and HT were assessed at rest and immediately following exercise (50 min at ~75% V˙O2max then 10 min intermittent efforts) and at 1 and 24 h post-exercise, before and after the 16-day supplementation protocol. Plasma HT under resting conditions was not detected pre-intervention, but increased to 6.3 ± 1.6 ng·mL−1 following OliP only (p < 0.001). OliP demonstrated modest antioxidant effects based on reduced SOD activity post-exercise (p = 0.016) and at 24 h (p ≤ 0.046), and increased GSH immediately post-exercise (p = 0.009) compared with PL. No differences were reported for MDA and CAT activity in response to the exercise protocol between conditions. The phenolic compounds within OliP, including HT, may have specific antioxidant benefits supporting acute exercise recovery. Further research is warranted to explore the impact of OliP following longer-term exercise training, and clinical domains pertinent to reduced oxidative stress.

Investigating the Opposing Effect of Two Different Green Tea Supplements on Oxidative Stress, Mitochondrial Function and Cell Viability in HepG2 Cells.

Green tea extract (GTE) improves exercise outcomes and reduces obesity. However, case studies indicate contradictory physiology regarding liver function and toxicity. We studied the effect of two different decaffeinated GTE (dGTE) products, from a non-commercial (dGTE1) and commercial (dGTE2) supplier, on hepatocyte function using the human cell model, HepG2. dGTE1 was protective against hydrogen peroxide (H2O2)-induced apoptosis and cell death by attenuating oxidative stress pathways. Conversely, dGTE2 increased cellular and mitochondrial oxidative stress and apoptosis. A bioavailability study with dGTE showed the major catechin in GTE, EGCG, reached 0.263 µg·ml-1. In vitro, at this concentration, EGCG mimicked the protective effect of dGTE1. GC/MS analysis identified steric acid and higher levels of palmitic acid in dGTE2 versus dGTE1 supplements. We demonstrate the significant biological differences between two GTE supplements which may have potential implications for manufacturers and consumers to be aware of the biological effects of supplementation.

Diet-induced iron deficiency in rats impacts small intestinal calcium and phosphate absorption.

AIMS: Recent reports suggest that iron deficiency impacts both intestinal calcium and phosphate absorption, although the exact transport pathways and intestinal segment responsible have not been determined. Therefore, we aimed to systematically investigate the impact of iron deficiency on the cellular mechanisms of transcellular and paracellular calcium and phosphate transport in different regions of the rat small intestine. METHODS: Adult, male Sprague-Dawley rats were maintained on a control or iron-deficient diet for 2 weeks and changes in intestinal calcium and phosphate uptake were determined using the in situ intestinal loop technique. The circulating levels of the hormonal regulators of calcium and phosphate were determined by ELISA, while the expression of transcellular calcium and phosphate transporters, and intestinal claudins were determined using qPCR and western blotting. RESULTS: Diet-induced iron deficiency significantly increased calcium absorption in the duodenum but had no impact in the jejunum and ileum. In contrast, phosphate absorption was significantly inhibited in the duodenum and to a lesser extent the jejunum, but remained unchanged in the ileum. The changes in duodenal calcium and phosphate absorption in the iron-deficient animals were associated with increased claudin 2 and 3 mRNA and protein levels, while levels of parathyroid hormone, fibroblast growth factor-23 and 1,25-dihydroxy vitamin D3 were unchanged. CONCLUSION: We propose that iron deficiency alters calcium and phosphate transport in the duodenum. This occurs via changes to the paracellular pathway, whereby upregulation of claudin 2 increases calcium absorption and upregulation of claudin 3 inhibits phosphate absorption.

Lutein and zeaxanthin attenuates VEGF-induced neovascularisation in human retinal microvascular endothelial cells through a Nox4-dependent pathway.

Age-related macular degeneration (AMD) and proliferative diabetic retinopathy (DR) are two of the most common and severe causes of vision loss in the population. Both conditions are associated with excessive levels of vascular endothelial growth factor (VEGF) in the eye which results in an increase in the formation of new blood vessels through a process called neovascularisation. As such, anti-VEGF therapies are currently utilised as a treatment for patients with AMD however they are associated with painful administration of injections and potential degeneration of healthy endothelium. There is therefore growing interest in alternate treatment options to reduce neovascularisation in the eye. The use of carotenoids, lutein (L) and zeaxanthin (Z), has been shown to improve vision loss parameters in patients with AMD, however the underlying mechanisms are not well-understood. We studied the impact of these compounds on neovascularisation processes using an in vitro cell model of the retinal microvascular endothelium. Our findings show that L and Z reduced VEGF-induced tube formation whilst, in combination (5:1 ratio), the compounds significantly blocked VEGF-induced neovascularisation. The carotenoids, individually and in combination, reduced VEGF-induced oxidative stress concomitant with increased activity of the NADPH oxidase, Nox4. We further demonstrated that the Nox4 inhibitor, GLX7013114, attenuated the protective effect of L and Z. Taken together, these findings indicate the protective effect of the carotenoids, L and Z, in reducing VEGF-mediated neovascularisation via a Nox4-dependent pathway. These studies implicate the potential for these compounds to be used as a therapeutic approach for patients suffering from AMD and proliferative DR.

Effect of α7 nicotinic acetylcholine receptor activation on cardiac fibroblasts: a mechanism underlying RV fibrosis associated with cigarette smoke exposure.

Right ventricular (RV) dysfunction is associated with numerous smoking-related illnesses, including chronic obstructive pulmonary disease (COPD), in which it is present even in the absence of pulmonary hypertension. It is unknown whether exposure to cigarette smoke (CS) has direct effects on RV function and cardiac fibroblast (CF) proliferation or collagen synthesis. In this study, we evaluated cardiac function and fibrosis in mice exposed to CS and determined mechanisms of smoke-induced changes in CF signaling and fibrosis. AKR mice were exposed to CS for 6 wk followed by echocardiography and evaluation of cardiac hypertrophy, collagen content, and pulmonary muscularization. Proliferation and collagen content were evaluated in primary isolated rat CFs exposed to CS extract (CSE) or nicotine. Markers of cell proliferation, fibrosis, and proliferative signaling were determined by immunoblot or Sircol collagen assay. Mice exposed to CS had significantly decreased RV function, as determined by tricuspid annular plane systolic excursion. There were no changes in left ventricular parameters. RV collagen content was significantly elevated, but there was no change in RV hypertrophy or pulmonary vascular muscularization. CSE directly increased CF proliferation and collagen content in CF. Nicotine alone reproduced these effects. CSE and nicotine-induced fibroblast proliferation and collagen content were mediated through α7 nicotinic acetylcholine receptors and were dependent on PKC-α, PKC-δ, and reduced p38-MAPK phosphorylation. CS and nicotine have direct effects on CFs to induce proliferation and fibrosis, which may negatively affect right heart function.

p18, a novel adaptor protein, regulates pulmonary endothelial barrier function via enhanced endocytic recycling of VE-cadherin.

Vascular permeability is a hallmark of several disease states including acute lung injury (ALI). Endocytosis of VE-cadherin, away from the interendothelial junction (IEJ), causes acute endothelial barrier permeability. A novel protein, p18, anchors to the endosome membrane and plays a role in late endosomal signaling via MAPK and mammalian target of rapamycin. However, the fate of the VE-cadherin-positive endosome has yet to be elucidated. We sought to elucidate a role for p18 in VE-cadherin trafficking and thus endothelial barrier function, in settings of ALI. Endothelial cell (EC) resistance, whole-cell ELISA, and filtration coefficient were studied in mice or lung ECs overexpressing wild-type or nonendosomal-binding mutant p18, using green fluorescent protein as a control. We demonstrate a protective role for the endocytic protein p18 in endothelial barrier function in settings of ALI in vitro and in vivo, through enhanced recycling of VE-cadherin-positive early endosomes to the IEJ. In settings of LPS-induced ALI, we show that Src tethered to the endosome tyrosine phosphorylates p18 concomitantly with VE-cadherin internalization and pulmonary edema formation. We conclude that p18 regulates pulmonary endothelial barrier function in vitro and in vivo, by enhancing recycling of VE-cadherin-positive endosomes to the IEJ.

On the mechanism of sequence-specific DNA-dependent acetylation of p53: the acetylation motif is exposed upon DNA binding.

P53 acetylation requires p300-docking to two contiguous sites in the activation domain that in turn mediates DNA-dependent acetylation of the tetramer. In an attempt to further define the mechanism of DNA-dependent acetylation of p53, an in vitro system has been reconstituted with distinct p53 isoforms and has been used to reveal conformational constraints on p53 acetylation. Two native p53 tetrameric isoforms purified from Sf9 cells differing by the extent of phosphorylation within the C-terminal acetylation site are both acetylated in a sequence-specific DNA-dependent manner. By contrast, p53 purified from an Escherichia coli expression system is in a largely denatured conformation and its acetylation is DNA-independent. Heating native p53 to destroy the folded structure restores DNA-independent acetylation similar to that seen with bacterially expressed p53. There are at least two sites of conformational flexibility in the p53 tetramer: the first in the flexible S10 beta-sheet within the MDM2 ubiquitination sequence and the second in the C-terminal regulatory domain. We analysed therefore whether DNA-dependent acetylation correlated with conformational changes in either of these two regions. DNA-dependent acetylation of p53 is maintained in a dose-dependent manner by low concentrations of consensus site DNA under conditions where flexibility in the S10 beta-sheet region is maintained. Oligonucleotide DNAs that promote acetylation stimulate the binding of monoclonal antibodies PAb421 and ICA-9; two antibodies whose contiguous epitopes overlap the C-terminal acetylation motif. By contrast, bent oligonucleotide DNAs that conceal both the S10 beta-sheet from binding of the monoclonal antibody DO-12 and attenuate binding of the monoclonal antibody PAb421 can preclude acetylation. These data suggest that, in the absence of DNA, the acetylation motif of p53 is in a cryptic state, but after DNA binding, allosteric effects mediate an exposure of the acetylation motif to allow DNA-dependent acetylation of the tetramer.