Cholesterol homeostasis in hair follicle keratinocytes is disrupted by impaired ABCA5 activity

The importance of cholesterol in hair follicle biology is underscored by its links to the pathogenesis of alopecias and hair growth disorders. Reports have associated defects in ABCA5, a membrane transporter, with altered keratinocyte cholesterol distribution in individuals with a form of congenital hypertrichosis, yet the biological basis for this defect in hair growth remains unknown. This study aimed to determine the impact of altered ABCA5 activity on hair follicle keratinocyte behaviour. Primary keratinocytes isolated from the outer root sheath of plucked human hair follicles were utilised as a relevant cell model. Following exogenous cholesterol loading, an increase in ABCA5 co-localisation to intracellular organelles was seen. Knockdown of ABCA5 revealed a dysregulation in cholesterol homeostasis, with LXR agonism leading to partial restoration of the homeostatic response. Filipin staining and live BODIPY cholesterol immunofluorescence microscopy revealed a reduction in endo-lysosomal cholesterol following ABCA5 knockdown. Analysis of oxysterols showed a significant increase in the fold change of 25-hydroxycholesterol and 7-β-hydroxycholesterol following cholesterol loading in ORS keratinocytes, after ABCA5 knockdown. These data suggest a role for ABCA5 in the intracellular compartmentalisation of free cholesterol in primary hair follicle keratinocytes. The loss of normal homeostatic response, following the delivery of excess cholesterol after ABCA5 knockdown, suggests an impact on LXR-mediated transcriptional activity. The loss of ABCA5 in the hair follicle could lead to impaired endo-lysosomal cholesterol transport, impacting pathways known to influence hair growth. This avenue warrants further investigation. [Abstract copyright: Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.

TNF-α-Mediated Endothelial Cell Apoptosis Is Rescued by Hydrogen Sulfide

Endothelial dysfunction is implicated in the development and aggravation of cardiovascular complications. Among the endothelium-released vasoactive factors, hydrogen sulfide (H2S) has been investigated for its beneficial effects on the vasculature through anti-inflammatory and redox-modulating regulatory mechanisms. Reduced H2S bioavailability is reported in chronic diseases such as cardiovascular disease, diabetes, atherosclerosis and preeclampsia, suggesting the value of investigating mechanisms, by which H2S acts as a vasoprotective gasotransmitter. We explored whether the protective effects of H2S were linked to the mitochondrial health of endothelial cells and the mechanisms by which H2S rescues apoptosis. Here, we demonstrate that endothelial dysfunction induced by TNF-α increased endothelial oxidative stress and induced apoptosis via mitochondrial cytochrome c release and caspase activation over 24 h. TNF-α also affected mitochondrial morphology and altered the mitochondrial network. Post-treatment with the slow-releasing H2S donor, GYY4137, alleviated oxidising redox state, decreased pro-caspase 3 activity, and prevented endothelial apoptosis caused by TNF-α alone. In addition, exogenous GYY4137 enhanced S-sulfhydration of pro-caspase 3 and improved mitochondrial health in TNF-α exposed cells. These data provide new insights into molecular mechanisms for cytoprotective effects of H2S via the mitochondrial-driven pathway

Distribution of plasma oxidised phosphatidylcholines in chronic kidney disease and periodontitis as a co-morbidity

Individuals with chronic kidney disease (CKD) and periodontitis as a co-morbidity have a higher mortality rate than individuals with CKD and no periodontitis. The inflammatory burden associated with both diseases contributes to an increased risk of cardiovascular and all-cause mortality. We previously demonstrated that periodontitis is associated with increasing circulating markers of inflammation and oxidative stress. We propose that inflammatory oxidised phosphocholines may contribute to the increased risk of cardiovascular disease in patients with CKD. However, the analysis of oxidised phospholipids has been limited by a lack of authentic standards for absolute quantification. Here, we have developed a comprehensive quantification liquid chromatography-mass spectrometry-based multiple reaction monitoring method for oxidised phospholipids (including some without available authentic species) that enables us to simultaneously measure twelve oxidised phosphatidylcholine species with high levels of sensitivity and specificity. The standard curves for commercial standards 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphatidylcholine (PGPC); 1-palmitoyl-2-(9′-oxo-nonanoyl)-sn-glycero-3-phosphatidylcholine (PONPC), 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphatidylcholine (PAzPC) and 1-palmitoyl-2-(5′-oxo-valeroyl)-sn-glycero-3-phosphatidylcholine (POVPC), were linear with a correlation coefficient greater than 0.99 for all analytes. The method is reproducible, with intra- and inter-day precision <15%, and accuracy within ±5% of nominal values for all analytes. This method has been successfully applied to investigate oxidised phosphatidylcholine in plasma from CKD patients with and without chronic periodontitis and the data that was obtained has been compared to plasma from healthy controls. Comparative analysis demonstrates altered chain fragmented phosphatidylcholine profiles in the plasma samples of patients with CKD and periodontitis as a co-morbidity compared to healthy controls

Paternal low protein diet perturbs inter-generational metabolic homeostasis in a tissue-specific manner in mice

The underlying mechanisms driving paternally-programmed metabolic disease in offspring remain poorly defined. We fed male C57BL/6 mice either a control normal protein diet (NPD; 18% protein) or an isocaloric low protein diet (LPD; 9% protein) for a minimum of 8 weeks. Using artificial insemination, in combination with vasectomised male mating, we generated offspring using either NPD or LPD sperm but in the presence of NPD or LPD seminal plasma. Offspring from either LPD sperm or seminal fluid display elevated body weight and tissue dyslipidaemia from just 3 weeks of age. These changes become more pronounced in adulthood, occurring in conjunction with altered hepatic metabolic and inflammatory pathway gene expression. Second generation offspring also display differential tissue lipid abundance, with profiles similar to those of first generation adults. These findings demonstrate that offspring metabolic homeostasis is perturbed in response to a suboptimal paternal diet with the effects still evident within a second generation

Sulforaphane restores cellular glutathione levels and reduces chronic periodontitis neutrophil hyperactivity in vitro

The production of high levels of reactive oxygen species by neutrophils is associated with the local and systemic destructive phenotype found in the chronic inflammatory disease periodontitis. In the present study, we investigated the ability of sulforaphane (SFN) to restore cellular glutathione levels and reduce the hyperactivity of circulating neutrophils associated with chronic periodontitis. Using differentiated HL60 cells as a neutrophil model, here we show that generation of extracellular O2 . - by the nicotinamide adenine dinucleotide (NADPH) oxidase complex is increased by intracellular glutathione depletion. This may be attributed to the upregulation of thiol regulated acid sphingomyelinase driven lipid raft formation. Intracellular glutathione was also lower in primary neutrophils from periodontitis patients and, consistent with our previous findings, patients neutrophils were hyper-reactive to stimuli. The activity of nuclear factor erythroid-2-related factor 2 (Nrf2), a master regulator of the antioxidant response, is impaired in circulating neutrophils from chronic periodontitis patients. Although patients' neutrophils exhibit a low reduced glutathione (GSH)/oxidised glutathione (GSSG) ratio and a higher total Nrf2 level, the DNA-binding activity of nuclear Nrf2 remained unchanged relative to healthy controls and had reduced expression of glutamate cysteine ligase catalytic (GCLC), and modifier (GCLM) subunit mRNAs, compared to periodontally healthy subjects neutrophils. Pre-treatment with SFN increased expression of GCLC and GCM, improved intracellular GSH/GSSG ratios and reduced agonist-activated extracellular O2 . - production in both dHL60 and primary neutrophils from patients with periodontitis and controls. These findings suggest that a deficiency in Nrf2-dependent pathways may underpin susceptibility to hyper-reactivity in circulating primary neutrophils during chronic periodontitis. © 2013 Dias et al

Mechanisms contributing to the enhanced respiratory burst of neutrophils observed in periodontitis

The aim of this thesis is to investigate possible mechanisms that may contribute to neutrophil hyperactivity and hyper-reactivity. One possibility is the presence of a neutrophil priming factors within the peripheral circulation of periodontitis patients. To examine this possibility differentiated HL-60 cells and primary neutrophils were studied in the presence and absence of plasma from periodontitis patients. In independent experiments, plasma was depleted of IL-8, GM-CSF, interferon-a, immunoglobulins and albumin. This work demonstrated that plasma factors such as IL-8, GM-CSF, and interferon-a present during periodontitis may contribute towards the reported hyperactive neutrophil phenotype. Furthermore, this work demonstrated that products from Pg may regulate neutrophil accumulation at infected periodontal sites by promoting gingipain-dependent modification of IL-8-77 into a more biologically active chemokine. To elucidate whether the oxidatively stressed environment that neutrophils are exposed to in periodontitis could influence hyperactivity and hyper-reactivity, neutrophils were depleted of glutathione. This work showed that during oxidative stress, where cellular redox-levels have been altered, neutrophils exhibit an increased respiratory burst. In conclusion, this work highlights the multiple mechanisms that may contribute to neutrophil hyperactivity and hyperreactivity including gingipain-modulated activity of IL-8 variants, the effect of host factors such as IL-8, GM-CSF, interferon-a on neutrophils priming and activation, and the shift of neutrophil GSH:GSSG ratio in favour of a more oxidised environment as observed in periodontitis.EThOS - Electronic Theses Online ServiceGBUnited Kingdo