Biology subjects

McKeown, L.

Publications and source records attributed to McKeown, L..

8 recordsLinked to original sources

bioRxiv2026

Diabetes impacts endothelial Weibel-Palade body biogenesis and VWF secretion

Diabetes is associated with endothelial dysfunction, impaired wound healing, and increased thrombotic risk, yet the impact of diabetes on endothelial secretory organelles remains poorly understood. Weibel-Palade bodies (WPBs) are specialised endothelial granules that store and release von Willebrand factor (VWF) and other vasoactive cargo essential for haemostasis, inflammation, and vascular repair. Here, we investigated how diabetic environments influence WPB biogenesis and VWF structure under physiologically relevant flow conditions. Acute exposure of endothelial cells to constant or fluctuating high glucose concentrations, designed to model diabetic glycaemic conditions, did not alter WPB number or morphology under either static or high laminar shear stress conditions. In contrast, primary endothelial cells derived from a diabetic donor exhibited reduced Akt and eNOS signalling, significantly fewer WPBs, reduced intracellular VWF content, and shorter stimulus-evoked VWF strings compared with non-diabetic endothelial cells. Although total cellular VWF levels were reduced, high molecular weight (HMW) VWF content within endothelial lysates was not significantly altered. Plasma from diabetic patients demonstrated elevated circulating VWF levels together with marked inter-patient heterogeneity in VWF multimer composition. These findings suggest that chronic diabetes-associated endothelial dysfunction, rather than hyperglycaemia alone, alters WPB biology and VWF handling. We propose that dysregulated basal endothelial secretion may deplete endothelial VWF stores, limiting appropriate stimulus-coupled WPB release during vascular injury and contributing to defective vascular repair in diabetes.

cell biology↗

bioRxiv2025

Two-pore channel protein 2-mediated calcium release promotes angiopoietin 2 secretion by regulating Rab46-dependent Weibel-Palade body trafficking

Angiopoietin2 (Ang2), a regulator of angiogenesis, is stored with other pro-inflammatory and pro-thrombotic mediators, in endothelial-specific vesicles called Weibel-Palade bodies (WPBs). The WPB secretagogue, histamine, delays Ang2 secretion by activating Rab46-specific trafficking of Ang2-positive WPBs to the microtubule organising centre (MTOC), where they persist until Ca2+ binds to the EF-hand of Rab46, enabling detachment. Here, using Ca2+ imaging and high-resolution light microscopy, we pharmacologically investigated the contribution of endolysosomal two-pore channels proteins (TPC) to the Ca2+ signal necessary for Ang2 secretion. We show an increase in the histamine-evoked clustering of Rab46 (and thus WPBs) at the MTOC in the presence of TPC inhibitors Ned19 and tetrandrine, and a decrease in the presence of a TPC2 agonist, TPC2-A1-N. Histamine-evoked secretion of Ang2 was decreased by pharmacological inhibition of TPC channels but potentiated in the presence of an TPC2 agonist. These data suggest that histamine-mediated Ca2+ release via TPC2 channels is necessary for the Rab46-dependent detachment of Ang2-positive WPBs from the MTOC and thus Ang2 secretion. SummaryCa2+ binding to the EF-hand of Rab46 in endothelial cells has previously been reported but the molecular mechanisms and functional relevance is unclear. Here the authors show that Ca2+ released from TPC2 regulates the detachment of Rab46 from the MTOC and thereby allows secretion of Ang2 from WPBs.

cell biology↗

bioRxiv2024

RNAseq analysis reveals transcriptome changes associated with the progression of non-alcoholic liver disease in livers from Efcab4b knockout mice

EFCAB4B is an evolutionarily conserved protein that encodes for the Rab GTPase Rab46, and the CRAC channel modulator, CRACR2A. Previous genome wide association studies have demonstrated the association of EFCAB4B variants in the progression of non-alcoholic liver disease (NAFLD). In this study we show that mice with global depletion of Efcab4b-/- have significantly larger livers than their wild-type (WT) counterparts. We performed RNA-sequencing (RNA-seq) analysis of liver tissues to investigate differential global gene expression among Efcab4b-/-and WT mice. Of the 69 differentially expressed genes (DEGs), analyses of biological processes found significant enrichment in liver and bile development, with 6 genes (Pck1, Aacs, Onecut1, E2f8, Xbp1, and Hes1) involved in both processes. Specific consideration of possible roles of DEGs or their products in NAFLD progression to (NASH) and hepatocarcinoma (HCC), demonstrated DEGs in the livers of Efcab4b-/- mice had roles in molecular pathways including lipid metabolism, inflammation, ER stress and fibrosis. The results in this study provide additional insights into molecular mechanisms responsible for increasing susceptibility of liver injuries associated with EFCAB4B.

genetics↗

bioRxiv2023

Low oscillatory shear stress regulates Weibel-Palade body size and vWF release.

Blood flow regulates vascular function by generating wall shear stress which impacts endothelial cell (EC) physiology. Whilst high laminar shear stress (HSS) maintains the functional integrity of the vasculature, low oscillatory shear stress (LOSS) evokes secretion of pro-thrombotic and pro-inflammatory components from ECs, thus promoting the development of cardiovascular disease (CVD). Pro-thrombotic and pro-inflammatory cargo are readily stored in endothelial-specific organelles termed Weibel-Palade bodies (WPBs). WPBs form purely due to the multimerization of the pro-thrombotic glycoprotein von Willebrand factor (vWF). Here we investigated if aberrant shear stress, induced by non-uniform oscillatory flow, modulates the biogenesis of WPBs, and the subsequent release of vWF. Ultimately, we demonstrate, using an in vitro model, that LOSS has no effect on the level of vWF expression, however, LOSS promotes endothelial thrombotic potential by increasing WPB size, which impacts the length of the vWF strings that are released upon stimulation. Thus, wall shear stress can confer functional plasticity to WPBs by manipulating their biogenesis. Further understanding of the underlying mechanisms could aid generation of novel therapeutics in CVD.

cell biology↗

bioRxiv2023

Rab46: a novel player in mast cell function

Mast cells are infamous for mediating allergic and inflammatory diseases due to their capacity of rapidly releasing a wide range of inflammatory mediators stored in cytoplasmic granules. However, mast cells also have several important physiological roles that involves selective and agonist-specific release of these active mediators. Whilst a filtering mechanism at the plasma membrane could regulate selective release of some cargo, the plethora of stored cargo and the diversity of mast cell functions suggests the existence of granule subtypes with distinct trafficking pathways. The molecular mechanisms underlying differential trafficking and exocytosis of these granules are not known, neither is it clear how granule trafficking is coupled to the stimulus. In endothelial cells, a Rab GTPase, Rab46, responds to histamine but not thrombin signals, and this regulates the trafficking of a subpopulation of endothelial-specific granules. Here, we sought to explore if Rab46 has a similar function in mast cells. We demonstrate, for the first time, that Rab46 is highly expressed in human and murine mast cells and Rab46 genetic deletion has an effect on mast cell degranulation that depends on both stimuli and mast cell developmental stage. Rab46 could therefore be an important regulator of stimuli-coupled responses in mast cells and future studies will seek to understand these mechanisms in order to develop novel and specific therapeutic targets for treatment of the diverse pathologies mediated by mast cells.

cell biology↗

bioRxiv2020

A systematic analysis of rodent models implicates adipogenesis and innate immunity in pathogenesis of fatty liver disease

Animal models of human disease are a key component of translational research and yet there is often no consensus on which model is optimal for a particular disease. Here, we generated a database of 3,920 rodent models of non-alcoholic fatty liver disease (NAFLD). Study designs were highly heterogeneous therefore few models had been cited more than once. Analysis of genetic models provided evidence for the role of adipose dysfunction and perturbation of the innate immune system in the progression of NAFLD. We identified that high-fat, high-fructose diets most closely recapitulate the human phenotype of NAFLD. There was substantial variability in the nomenclature of animal models; a consensus on terminology of specialist diets is needed. More broadly, this analysis demonstrates the variability in preclinical study design, which has implications for the reproducibility of in vivo experiments.

physiology↗

bioRxiv2020

Identification of novel Rab46 effector proteins

Rab46 is a novel Ca2+- sensing Rab GTPase shown to have important functions in endothelial and immune cells. The presence of functional Ca2+- binding, coiled-coil and Rab domains suggest that Rab46 will be important for coupling rapid responses to signalling in many cell types. The molecular mechanisms underlying Rab46 function are currently unknown. Here we provide the first resource for studying Rab46 interacting proteins. Using liquid chromatography mass spectrometry (LC-MS/MS) to identify affinity purified proteins that bind to constitutively active GFP-Rab46 or inactive GFP-Rab46 expressed in endothelial cells, we have revealed 922 possible interacting proteins. Further comparative analyses between nucleotide-locked Rab46 proteins enriched this dataset to confidently identify 29 effector proteins. Importantly, through biochemical and imaging approaches we have validated two potential effector proteins; dynein and the Na2+/ K+ ATPase subunit alpha 1 (ATP1α1). Hence, our use of affinity purification and LC-MS/MS to identify Rab46 neighbouring proteins provides a valuable resource for detecting Rab46 effector proteins and analysing Rab46 functions.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsLC-MS/MSliquid chromatography mass spectrometryGEFsliquid chromatography mass spectrometrGAPsGTPase activating proteinsWPBsWeibel-Palade bodiesECsendothelial cellsMTOCmicrotubule organising centreFDRfalse discovery rateDHCdynein heavy chainATP1α1Na2+/ K+ ATPase subunit α1View Full Text

cell biology↗

bioRxiv2019

What influences treatment response in animal models of non-alcoholic fatty liver disease? A meta-analysis with meta-regression.

The classical drug development pipeline necessitates studies using animal models of human disease to gauge future efficacy in humans, however, there is a comparatively low conversion rate from success in animals to in humans. Non-alcoholic fatty liver disease (NAFLD) is a complex chronic disease without any licensed therapies and hence a major field of animal research. We performed a meta-analysis of 414 interventional rodent studies (6,575 animals) in NAFLD to assess the mean difference in hepatic triglyceride content. 20 of 21 studied drug classes had similar efficacy with a mean difference of -30% hepatic triglyceride. However, when publication bias was accounted for, this reduced to -16% difference. Study characteristics were only able to account for a minority of variability on meta-regression, and we replicated previous findings of high risk of bias across 82% of cohorts. These findings build on previous work in preclinical neuroscience and help to explain the challenge of reproducibility and translation within the field of metabolism.

physiology↗