Biology subjects

Rye, K.-A.

Publications and source records attributed to Rye, K.-A..

6 recordsLinked to original sources

bioRxiv2025

Shear Difference: Flow Type Dictates Endothelial Flow-Responsive Gene Programs in a 3D-Printed in vitro Model

BackgroundEndothelial cells (ECs) are mechanosensitive and adopt distinct phenotypes in response to hemodynamic forces. These phenotypes are rarely seen in shear-responsive signalling studies because commonly used in vitro platforms rarely reproduce the true scale of vessel geometries or physiologic waveforms. AimTo determine how the presence and temporal pattern of flow impact endothelial morphology and transcriptional activity in a 3D macrofluidic model. MethodsIdealised vessels were 3D-printed using a water-soluble polyvinyl alcohol filament and cast in polydimethylsiloxane. The polyvinyl alcohol cores were dissolved leaving a polydimethylsiloxane lumen on which HMEC-1 cells were grown and perfused for 24 h under static, continuous flow, or pulsatile flow. Cell morphology was assessed using immunofluorescence. Bulk RNA-seq was performed with Hallmark pathway enrichment using gene set enrichment analysis. ResultsRelative to static culture, continuous flow increased cell eccentricity (0.74 vs 0.48, p <0.0001) and reduced variability in cell orientation ({Delta} = -47.1{degrees}, p <0.0001). At the transcriptome level, differential gene expression was extensive (continuous vs static: 2,103 genes; pulsatile vs static: 2,643 genes; pulsatile vs continuous: 384 genes). Continuous flow favoured oxidative-metabolic and barrier-maintenance pathways. By contrast, pulsatile shear of the same mean load promoted cell-cycle/checkpoint signalling rather than oxidative-metabolic programmes. Head-to-head, pulsatile flow emphasized MYC proto-oncogene (MYC) and E2F transcription factor (E2F), whereas continuous flow preferentially engaged oxidative phosphorylation and phosphoinositide 3-kinase (PI3K)-AKT serine/threonine kinase (AKT)-mechanistic target of rapamycin (MTOR) (with higher tumor protein p53and transforming growth factor-{beta} signaling). ConclusionUnder matched mean shear, pulsatile versus continuous flow drive distinct endothelial morphological and transcriptional programs, enriching pathways central to blood vessel function, remodelling, and disease. 3D macrofluidic platforms can replicate flow-specific endothelial cell mechanobiology, providing a translational tool to better understand vascular outcomes.

bioengineering↗

bioRxiv2023

ApoA-I Protects Pancreatic β-cells from Cholesterol-induced Mitochondrial Damage and Restores their Ability to Secrete Insulin

Objective: High cholesterol levels in pancreatic {beta}-cells cause oxidative stress and decrease insulin secretion. {beta}-cells can internalize apolipoprotein (apo) A-I, which increases insulin secretion. This study asks whether internalization of apoA-I improves {beta}-cell insulin secretion by reducing oxidative stress. Approach: Ins-1E cells were cholesterol-loaded by incubation with cholesterol-methyl-{beta}-cyclodextrin. Insulin secretion in the presence of 2.8 or 25 mM glucose was quantified by radioimmunoassay. Internalization of fluorescently labelled apoA-I by {beta}-cells was monitored by flow cytometry. The effects of apoA-I internalization on {beta}-cell gene expression was evaluated by RNA sequencing. ApoA-I binding partners on the {beta}-cell surface were identified by mass spectrometry. Mitochondrial oxidative stress was quantified in {beta}-cells and isolated islets with MitoSOX and confocal microscopy. Results: An F1-ATPase {beta}-subunit on the {beta}-cell surface was identified as the main apoA-I binding partner. {beta}-cell internalization of apoA-I was time-, concentration-, temperature-, cholesterol- and F1-ATPase {beta}-subunit-dependent. {beta}-cells with internalized apoA-I (apoA-I+ cells) had higher cholesterol and cell surface F1-ATPase {beta}-subunit levels than {beta}-cells without internalized apoA-I (apoA-I- cells). The internalized apoA-I co-localized with mitochondria and was associated with reduced oxidative stress and increased insulin secretion. The ATPase inhibitory factor 1, IF1, attenuated apoA-I internalization and increased oxidative stress in Ins-1E {beta}-cells and isolated mouse islets. Differentially expressed genes in apoA-I+ and apoA-I- Ins-1E cells were related to protein synthesis, the unfolded protein response, insulin secretion and mitochondrial function. Conclusions: These results establish that {beta}-cells are functionally heterogeneous and apoA-I restores insulin secretion in {beta}-cells with elevated cholesterol levels by improving mitochondrial redox balance.

cell biology↗

bioRxiv2022

Effect of insulin insufficiency on ultrastructure and function in skeletal muscle

BackgroundDecreased insulin availability and high blood glucose levels, the hallmark features of poorly controlled diabetes, drive disease progression and are associated with decreased skeletal muscle mass. We have shown that mice with {beta}-cell dysfunction and normal insulin sensitivity have decreased skeletal muscle mass. This project asks how insulin deficiency impacts on the structure and function of the remaining skeletal muscle in these animals. MethodsSkeletal muscle function was determined by measuring exercise capacity and specific muscle strength prior to and after insulin supplementation for 28 days in 12-week-old mice with conditional {beta}-cell deletion of the ATP binding cassette transporters ABCA1 and ABCG1 ({beta}-DKO mice). Abca1 and Abcg1 floxed (fl/fl) mice were used as controls. RNAseq was used to quantify changes in transcripts in soleus and extensor digitorum longus muscles. Skeletal muscle and mitochondrial morphology were assessed by transmission electron microscopy. Myofibrillar Ca2+ sensitivity and maximum isometric single muscle fibre force were assessed using MyoRobot biomechatronics technology. ResultsRNA transcripts were significantly altered in {beta}-DKO mice compared to fl/fl controls (32 in extensor digitorum longus and 412 in soleus). Exercise capacity and muscle strength were significantly decreased in {beta}-DKO mice compared to fl/fl controls (p = 0.012), and a loss of structural integrity was also observed in skeletal muscle from the {beta}-DKO mice. Supplementation of {beta}-DKO mice with insulin restored muscle integrity, strength and expression of 13 and 16 of the dysregulated transcripts in and extensor digitorum longus and soleus muscles, respectively. ConclusionsInsulin insufficiency due to {beta}-cell dysfunction perturbs the structure and function of skeletal muscle. These adverse effects are rectified by insulin supplementation.

physiology↗

bioRxiv2020

Use of high-refractive index hydrogels and tissue clearing for large biological sample imaging

Recent advances in tissue clearing and light sheet fluorescence microscopy have improved insights into and understanding of tissue morphology and disease pathology by imaging large samples without the requirement of histological sectioning. However, sample handling and conservation of sample integrity during lengthy staining and acquisition protocols remains a challenge. This study overcomes these challenges with acrylamide hydrogels synthesised to match the refractive index of solutions typically utilised in aqueous tissue clearing protocols. These hydrogels have a high-water content (82.0{+/-}3.7% by weight). The gels are stable over time and FITC-IgG readily permeated into, and effluxed out of them. Whilst the gels deformed and/or swelled over time in some commonly used solutions, this was overcome by using a previously described custom refractive index matched solution. To validate their use, CUBIC cleared mouse tissues and whole embryos were embedded in hydrogels, stained using fluorescent small molecule dyes, labels and antibodies and successfully imaged using light sheet fluorescence microscopy. In conclusion, the high-water content, high refractive index hydrogels described in this study have a broad applicability to research that delves into pathophysiological processes by stabilising and protecting large and fragile samples.

pathology↗

bioRxiv2018

The association of plasma lipids with white blood cell counts: Results from the Multi-Ethnic Study of Atherosclerosis

Background and aimsPrevious studies have demonstrated that elevated cholesterol results in increased white blood cell counts in mouse models. However, there is insufficient evidence to support this in humans. We, therefore, investigated the relationship of plasma lipids with white blood cell counts (basophils, eosinophils, monocytes, neutrophils and lymphocytes) in the Multi-Ethnic Study of Atherosclerosis (MESA). MethodsThe analysis included 2873 MESA participants at visit 5 with a complete white blood count and differential analysis. The cross-sectional association of total cholesterol, LDL cholesterol, HDL cholesterol and triglyceride levels with different white blood cell counts was analyzed by multivariable linear regression. ResultsAfter adjusting sociodemographic and confounding factors including red blood cells counts, platelet counts, use of lipid-lowering medication, CVD risk factors and other lipid measures, and multiple testing correction, a 1-SD increment in total cholesterol and LDL cholesterol was associated with 2.8% and 2.3% (both p<0.001) lower total white blood cell counts. The same increment in ln-transformed triglyceride levels was associated with 2.3% higher total white blood cell counts, 2.9% higher lymphocyte counts and 2.7% lower monocyte counts (all p<0.001). HDL cholesterol was not associated with any white blood cell counts. Similar results were obtained after excluding participants taking lipid-lowering medication. ConclusionsWhilst significant associations were observed, the heterogenous and modest nature of the relationships between plasma lipid levels and white blood cell populations make it hard to support the hypothesis that lipids are in the causal pathway for leukogenesis.

epidemiology↗

bioRxiv2018

Translocator Protein Localises To CD11b+ Macrophages In Atherosclerosis

BackgroundAtherosclerosis is characterized by lipid deposition, monocyte infiltration and foam cell formation in the artery wall. Translocator protein (TSPO) is abundantly expressed in lipid rich tissues. Recently, TSPO has been identified as a potential diagnostic tool in cardiovascular disease. The purpose of this study was to determine if the TSPO ligand, 18F-PBR111, can identify early atherosclerotic lesions and if TSPO expression can be used to identify distinct macrophage populations during lesion progression.\n\nMethods and ResultsApoE-/- mice were maintained on a high-fat diet for 3 or 12 weeks. C57BL/6J mice maintained on chow diet served as controls. Mice were administered 18F-PBR111 intravenously and PET/CT imaged. After euthanasia, aortas were isolated, fixed and optically cleared. Cleared aortas were immunostained with DAPI, and fluorescently labelled with antibodies to-TSPO, the tissue resident macrophage marker F4/80 and the monocyte-derived macrophage marker CD11b. TSPO expression and the macrophage markers were visualised in fatty streaks and mature lesions by light sheet microscopy. While tissue resident F4/80+ macrophages were evident in the arteries of animals without atherosclerosis, no CD11b+ macrophages were observed in these animals. In contrast, mature plaques had high CD11b and low F4/80 expression. A ~3-fold increase in the uptake of 18F-PBR111 was observed in the aortas of atherosclerotic mice relative to controls.\n\nConclusionsImaging of TSPO expression is a new approach for studying atherosclerotic lesion progression and inflammatory cell infiltration. The TSPO ligand, 18F-PBR111, is a potential clinical diagnostic tool for the detection and quantification of atherosclerotic lesion progression in humans.

pathology↗