Biology subjects

Turner-Brannen, E.

Publications and source records attributed to Turner-Brannen, E..

4 recordsLinked to original sources

bioRxiv2022

Development of a 3D bioprinted airway smooth muscle model for manipulating structure and measuring contraction

The contractile function of airway smooth muscle (ASM) is inextricably linked to its mechanical properties and interaction with the surrounding mechanical environment. As tissue engineering approaches become more commonplace for studying lung biology, the inability to replicate realistic mechanical contexts for ASM will increasingly become a barrier to a fulsome understanding of lung health and disease. To address this knowledge gap, we describe the use of 3D bioprinting technology to generate a novel experimental model of ASM with a wide scope for modulating tissue mechanics. Using a stiffness modifiable alginate-collagen-fibrinogen bioink, we demonstrate that modulating the stiffness of free-floating ASM bare rings is unfeasible; bioink conditions favorable for muscle formation produce structures that rapidly collapse. However, the creation of novel sandwich and spiderweb designs that encapsulate the ASM bundle within stiff acellular load bearing frames successfully created variable elastic loads opposing tissue collapse and contraction. Sandwich and spiderweb constructs demonstrated realistic actin filament organisation, generated significant baseline tone, and responded appropriately to acetylcholine, potassium chloride and cytochalasin D. Importantly, the two designs feasibly simulate different mechanical contexts within the lung. Specifically, the sandwich was relatively compliant and subject to plastic deformation under high contractile loads, whereas the stiffer spiderweb was more robust and only deformed minimally after repeated maximal contractions. Thus, our model represents a new paradigm for studying ASM contractile function in a realistic mechanical context. Moreover, it holds significant capacity to study the effects of ECM composition, multiple cell types and fibrosis on lung health and disease. GRANTSO_LINatural Sciences and Engineering Research Council, Discovery Grant (Adrian West) C_LIO_LIResearch Manitoba, New Investigator Operating Grant (Adrian West) C_LIO_LIChildrens Hospital Research Institute of Manitoba, Operating Grant (Adrian West) C_LIO_LICanadian Foundation for Innovation, John R. Evans Leaders Fund (Adrian West) C_LIO_LIUniversity of Manitoba, Manitoba Graduate Scholarship (Jeffery Osagie) C_LIO_LIResearch Manitoba, Masters Studentship Award (Jeffery Osagie) C_LIO_LIResearch Manitoba, Masters Studentship Award (Sanjana Syeda) C_LIO_LIChildrens Hospital Research Institute of Manitoba, Summer Studentship (Michelle Guimond) C_LIO_LIUniversity of Manitoba, Jack Prior Memorial Undergraduate Student Research Award (Lumiere Parrenas) C_LIO_LIUniversity of Manitoba, Undergraduate Research Award (Ahsen Haroon) C_LIO_LIUniversity of Manitoba, UMSU Undergraduate Research Award (Philip Imasuen) C_LI The grant bodies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

physiology↗

bioRxiv2022

Skeletal muscle-derived extracellular vesicles are altered with chronic contractile activity

Extracellular vesicles (EVs) are small lipid bilayer-delimited particles that are secreted by all cells, playing a central role in cellular communication. EVs are released from skeletal muscle during exercise, but the effects of contractile activity on skeletal muscle-derived EVs (Skm-EVs) are poorly understood due to the challenges in distinguishing Skm-EVs derived from exercising muscle in vivo. Using tunable resistive pulse sensing (TRPS), we previously demonstrated that chronic contractile activity (CCA) increased the secretion of Skm-EVs from C2C12 myotubes, while their size and zeta potential remained unchanged. Here, we aimed to determine whether similar results could be obtained using an alternative method of EV characterization, dynamic light scattering (DLS). C2C12 myoblasts were differentiated into myotubes, and electrically paced (3h/day x 4days @14V, C-PACE EM, IonOptix) to mimic chronic exercise in vitro. EVs were isolated from conditioned media of control and stimulated myotubes using differential ultracentrifugation, and characterized based on size and zeta potential. The mean size of EVs from chronically stimulated myotubes (CCA-EVs, 132 nm) was 26% smaller than control (CON-EVs, 178 nm). Size distribution analysis revealed that CCA-EVs were enriched in small EVs (100-150 nm), while CON-EVs were largely composed of 200-250 nm sized vesicles. Additionally, zeta potential was 27% lower in CCA-EVs compared to CON-EVs. Our data indicate that the effect of CCA on facilitating the release of smaller, more stable EVs, is a robust finding, reproducible by multiple methods of EV characterization. Future studies investigating the mechanisms by which CCA influences Skm-EV biogenesis and secretion are warranted.

cell biology↗

bioRxiv2019

Development of an in vitro Experimental Model for Investigating the Effect of Matrix Stiffness on Epithelial Barrier Permeability

Epithelial cells are well-known to be modulated by extracellular mechanical factors including substrate stiffness. However, the effect of substrate stiffness on an epithelial cells principal function -creating an effective barrier to protect the underlying tissue - cannot be directly measured using existing experimental techniques. We developed a strategy involving ethylenediamine aminolysis and glutaraldehyde crosslinking to chemically graft polyacrylamide hydrogels with tunable stiffness to PET Transwell membranes. Grafting success was evaluated using spectroscopic methods, scrape tests, and extended incubation in culture. By assessing apical to basolateral transfer of fluorescent tracers, we demonstrated that our model is permeable to biologically relevant molecules and usable for direct measurement of barrier function by calculating paracellular permeability.\n\nWe found that BEAS-2B epithelial cells form a more effective barrier on stiff substrates, likely attributable to increased cell spreading. We also observed barrier impairment after treatment with transforming growth factor beta, indicating loss of cell-cell junctions, validating our models ability to detect biologically relevant stimuli. Thus, we have created an experimental model that allows explicit measurement of epithelial barrier function for cells grown on different substrate stiffnesses. This model will be a valuable tool to study mechanical regulation of epithelial and endothelial barrier function in health and disease.

bioengineering↗

bioRxiv2019

Oxidised phosphatidylcholines induce multiple functional defects in airway epithelial cells

Oxidative stress is a hallmark of numerous airway diseases, contributing to extensive cell and tissue damage. Cell membranes and the airway mucosal lining are rich in phospholipids that are particularly susceptible to oxidative attack, producing bioactive molecules including oxidized phosphatidylcholines (OxPC). With the recent discovery of elevated OxPC in asthmatic patients after allergen challenge, we hypothesized that OxPC directly contribute to disease by inducing airway epithelial cell dysfunction. We found that OxPC induced dose-dependent cell stress and loss of viability in BEAS-2B and Calu-3 cell lines and primary human epithelial cells. These responses corresponded with significant epithelial barrier dysfunction, which was further compounded when combining OxPC with an epithelial wound. OxPC inhibited DNA synthesis and migration required to re-establish barrier function, but cells recovered if OxPC were washed off soon after treatment. OxPC induced generation of reactive oxygen species, lipid peroxidation and mitochondrial dysfunction, raising the possibility that OxPC cause pathological lipid metabolism in a self-propagating cycle. The oxidative stress induced by OxPC could not be abrogated by putative OxPC receptor blockers, but partial recovery of barrier function, proliferation and lipid peroxidation could be achieved with the antioxidant n-acetyl cysteine. In summary, we have identified OxPC as a group of bioactive molecules that significantly impair multiple facets of epithelial cell function, consistent with pathological features of asthma. Further characterisation of the mechanisms by which OxPC affect epithelial cells could yield new insights into how oxidative stress contributes to the pathogenesis of airway disease.

cell biology↗