Biology subjects

Gardiner, E. E.

Publications and source records attributed to Gardiner, E. E..

7 recordsLinked to original sources

bioRxiv2025

Treatment with the ribosome biogenesis inhibitor CX-5461 increases platelet count in humans and enhances murine megakaryopoiesis

Thrombocytopenia is a common and serious complication of anticancer therapies. Here, we identify a novel thrombopoietic activity of the first-in-class ribosome biogenesis inhibitor CX-5461. In a phase I trial, 56% (9/16) of patients exhibited up to a 34% increase in platelet count following a single dose of CX-5461. In mice, CX-5461 elicited a rapid, reversible, and sustained [~]1.7-fold increase in platelet numbers without altering platelet function, lifespan, or inflammatory cytokines. Bone marrow analysis revealed a specific expansion of megakaryocytes (MKs), increased Sca1 MKs, and selective enrichment of MK-biased multipotent progenitor 2, independent of thrombopoietin (TPO) or c-mpl signalling. CX-5461 also mitigated carboplatin-induced thrombocytopenia, accelerating platelet recovery. Single-cell RNA sequencing and RNA velocity analysis confirmed enhanced differentiation of MK progenitors. These findings demonstrate that inhibition of ribosome biogenesis promotes TPO-independent megakaryopoiesis and identifies a previously unrecognised therapeutic opportunity to support platelet recovery in cancer treatment and potentially other thrombocytopenic states.

cell biology↗

bioRxiv2023

Glycoprotein VI is Critical for the Detection and Progression of Abdominal Aortic Aneurysms

A common feature in patients with abdominal aortic aneurysms (AAA) is the formation of a nonocclusive intraluminal thrombus (ILT) in regions of aortic dilation. Platelets are known to maintain hemostasis and propagate thrombosis through several redundant activation mechanisms, yet the role of platelet activation in the pathogenesis of AAA associated ILT is still poorly understood. Thus, we sought to investigate how platelet activation impacts the pathogenesis of AAA. Using RNA-sequencing, we identify that the platelet-associated transcripts are significantly enriched in the ILT compared to the adjacent aneurysm wall and healthy control aortas. We found that the platelet specific receptor glycoprotein VI (GPVI) is among the top enriched genes in AAA ILT and is increased on the platelet surface of AAA patients. Examination of a specific indicator of platelet activity, soluble GPVI (sGPVI), in two independent AAA patient cohorts is highly predictive of a AAA diagnosis and associates more strongly with aneurysm growth rate when compared to D-dimer in humans. Finally, intervention with the anti-GPVI antibody (J) in mice with established aneurysms blunted the progression of AAA in two independent mouse models. In conclusion, we show that levels of sGPVI in humans can predict a diagnosis of AAA and AAA growth rate, which may be critical in the identification of high-risk patients. We also identify GPVI as a novel platelet-specific AAA therapeutic target, with minimal risk of adverse bleeding complications, where none currently exist. KEY POINTSO_LISoluble glycoprotein VI, which is a platelet-derived blood biomarker, predicts a diagnosis of AAA, with high sensitivity and specificity in distinguishing patients with fast from slow-growing AAA. C_LIO_LIBlockade of glycoprotein VI in mice with established aneurysms reduces AAA progression and mortality, indicating therapeutic potential. C_LI

pathology↗

bioRxiv2022

Combined scattering, interferometry and fluorescence oblique illumination for live cell nanoscale imaging

To determine the molecular and/or mechanical basis of cell migration using live cell imaging tools, it is necessary to correlate multiple 3D spatiotemporal events simultaneously. Fluorescence nanoscopy and label free nanoscale imaging can complement each other by providing both molecular specificity and structural dynamics of sub-cellular structure. In doing so, a combined imaging system would permit quantitative 3D spatial temporal detail of individual cellular components. In this paper, we empirically determined a series of optimal azimuthal scanning angles and rotating beam to achieve simultaneous and label-free nanoscale and fluorescence imaging. Label-free nanoscale imaging here refers to interferometric, brightfield (BF) and darkfield (DF) rotating coherence scattering (ROCS) microscopy, while fluorescence refers to high inclined Laminated Oblique (HiLO) and total internal reflection fluorescence (TIRF) imaging. The combined capabilities of interferometric, scattering and fluorescence imaging enables (1) the identification of molecular targets (substrate or organelle), (2) quantification of 3D cell morphodynamics, and (3) tracking of intracellular organelles in 3D. This combined imaging tool was then used to characterize migrating platelets and adherent endothelial cells, both critical to the process of infection and wound healing. The combined imaging results of over [~]1000 platelets, suggested that serum albumin (bovine) was necessary for platelets to migrate and scavenge fibrin/fibrinogen. Furthermore, we determine new asynchronous membrane fluctuations between the leading and rear edge of a migrating platelet. We further demonstrated that interferometric imaging permitted the quantification of mitochondria dynamics on lung microvascular cells (HMVEC). Our data suggests that axial displacement of mitochondria is minimized when it is closer to the nucleus or the leading edge of a cell membrane that exhibits retrograde motion. Taken together, this combined imaging platform has proven to quantify multiple spatial temporal events of a migrating cell, that will undoubtedly open ways to new quantitative correlative nanoscale live cell imaging.

biophysics↗

bioRxiv2022

Patterning Fluid Shear Stress Landscapes with Multiphoton Inner Laser Lithography (MILL) for Live Cell Adhesion and Translocation

Heterogenous fluid shear stress is known to provide mechanical cues for cell adhesion and translocation. To assemble 3D microstructures using current fabrication methods in a single channel and recapitulate in vivo heterogenous fluid flow would require hours of fabrication and specialized equipment. Inspired by the traditional art form of inside painting, we developed a technique for 3D fabrication of micro-patterned flow channels and mixed in vivo fluid flow in a matter of minutes. We termed this technique Multiphoton Inner Laser Lithography (MILL). We further showed that when combined with adaptive optics, MILL is compatible with both flat and curved channel shapes. MILL recapitulated in vivo tissue topology and 3D fluid flow within tissue stroma (low fluid shear, 0 - 3.5 dynes/cm2) and blood vessel (high fluid shear, 0 - 80 dynes/cm2). We demonstrate fibroblast cell and platelets adhere and translocate differently between laminar flow patterns that are homogenous versus heterogeneous in real time. Parallel strips of MILL channels were assembled for simultaneous platelet function test to quantify the efficacy of an antithrombotic GPVI Fab (~2000 microthrombi per test). The MILL technique can be readily reproduced in vivo fluid flow in minutes and benefit preclinical screening of drug pharmacokinetics. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/496569v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1efbe9eorg.highwire.dtl.DTLVardef@cbab5aorg.highwire.dtl.DTLVardef@15ed255org.highwire.dtl.DTLVardef@42d14e_HPS_FORMAT_FIGEXP M_FIG C_FIG Significant pointsO_LIMILL channels are made from commercially available off the shelf components using a standard multiphoton imaging system C_LIO_LIVarying degrees of in vivo heterogenous laminar flow is shown to directly influence cells translocating on thinly coated collagen surfaces C_LIO_LIParallel strips of MILL channels were assembled for simultaneous platelet function tests (~2000 microthrombi per test). C_LI

bioengineering↗

bioRxiv2021

Platelets are dispensable for the ability of CD8+ T cells to accumulate, patrol, kill and reside in the liver

Effector and memory CD8+ T cells accumulate in large numbers in the liver where they play key roles in the control of liver pathogens including Plasmodium. It has also been proposed that liver may act as the main place for elimination of effector CD8+ T cells at the resolution of immune responses. Platelets and the integrin LFA-1 have been proposed to be critical for the accumulation of protective CD8+ T cells in the liver; conversely, asialo-glycoprotein (ASGP) expression on the surface of CD8+ T cells has been proposed to assist in elimination of effector T cells in the liver. Here we investigated the contributions of these interactions in the accumulation of CD8+ T cells activated in vitro or in vivo by immunization with Plasmodium parasites. Using Mpl-/- mice with constitutive thrombocytopaenia and antibody-mediated platelet depletion models we found that severe reduction in platelet concentration in circulation did not strongly influence the accumulation and protective function of CD8+ T cells in the liver in these models. Surprisingly, inhibition of ASGP receptors did not inhibit the accumulation of effector cells in the liver, but instead prevented these cells from accumulating in the spleen. We further found that enforced expression of ASGP on effector CD8+ T cells using ST3GalI knockout cells lead to their loss from the spleen. These data suggest that platelets play a marginal role in CD8+ T cell function in the liver. Furthermore, ASGP-expressing effector CD8+ T cells are retained in the liver but are lost from the spleen.

immunology↗

bioRxiv2020

COSI for label-free multiparameter quantitative imaging of intra-thrombus stability in vitro.

Although existing microfluidics in vitro assays recapitulate blood vessel microenvironment using surface-immobilized agonists under biofluidic flows, these assays do not quantify intra-thrombus mass and activities of adhesive platelets at agonist margin and uses fluorescence labeling, therefore limiting clinical translation potential. Here, we describe a real time label-free in vitro quantitative imaging flow assay called Coherent Optical Scattering and phase Interferometry (COSI) that evaluates both intra-thrombus and adhesive-only platelet dynamics using only changes in refractive index. By combining coherent optical scattering and optical interferometry, we evaluated and quantified both intra-thrombus mass with picogram accuracy and adhesive platelet-only events/dynamics with high spatial-temporal resolution (400 nm/s) under fluid shear stress using only changes in refractive index. Using oblique illumination, COSI provide a [~] 4 {micro}m thin axial slice that quantifies the magnitude of physical of surface adhesive platelets (spreading, adhesion and consolidation) in a developing thrombus without labelling under fluid shear stress. We achieve real time visualization of recruitment of single platelet into thrombus and further correlate it to the developing mass of a thrombus. The adhesive platelet activity exhibit stabilized surface activity of around 2 {micro}m/s and intra-thrombus mass exchange were balanced at around 1 picogram after treatment of a broad range metalloproteinase inhibitor (250 {micro}M GM6001). SignificanceThe combination of phase imaging with transmitted light and backscattering imaging via oblique illumination in COSI unpicked intra-thrombus mass and adhesive platelet-only activity events at picogram and sub-micrometer precision with millisecond time resolution under fluid shear stress. COSI maps the longitudinal time dynamics of adhesive platelets along changing thrombus mass under metalloproteinase inhibition, and demonstrates potential for real-time correlative microfluidic label-free imaging for flow-dependent biological adhesive events.

bioengineering↗

bioRxiv2020

Immobilised collagen prevents shedding and induces sustained GPVI clustering and signalling in platelets.

Collagen, the most thrombogenic constituent of blood vessel walls, activates platelets through glycoprotein VI (GPVI). In suspension, following platelet activation by collagen, GPVI is cleaved by A Disintegrin And Metalloproteinase (ADAM)10 and ADAM17. In this study, we use single-molecule localization microscopy and a 2-level DBSCAN-based clustering tool to show that GPVI remains clustered along immobilised collagen fibres for at least 3 hours in the absence of significant shedding. Tyrosine phosphorylation of spleen tyrosine kinase (Syk) and Linker of Activated T cells (LAT), and elevation of intracellular Ca2+, are sustained over this period. Syk, but not Src kinase-dependent signalling is required to maintain clustering of the collagen integrin α2β1, whilst neither is required for GPVI. We propose that clustering of GPVI on immobilised collagen protects GPVI from shedding in order to maintain sustained Src and Syk-kinases dependent signalling, activation of integrin α2β1 and continued adhesion.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cell biology↗