160 research outputs found
Fluid shear stress modulation of hepatocyte like cell function
Freshly isolated human adult hepatocytes are considered to be the gold standard tool for in vitro studies. However, primary hepatocyte scarcity, cell cycle arrest and the rapid loss of cell phenotype limit their widespread deployment. Human embryonic stem cells and induced pluripotent stem cells provide renewable sources of hepatocyte-like cells (HLCs). Despite the use of various differentiation methodologies, HLCs like primary human hepatocytes exhibit unstable phenotype in culture. It has been shown that the functional capacity can be improved by adding back elements of human physiology, such as cell co-culture or through the use of natural and/or synthetic surfaces. In this study, the effect of fluid shear stress on HLC performance was investigated. We studied two important liver functions, cytochrome P450 drug metabolism and serum protein secretion, in static cultures and those exposed to fluid shear stress. Our study demonstrates that fluid shear stress improved Cyp1A2 activity by approximately fivefold. This was paralleled by an approximate ninefold increase in sensitivity to a drug, primarily metabolised by Cyp2D6. In addition to metabolic capacity, fluid shear stress also improved hepatocyte phenotype with an approximate fourfold reduction in the secretion of a foetal marker, alpha-fetoprotein. We believe these studies highlight the importance of introducing physiologic cues in cell-based models to improve somatic cell phenotype
Actin polymerization stabilizes α4β1 integrin anchors that mediate monocyte adhesion
Leukocytes arrested on inflamed endothelium via integrins are subjected to force imparted by flowing blood. How leukocytes respond to this force and resist detachment is poorly understood. Live-cell imaging with Lifeact-transfected U937 cells revealed that force triggers actin polymerization at upstream α4β1 integrin adhesion sites and the adjacent cortical cytoskeleton. Scanning electron microscopy revealed that this culminates in the formation of structures that anchor monocyte adhesion. Inhibition of actin polymerization resulted in cell deformation, displacement, and detachment. Transfection of dominant-negative constructs and inhibition of function or expression revealed key signaling steps required for upstream actin polymerization and adhesion stabilization. These included activation of Rap1, phosphoinositide 3-kinase γ isoform, and Rac but not Cdc42. Thus, rapid signaling and structural adaptations enable leukocytes to stabilize adhesion and resist detachment forces
Myocardial perfusion imaging with 99 mTc - tetrofosmin SPECT in breast cancer patients that received postoperative radiotherapy: a case-control study
<p>Abstract</p> <p>Purpose</p> <p>To evaluate the cardiac toxicity of radiotherapy (RT) in breast cancer (BC) patients employing myocardial perfusion imaging (MPI) with Tc-99 m Tetrofosmin - single photon emission computer tomography (T-SPECT).</p> <p>Materials and methods</p> <p>We studied 46 BC female patients (28 patients with left and 18 patients with right BC) treated with postoperative RT compared to a control group of 85 age-matched females. The median time of RT to SPECT was 40 months (6-263).</p> <p>Results</p> <p>Abnormalities in the summed stress score (SSS) were found in 54% of left BC patients, 44.4% of right BC patients, and 32.9% of controls. In left BC patients there were significantly more SSS abnormalities compared to controls (4.0 ± 3.5 vs 2.6 ± 2.0, p = 0.05) and possible trend of increased abnormalities of right BC patients (3.7 ± 3.0 vs 2.6 ± 2.0, p = 0.14). Multiple regression analysis showed more abnormalities in the MPI of left BC patients compared to controls (SSS, p = 0.0001); Marginal toxicity was also noted in right BC patients (SSS, p = 0.045). No additional toxicity was found in patients that received adjuvant cardiotoxic chemotherapy. All T-SPECT abnormalities were clinically silent.</p> <p>Conclusion</p> <p>The study suggests that radiation therapy to BC patients result in MPI abnormalities but without apparent clinical consequences.</p
Disturbed flow induces a sustained, stochastic NF-κB activation which may support intracranial aneurysm growth in vivo
Intracranial aneurysms are associated with disturbed velocity patterns, and chronic inflammation, but the relevance for these findings are currently unknown. Here, we show that (disturbed) shear stress induced by vortices is a sufficient condition to activate the endothelial NF-kB pathway, possibly through a mechanism of mechanosensor de-activation. We provide evidence for this statement through in-vitro live cell imaging of NF-kB in HUVECs exposed to different flow conditions, stochastic modelling of flow induced NF-kB activation and induction of disturbed flow in mouse carotid arteries. Finally, CFD and immunofluorescence on human intracranial aneurysms showed a correlation similar to the mouse vessels, suggesting that disturbed shear stress may lead to sustained NF-kB activation thereby offering an explanation for the close association between disturbed flow and intracranial aneurysms
Unique domain appended to vertebrate tRNA synthetase is essential for vascular development
New domains were progressively added to cytoplasmic aminoacyl transfer RNA (tRNA) synthetases during evolution. One example is the UNE-S domain, appended to seryl-tRNA synthetase (SerRS) in species that developed closed circulatory systems. Here we show using solution and crystal structure analyses and in vitro and in vivo functional studies that UNE-S harbours a robust nuclear localization signal (NLS) directing SerRS to the nucleus where it attenuates vascular endothelial growth factor A expression. We also show that SerRS mutants previously linked to vasculature abnormalities either deleted the NLS or have the NLS sequestered in an alternative conformation. A structure-based second-site mutation, designed to release the sequestered NLS, restored normal vasculature. Thus, the essential function of SerRS in vascular development depends on UNE-S. These results are the first to show an essential role for a tRNA synthetase-associated appended domain at the organism level, and suggest that acquisition of UNE-S has a role in the establishment of the closed circulatory systems of vertebrates
Dynamics of Mechanical Signal Transmission through Prestressed Stress Fibers
Transmission of mechanical stimuli through the actin cytoskeleton has been proposed as a mechanism for rapid long-distance mechanotransduction in cells; however, a quantitative understanding of the dynamics of this transmission and the physical factors governing it remains lacking. Two key features of the actin cytoskeleton are its viscoelastic nature and the presence of prestress due to actomyosin motor activity. We develop a model of mechanical signal transmission through prestressed viscoelastic actin stress fibers that directly connect the cell surface to the nucleus. The analysis considers both temporally stationary and oscillatory mechanical signals and accounts for cytosolic drag on the stress fibers. To elucidate the physical parameters that govern mechanical signal transmission, we initially focus on the highly simplified case of a single stress fiber. The results demonstrate that the dynamics of mechanical signal transmission depend on whether the applied force leads to transverse or axial motion of the stress fiber. For transverse motion, mechanical signal transmission is dominated by prestress while fiber elasticity has a negligible effect. Conversely, signal transmission for axial motion is mediated uniquely by elasticity due to the absence of a prestress restoring force. Mechanical signal transmission is significantly delayed by stress fiber material viscosity, while cytosolic damping becomes important only for longer stress fibers. Only transverse motion yields the rapid and long-distance mechanical signal transmission dynamics observed experimentally. For simple networks of stress fibers, mechanical signals are transmitted rapidly to the nucleus when the fibers are oriented largely orthogonal to the applied force, whereas the presence of fibers parallel to the applied force slows down mechanical signal transmission significantly. The present results suggest that cytoskeletal prestress mediates rapid mechanical signal transmission and allows temporally oscillatory signals in the physiological frequency range to travel a long distance without significant decay due to material viscosity and/or cytosolic drag
An Oscillatory Contractile Pole-Force Component Dominates the Traction Forces Exerted by Migrating Amoeboid Cells
We used principal component analysis to dissect the mechanics of chemotaxis of amoeboid cells into a reduced set of dominant components of cellular traction forces and shape changes. The dominant traction force component in wild-type cells accounted for ~40% of the mechanical work performed by these cells, and consisted of the cell attaching at front and back contracting the substrate towards its centroid (pole-force). The time evolution of this pole-force component was responsible for the periodic variations of cell length and strain energy that the cells underwent during migration. We identified four additional canonical components, reproducible from cell to cell, overall accounting for an additional ~20% of mechanical work, and associated with events such as lateral protrusion of pseudopodia. We analyzed mutant strains with contractility defects to quantify the role that non-muscle Myosin II (MyoII) plays in amoeboid motility. In MyoII essential light chain null cells the polar-force component remained dominant. On the other hand, MyoII heavy chain null cells exhibited a different dominant traction force component, with a marked increase in lateral contractile forces, suggesting that cortical contractility and/or enhanced lateral adhesions are important for motility in this cell line. By compressing the mechanics of chemotaxing cells into a reduced set of temporally-resolved degrees of freedom, the present study may contribute to refined models of cell migration that incorporate cell-substrate interactions
Caveolin-1-Enhanced Motility and Focal Adhesion Turnover Require Tyrosine-14 but Not Accumulation to the Rear in Metastatic Cancer Cells
Caveolin-1 is known to promote cell migration, and increased caveolin-1 expression is associated with tumor progression and metastasis. In fibroblasts, caveolin-1 polarization and phosphorylation of tyrosine-14 are essential to promote migration. However, the role of caveolin-1 in migration of metastatic cells remains poorly defined. Here, caveolin-1 participation in metastatic cell migration was evaluated by shRNA targeting of endogenous caveolin-1 in MDA-MB-231 human breast cancer cells and ectopic expression in B16-F10 mouse melanoma cells. Depletion of caveolin-1 in MDA-MB-231 cells reduced, while expression in B16-F10 cells promoted migration, polarization and focal adhesion turnover in a sequence of events that involved phosphorylation of tyrosine-14 and Rac-1 activation. In B16-F10 cells, expression of a non-phosphorylatable tyrosine-14 to phenylalanine mutant failed to recapitulate the effects observed with wild-type caveolin-1. Alternatively, treatment of MDA-MB-231 cells with the Src family kinase inhibitor PP2 reduced caveolin-1 phosphorylation on tyrosine-14 and cell migration. Surprisingly, unlike for fibroblasts, caveolin-1 polarization and re-localization to the trailing edge were not observed in migrating metastatic cells. Thus, expression and phosphorylation, but not polarization of caveolin-1 favor the highly mobile phenotype of metastatic cells
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