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Atherosclerosis, a chronic inflammatory disorder characterized by endothelial dysfunction and blood vessel narrowing, is the leading cause of cardiovascular diseases including heart attack and stroke. Herein, we present a novel tunable microfluidic atherosclerosis model to study vascular inflammation and leukocyte-endothelial interactions in 3D vessel stenosis. Flow and shear stress profiles were characterized in pneumatic-controlled stenosis conditions (0%, 50% and 80% constriction) using fluid simulation and experimental beads perfusion. Due to non-uniform fluid flow at the 3D stenosis, distinct monocyte (THP-1) adhesion patterns on inflamed [tumor necrosis factor-α (TNF-α) treated] endothelium were observed, and there was a differential endothelial expression of intercellular adhesion molecule-1 (ICAM-1) at the constriction region. Whole blood perfusion studies also showed increased leukocyte interactions (cell rolling and adherence) at the stenosis of healthy and inflamed endothelium, clearly highlighting the importance of vascular inflammation, flow disturbance, and vessel geometry in recapitulating atherogenic microenvironment. To demonstrate inflammatory risk assessment using leukocytes as functional biomarkers, we perfused whole blood samples into the developed microdevices (80% constriction) and observed significant dose-dependent effects of leukocyte adhesion in healthy and inflamed (TNF-α treated) blood samples. Taken together, the 3D stenosis chip facilitates quantitative study of hemodynamics and leukocyte-endothelial interactions, and can be further developed into a point-of-care blood profiling device for atherosclerosis and other vascular diseases.

BackgroundEffects of shear stress on endothelium are important for the normal physiology of blood vessels and are implicated in the pathogenesis of atherosclerosis. They have been extensively studied in vitro. In one paradigm, endothelial cells are cultured in devices that produce spatially varying shear stress profiles, and the local profile is compared with the properties of cells at the same position. A flaw in this class of experiments is that cells exposed to a certain shear profile in one location may release mediators into the medium that alter the behaviour of cells at another location, experiencing different shear, thus obscuring or corrupting the true relation between shear and cell properties.MethodsSurface coating methods were developed for attaching cells only to some areas of culture-ware and preventing them from spreading into other regions even during prolonged culture.ResultsSegmenting the growth of cells had no effect on cell shape, alignment and number per unit area compared to culturing cells in the whole well, but there were differences in tumour-necrosis-factor-α (TNF-α)-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), and monocyte adherence to the monolayer.ConclusionsThe results are consistent with the release of a mediator from cells exposed to high-magnitude uniaxial shear stress that has anti-inflammatory effects on activated endothelium; the mediator may be of importance in atherogenesis. Hence the new methods revealed an important property that would not have been observed without growth segmentation, suggesting that they could find more widespread application.

Blood vessel narrowing and arterial occlusion are pathological hallmarks of atherosclerosis, which involves a complex interplay of perturbed hemodynamics, endothelial dysfunction and inflammatory cascade. Herein, we report a novel circular microfluidic stenosis model that recapitulates atherogenic flow-mediated endothelial dysfunction and blood-endothelial cell (EC) interactions in vitro. 2D and 3D stenosis microchannels with different constriction geometries were fabricated using 3D printing to study flow disturbances under varying severity of occlusion and wall shear stresses (100 to 2000 dynecm −2 ). Experimental and fluid simulation results confirmed the presence of pathological shear stresses in the stenosis region, and recirculation flow post stenosis. The resultant pathological flow profile induced pro-inflammatory and pro-thrombotic EC state as demonstrated by orthogonal EC alignment, enhanced platelet adhesion at the stenosis, and aberrant leukocyte-EC interactions post stenosis. Clinical utility of the vascular model was further investigated by testing anti-thrombotic and immunomodulatory efficacy of aspirin and metformin, respectively. Overall, the platform enables multi-factorial analysis of critical atherogenic events including endothelial dysfunction, platelets and leukocyte adhesion, and can be further developed into a liquid biopsy tool for cardiovascular risk stratification.

Phosphorylation of the eukaryotic translation initiation factor, eIF2α, by stress-activated protein kinases and dephosphorylation by the growth arrest and DNA damage-inducible protein (GADD34)-containing phosphatase is a central node in the integrated stress response. Mass spectrometry demonstrated GADD34 acetylation at multiple lysines. Substituting K and K with alanines or glutamines did not impair GADD34's ability to recruit protein phosphatase 1α (PP1α) or eIF2α, suggesting that GADD34 acetylation did not modulate eIF2α phosphatase activity. Arsenite (Ars)-induced oxidative stress increased cellular GADD34 levels and enhanced Sirtuin 1 (SIRT1) recruitment to assemble a cytoplasmic complex containing GADD34, PP1α, eIF2α and SIRT1. Induction of GADD34 in WT MEFs paralleled the dephosphorylation of eIF2α (phosphoserine-51) and SIRT1 (phosphoserine-47). By comparison, eIF2α and SIRT1 were persistently phosphorylated in Ars-treated GADD34-/- MEFs. Expressing WT GADD34, but not a mutant unable to bind PP1α in GADD34-/- MEFs restored both eIF2α and SIRT1 dephosphorylation. SIRT1 dephosphorylation increased its deacetylase activity, measured in vitro and in cells. Loss of function of GADD34 or SIRT1 enhanced cellular p-eIF2α levels and attenuated cell death following Ars exposure. These results highlighted a novel role for the GADD34/PP1α complex in coordinating the dephosphorylation and reactivation of eIF2α and SIRT1 to determine cell fate following oxidative stress.

In spike-timing-dependent plasticity (STDP), the direction and degree of synaptic modification are determined by the coherence of pre-and postsynaptic activities within a neuron. However, in the adult rat hippocampus, it remains unclear whether STDP-like mechanisms in a neuronal population induce synaptic potentiation of a long duration. Thus, we asked whether the magnitude and maintenance of synaptic plasticity in a population of CA1 neurons differ as a function of the temporal order and interval between pre-and postsynaptic activities. Modulation of the relative timing of Schaffer collateral fibers (presynaptic component) and CA1 axons (postsynaptic component) stimulations resulted in an asymmetric population STDP (pSTDP). The resulting potentiation in response to 20 pairings at 1 Hz was largest in magnitude and most persistent (4 h) when presynaptic activity coincided with or preceded postsynaptic activity. Interestingly, when postsynaptic activation preceded presynaptic stimulation by 20 ms, an immediate increase in field excitatory postsynaptic potentials was observed, but it eventually transformed into a synaptic depression. Furthermore, pSTDP engaged in selective forms of late-associative activity: It facilitated the maintenance of tetanization-induced early long-term potentiation (LTP) in neighboring synapses but not early long-term depression, reflecting possible mechanistic differences with classical tetanization-induced LTP. The data demonstrate that a pairing of pre-and postsynaptic activities in a neuronal population can greatly reduce the required number of synaptic plasticity-evoking events and induce a potentiation of a degree and duration similar to that with repeated tetanization. Thus, pSTDP determines synaptic efficacy in the hippocampal CA3-CA1 circuit and could bias the CA1 neuronal population toward potentiation in future events. STDP | synaptic tagging | synaptic capture | area CA1 | synaptic plasticity I t is widely accepted that learning and memory are subserved by long-term modifications of synaptic efficacy in neural circuits (1). The most studied forms of such synaptic plasticity include long-term potentiation (LTP) and long-term depression (LTD). Both LTP and LTD are dissociated into different temporal phases (early LTP or LTD and late LTP or LTD) that are governed by distinct but related molecular mechanisms (2-4). The induction of both LTP and LTD is mediated by the dynamics of postsynaptic calcium influx and calcium-dependent signaling cascades (2, 5, 6). Furthermore, cellular consolidation of LTP and LTD is also determined by other neural activities occurring near the time of induction, as outlined by the synaptic tagging and capture (STC) hypothesis (7-9). The STC hypothesis points out that a strong activation (e.g., strong tetanization) of synapses potentiates those synapses by a dual mechanismsetting synapse-specific "tags" and triggering the synthesis of plasticity-related products (PRPs). In addition, other synapses that form a synaptic tag but express a transient synaptic p...

Background and aims: When endothelium is cultured in wells swirled on an orbital shaker, cells at the well centre experience putatively atherogenic flow whereas those near the edge experience putatively atheroprotective flow. Transcellular transport is decreased equally in both regions, consistent with it being reduced by a mediator released from cells in one part of the well and mixed in the swirling medium. Similar effects have been inferred for pro-inflammatory changes. Here we identify the mediator and flow characteristics stimulating its release. Methods and results: Medium conditioned by cells swirled at the edge, but not by cells swirled at the centre or cultured under static conditions, significantly reduced transendothelial transport of a low density lipoprotein (LDL)-sized tracer and tumor necrosis factor α (TNF-α)-induced activation and translocation of nuclear factor κB (NF-κB), adhesion molecule expression and monocyte adhesion. Inhibiting transcytosis similarly decreased tracer transport. Unbiased proteomics revealed that cells from the swirled edge secreted substantially more follistatinlike 1 (FSTL1) than cells from the swirled centre or from static wells. Exogenous FSTL1 reduced transport of the LDL-sized tracer and of LDL itself, as well as TNF-α-induced adhesion molecule expression. Bone morphogenetic protein 4 (BMP4) increased transport of the LDL-sized tracer and adhesion molecule expression; FSTL1 abolished these effects. Conclusions: Putatively atheroprotective flow stimulates secretion of FSTL1 by cultured endothelial cells. FSTL1 reduces transcellular transport of LDL-sized particles and of LDL itself, and inhibits endothelial activation. If this also occurs in vivo, it may account for the atheroprotective nature of such flow.

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