Low Shear Stress Conditions Research Articles

Low fluid shear stress stimulates the uptake of noxious endothelial extracellular vesicles via MCAM and PECAM-1 cell adhesion molecules.

Atherosclerotic lesions mainly form in arterial areas exposed to low shear stress (LSS), where endothelial cells express a senescent and inflammatory phenotype. Conversely, areas exposed to high shear stress (HSS) are protected from plaque development. Endothelial extracellular vesicles (EVs) have been shown to regulate inflammation and senescence, and therefore play a crucial role in vascular homeostasis. Whilst previous studies have shown links between hemodynamic forces and EV release, the effects of shear stress on the release and uptake of endothelial EVs remains elusive. We aim to decipher the interplay between these processes in endothelial cells exposed to atheroprone or atheroprotective shear stress. Confluent HUVECs were exposed to LSS or HSS for 24h. Large and small EVs were isolated from conditioned medium by centrifugation and size exclusion chromatography. They were characterised by TEM, Western blot, tunable resistive pulse sensing, flow cytometry and proteomics. Uptake experiments were performed using fluorescently-labelled EVs and differences between groups were assessed by flow cytometry and confocal microscopy. We found that levels of large and small EVs in conditioned media were fifty and five times higher in HSS than in LSS conditions, respectively. In vivo and in vitro uptake experiments revealed greater EV incorporation by cells exposed to LSS conditions. Additionally, endothelial LSS-EVs have a greater affinity for HUVECs than HSS-EVs or EVs derived from platelets, erythrocytes and leukocytes. Proteomic analysis revealed that LSS-EVs were enriched in adhesion proteins (PECAM1, MCAM), participating in EV uptake by endothelial cells. LSS-EVs also carried mitochondrial material, which may be implicated in elevating ROS levels in recipient cells. These findings suggest that shear stress influences EV biogenesis and uptake. Given the major role of EVs and shear stress in vascular health, deciphering the relation between these processes may yield innovative strategies for the early detection and treatment of endothelial dysfunction.

Low shear stress-induced blockage of autophagic flux impairs endothelial barrier and facilitates atherosclerosis in mice

Atherosclerosis preferentially occurs in areas with low shear stress (LSS) and oscillatory flow. LSS has been demonstrated to correlate with the development of atherosclerosis. The sphingosine 1-phosphate receptor 1 (S1PR1), involving intravascular blood flow sensing, regulates vascular development and vascular barrier function. However, whether LSS affects atherosclerosis via regulating S1PR1 remains incompletely clear. In this study, immunostaining results of F-actin, β-catenin, and VE-cadherin indicated that LSS impaired endothelial barrier function in human umbilical vein endothelial cells (HUVECs). Western blot analysis showed that LSS resulted in blockage of autophagic flux in HUVECs. In addition, autophagy agonist Rapamycin (Rapa) antagonized LSS-induced endothelial barrier dysfunction, whereas autophagic flux inhibitor Bafilomycin A1 (BafA1) exacerbated it, indicating that LSS promoted endothelial barrier dysfunction by triggering autophagic flux blockage. Notably, gene expression analysis revealed that LSS downregulated S1PR1 expression, which was antagonized by Rapa. Selective S1PR1 antagonist W146 impaired endothelial barrier function of HUVECs under high shear stress (HSS) conditions. Moreover, our data showed that expression of GAPARAPL2, a member of autophagy-related gene 8 (Atg8) proteins, was decreased in HUVECs under LSS conditions. Autophagic flux blockage induced by GAPARAPL2 knockdown inhibited S1PR1, aggravated endothelial barrier dysfunction of HUVECs in vitro, and promoted aortic atherosclerosis in ApoE−/− mice in vivo. Our study demonstrates that autophagic flux blockage induced by LSS downregulates S1PR1 expression and impairs endothelial barrier function. GABARAPL2 inhibition is involved in LSS-induced autophagic flux blockage, which impairs endothelial barrier function via downregulation of S1PR1.

Differential Distribution and Expression of α1-adrenergic Receptors in Low Shear Stress Induced Neointimal Formation

Low fluid shear stress in conduit arteries has emerged as a determinant of neointimal formation—an accumulation of vascular smooth muscle cells on the surface of the intima—the most challenging component of atherosclerosis to treat. α1-adrenergic receptor (α1-AR) antagonists have been well evaluated in vascular injury models yielding reduced neointimal formation. However, the α1-AR subtypes’ (α1A-AR, α1B-AR, α1D-AR) role in neointimal formation have not been determined under low shear stress conditions. α1-AR subtypes are expressed in various tissues and evoke different responses to the same ligand resulting in both adaptive and maladaptive responses. This study was designed to identify and characterize the α1-AR subtypes in low shear stress induced neointimal formation. An in vivo partial ligation mouse model was utilized to actuate low fluid shear stress and create neointimal formation in the left carotid artery in 14 days. The right carotid artery remained uninjured as the control. For tissue collection and processing, the left and right carotid arteries were harvested, snap-frozen in liquid nitrogen, and cryosectioned. Immunofluorescence staining was performed using primary antibodies for α1B-AR or α1A-AR and CD31 (endothelial cell marker). Confocal microscopy combined with processing and analysis was utilized for the detection, location, and distribution of cell markers. Histomorphometric analysis revealed significant structural changes, including the medial area and neointimal area, between the injured and non-injured arteries at the 14-day timepoint. Moreover, the injured arteries showed an increase in percent area stenosis, compared to the non-injured control. Histological observation of fluorescently labeled images revealed α1B-AR is densely distributed in the neointimal and medial area of the injured carotid arteries compared to the non-injured control. NucBlue (nuclear) staining indicated an increase in cell number in the medial and neointimal areas of the injured artery. Together, these results offer insight into changes in vascular α1-ARs under low shear stress conditions suggesting that α1B-AR may be upregulated in response to injury and contribute to neointimal formation. NIH HL155288. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Detecting normal and cancer skin cells via glycosylation and adhesion signatures: A path to enhanced microfluidic phenotyping

Recruiting circulating cells based on interactions between surface receptors and corresponding ligands holds promise for capturing cells with specific adhesive properties. Our study investigates the adhesion of skin cells to specific lectins, particularly focusing on advancements in lectin-based biosensors with diagnostic potential. We explore whether we can successfully capture normal skin (melanocytes and keratinocytes) and melanoma (WM35, WM115, WM266-4) cells in a low-shear flow environment by coating surfaces with lectins. Specifically, we coated surfaces with Dolichos biflorus (DBA) and Maackia Amurensis (MAL) lectins, which were used to detect and capture specific skin cells from the flow of cell mixture. Alterations in glycan expression (confirmed by fluorescent microscopy) demonstrated that DBA binds predominantly to normal skin cells, while MAL interacts strongly with melanoma cells. Assessing adhesion under static and dynamic low-shear stress conditions (up to 30 mPa) underscores the reliability of DBA and MAL as markers for discriminating specific cell type. Melanocytes and keratinocytes adhere to DBA-coated surfaces, while melanoma cells prefer MAL-coated surfaces. A comprehensive analysis encompassing cell shape, cytoskeleton, and focal adhesions shows the independence of our approach from the inherent characteristics of cells, thus demonstrating its robustness. Our results carry practical implications for lectin-biosensor designs, emphasizing the significance of glycan-based discrimination of pathologically altered cells. Combined with microfluidics, it demonstrates the value of cell adhesion as a discriminant of cancer-related changes, with potential applications spanning diagnostics, therapeutic interventions, and advanced biomedical technologies.

Low shear stress induces macrophage infiltration and aggravates aneurysm wall inflammation via CCL7/CCR1/TAK1/ NF-κB axis

BackgroundThis study aimed to elucidate the mechanism by which wall shear stress (WSS) influences vascular walls, accounting for the susceptibility of intracranial aneurysms (IAs) to rupture. MethodWe collected blood samples from the sacs of 24 ruptured and 28 unruptured IAs and analyzed the expression of chemokine CCL7 using enzyme-linked immunosorbent assay (ELISA). Univariate and multivariate logistic regression analyses were employed to assess clinical data, aneurysm morphology, and hemodynamics in both groups. Pearson correlation analysis investigated the relationship between CCL7 expression in aneurysm sac blood and WSS. Additionally, we established a bionic cell parallel plate co-culture shear stress model and a mouse low shear stress (LSS) model. The model was modulated using CCL7 recombinant protein, CCR1 inhibitor, and TAK1 inhibitor. We further evaluated CCL7 expression in endothelial cells and the levels of TAK1, NF-κB, IL-1β, and TNF-α in macrophages. Subsequently, the intergroup differences in expression were calculated. ResultsCCL7 expression was significantly higher in the ruptured group compared to the unruptured group. Hemodynamic analysis indicated that WSS was an independent predictor of the risk of aneurysm rupture. A negative linear correlation was observed between CCL7 expression and WSS. Upon addition of CCL7 recombinant protein, upregulation of CCR1 expression and increased levels of p-TAK1 and p-p65 were observed. Treatment with CCR1 and TAK1 inhibitors reduced inflammatory cytokine expression in macrophages under LSS conditions. Overexpression of TAK1 significantly alleviated the inhibitory effects of CCR1 inhibitors on p-p65 and inflammatory cytokines. ConclusionLSS prompts endothelial cells to secrete CCL7, which, upon binding to the macrophage surface receptor CCR1, stimulates the release of macrophage inflammatory factors via the TAK1/NF-κB signaling pathway. This process exacerbates aneurysm wall inflammation and increases the risk of aneurysm rupture.

Endothelial Dickkopf-1 Promotes Smooth Muscle Cell-derived Foam Cell Formation via USP53-mediated Deubiquitination of SR-A During Atherosclerosis.

Background: Shear stress-induced Dickkopf-1 (DKK1) secretion by endothelial cells (ECs) promotes EC dysfunction and accelerates atherosclerosis (AS). However, the paracrine role of endothelial DKK1 in modulating adjacent smooth muscle cells (SMCs) in atherosclerosis remains unclear. This study investigated the role of EC-secreted DKK1 in SMC-derived foam cell formation under shear stress, in vitro and in vivo. Methods: Parallel-plate co-culture flow system was used to explore the cellular communication between ECs and SMCs under shear stress in vitro. Endothelium-specific knockout of DKK1 (DKK1ECKO/APOE-/-) and endothelium-specific overexpression of DKK1 (DKK1ECTg) mice were constructed to investigate the role of endothelial DKK1 in atherosclerosis and SMC-derived foam cell formation in vivo. RNA sequencing (RNA-seq) was used to identify the downstream targets of DKK1. Reverse transcription quantitative polymerase chain reaction (RT-qPCR), western blot, coimmunoprecipitation (Co-IP) assays and chromatin immunoprecipitation (ChIP) experiments were conducted to explore the underlying regulatory mechanisms. Results: DKK1 is transcriptionally upregulated in ECs under conditions of low shear stress, but not in co-cultured SMCs. However, DKK1 protein in co-cultured SMCs is increased via uptake of low shear stress-induced endothelial DKK1, thereby promoting lipid uptake and foam cell formation in co-cultured SMCs via the post-translational upregulation of scavenger receptor-A (SR-A) verified in parallel-plate co-culture flow system, DKK1ECKO and DKK1ECTg mice. RNA sequencing revealed that DKK1-induced SR-A upregulation in SMCs is dependent on Ubiquitin-specific Protease 53 (USP53), which bound to SR-A via its USP domain and cysteine at position 41, exerting deubiquitination to maintain the stability of the SR-A protein by removing the K48 ubiquitin chain and preventing proteasomal pathway degradation, thereby mediating the effect of DKK1 on lipid uptake in SMCs. Moreover, DKK1 regulates the transcription of USP53 by facilitating the binding of transcription factor CREB to the USP53 promoter. SMC-specific overexpression of USP53 via adeno-associated virus serotype 2 vectors in DKK1ECKO/APOE-/- mice reversed the alleviation of atherosclerotic plaque burden, SR-A expression and lipid accumulation in SMCs within plaques resulting from DKK1 deficiency. Conclusions: Our findings demonstrate that, endothelial DKK1, induced by pathological low shear stress, acts as an intercellular mediator, promoted the foam cell formation of SMCs. These results suggest that targeted intervention with endothelial DKK1 may confer beneficial effects on atherosclerosis.

Influence of lattice strain on the mechanical properties of CoSb3 skutterudites

Elemental doping has been widely employed to manipulate the electrical and thermal transport properties of thermoelectric materials, without considering the influence to mechanical properties. In skutterudites, electronegative S-dopants have been found to reduce the lattice thermal conductivity. However, the effect of the covalent S-Sb bond on the mechanical properties of CoSb3 skutterudites remains unexplored. Compressive strength, in particular, exhibits a significant reduction in S-doped skutterudites, measuring only 249 MPa, representing a nearly 50% decrease compared to binary skutterudites. Additionally, there is a noticeable drop in the elastic, bulk and shear modulus. With molecular dynamics calculations, the shear strength of S-doped skutterudites also follows a sharp drop at a small ultimate strain. The evolutions of atomic configurations reveal that the lattice strain induced by S-dopants softens the Sb-Sb bond, ultimately leading to structure collapse under low shear stress conditions. These findings underscore the necessity for a comprehensive approach to elemental doping in thermoelectric community.

Shear thickening in dense bidisperse suspensions

Discrete-particle simulations of bidisperse shear thickening suspensions are reported. The work considers two packing parameters, the large-to-small particle radius ratio ranging from δ=1.4 (nearly monodisperse) to δ=4, and the large particle fraction of the total solid loading with values ζ=0.15, 0.5, and 0.85. Particle-scale simulations are performed over a broad range of shear stresses using a simulation model for spherical particles accounting for short-range lubrication forces, frictional interaction, and repulsion between particles. The variation of rheological properties and the maximum packing fraction ϕJ with shear stress σ are reported. At a fixed volume fraction ϕ, bidispersity decreases the suspension relative viscosity ηr=ηs/η0, where ηs is the suspension viscosity and η0 is the suspending fluid viscosity, over the entire range of shear stresses studied. However, under low shear stress conditions, the suspension exhibits an unusual rheological behavior: the minimum viscosity does not occur as expected at ζ≈0.5, but instead decreases with further increase of ζ to 0.85. The second normal stress difference N2 acts similarly. This behavior is caused by particles ordering into a layered structure, as is also reflected by the zero slope with respect to time of the mean-square displacement in the velocity gradient direction. The relative viscosity ηr of bidisperse rate-dependent suspensions can be predicted by a power law linking it to ϕJ, ηr=(1−ϕ/ϕJ)−2 in both low and high shear stress regimes. The agreement between the power law and experimental data from literature demonstrates that the model captures well the effect of particle size distribution, showing that viscosity roughly collapses onto a single master curve when plotted against the reduced volume fraction ϕ/ϕJ.

Shear-Induced ITGB4 Promotes Endothelial Cell Inflammation and Atherosclerosis.

The local heterogeneity in the distribution of atherosclerotic lesions is caused by local flow patterns. The integrin family plays crucial regulatory roles in diverse biological processes, but knowledge of integrin β4 (ITGB4) in shear stress-induced atherosclerosis is limited. This study clarified that low shear stress (LSS) regulates the generation of ITGB4 in endothelial cells with atheroprone phenotype to identify ITGB4's role in atherosclerosis. We found that LSS led to an increase in ITGB4 protein expression both in vitro and in vivo. ITGB4 knockdown attenuated inflammation and ROS generation in human umbilical vein endothelial cells (HUVECs) and reduced atherosclerotic lesion areas in ApoE-/- mice fed with HFD, largely independent of effects on the lipid profile. Mechanistically, ITGB4 knockdown altered the phosphorylation levels of SRC, FAK, and NFκB in HUVECs under LSS conditions. In addition, the knockdown of NFκB inhibited the production of ITGB4 and SRC phosphorylation, and the knockdown of SRC downregulated ITGB4 protein expression and NFκB activation. These data demonstrate a critical role of ITGB4 in atherosclerosis via modulation of endothelial cell inflammation, and ITGB4/SRC/NFκB might form a positive feedback loop in the regulation of endothelial cell inflammation.

Coupling Supramolecular Assemblies and Reactive Oxygen Species (ROS) with Megasonic Action for Applications in Shallow Trench Isolation (STI) Post-Chemical Mechanical Planarization (p-CMP) Cleaning.

Due to the continued miniaturization of semiconductor devices, slurry formulations utilized in the chemical mechanical planarization (CMP) process have become increasingly complex to meet stringent manufacturing specifications. Traditionally, in shallow trench isolation (STI), CMP, a contact cleaning method involving a poly(vinyl alcohol) (PVA) brush, is used to effectively transfer cleaning chemistry to the oxide substrate. This PVA brush can cause nonuniform cleaning chemistry transport, increased interfacial shear force, and cleaning-induced defectivity from brush loading. Previous work with traditional cleaning processes has shown that using “soft” supramolecular cleaning chemistries has dramatically improved cleaning efficacy while also minimizing the number of induced p-CMP defects. To minimize these effects, noncontact cleaning via the implementation of megasonic action has gained attention. This work employs “soft” cleaning chemistries with Cu2+–amino acid complexes, which can catalyze the formation of critical reactive oxygen species (ROS), and evaluates the p-CMP performance under megasonic action. Results from a second-order kinetic model indicate that megasonic conditions (i.e., time and power), “soft” cleaning chemistry structure (i.e., shape and charge), and the generation of ROS all play a critical role in cleaning efficacy under low shear stress conditions.

Assessment of on-treatment platelet reactivity at high and low shear stress and platelet activation status after the addition of dipyridamole to aspirin in the early and late phases after TIA and ischaemic stroke

BackgroundData are limited on the ability of dipyridamole to additionally inhibit platelet function/reactivity in ischaemic cerebrovascular disease (CVD) patients on aspirin. AimsTo assess inhibition of platelet function/reactivity and platelet activation with dipyridamole in CVD. MethodsThis prospective, observational study assessed TIA/ischaemic stroke patients before (baseline; N = 60), at 14 ±7 days (14d, N = 39) and ≥ 90 days (90d, N = 31) after adding dipyridamole to aspirin. Platelet function/reactivity at high shear stress (PFA-100® C-ADP) and low shear stress (VerifyNow® P2Y12 and Multiplate® ADP assays), and platelet activation status (% expression of CD62P, CD63 and leucocyte-platelet complexes on whole blood flow cytometry) were quantified. ‘Dipyridamole-high on-treatment platelet reactivity (HTPR)’ was defined as failure to inhibit ADP-induced platelet aggregation +/- adhesion compared with the patient's baseline on aspirin monotherapy by more than twice the coefficient-of-variation of the assay after adding dipyridamole to aspirin. ResultsDipyridamole-HTPR was identified in 71.4–75% of patients on PFA-100 C-ADP, 83.9–86.8% of patients on VerifyNow P2Y12, and 81.5–83.3% of patients on Multiplate ADP assays. There were no changes in CD62P/CD63 expression (P ≥ 0.18), or consistent changes in leucocyte-platelet complexes in CVD patients overall at 14d or 90d vs. baseline after commencing dipyridamole. Monocyte-platelet complexes increased in the patient subgroup with dipyridamole-HTPR at 14d and 90d on PFA-100, and at 14d on VerifyNow (P ≤ 0.04), but not in those without dipyridamole-HTPR. DiscussionAdditional antiplatelet effects of dipyridamole are detectable under high and low shear stress conditions with user-friendly platelet function/reactivity tests ex vivo. Increasing circulating monocyte-platelet complexes over time are associated with dipyridamole-HTPR.

A visual method for threshold detection of sediment motion in a flume experiment without human interference

AbstractAn experimental investigation of the entrainment threshold of sediment grains conducted in a non‐tiltable flume is reported and an original processing method based on image correlation suitable for detection of sediment threshold of motion is presented. Unlike existing visual measurement methods, the proposed method is not affected by the individual judgement of the experimentalists, which may strongly impact the estimation of incipient motion. Three entrainment thresholds were exhibited. The method highlighted a first threshold for very low shear stress conditions that was interpreted as a nascent movement threshold. The other two thresholds of motion were related to distinct stages of surface mobility of the bed: one related to bedload threshold and the other to bedform threshold. Series of tests were first performed to study the sensitivity of the method to different parameters: angle of view of the camera, size of the image and acceleration rate of the linear ramp of flow velocity. A ratio of the image surface and the grain surface equal to 3000 was found to be the minimum size to perform reliable estimations of entrainment thresholds for all the sediments in the range of 0.328–2.648 mm tested. It was the only condition for the method to work. Once the method was validated, tests were performed with homogeneous sands and compared to results based on reference‐based and visual methods. The correlation method yields similar values for the bedload threshold to those obtained from reference‐based methods. Thus it could be an alternative to other methodologies by the simplicity of its implementation, precision, no requirement for human interference and direct correlation between threshold velocity and bed mobility without extrapolation.

A peptide from the staphylococcal protein Efb binds P‐selectin and inhibits the interaction of platelets with leukocytes

AimsP‐selectin is a key surface adhesion molecule for the interaction of platelets with leukocytes. We have shown previously that the N‐terminal domain of Staphylococcus aureus extracellular fibrinogen‐binding protein (Efb) binds to P‐selectin and interferes with platelet‐leukocyte aggregate formation. Here, we aimed to identify the minimal Efb motif required for binding platelets and to characterize its ability to interfering with the formation of platelet‐leukocyte aggregates. Methods and ResultsUsing a library of synthetic peptides, we mapped the platelet‐binding site to a continuous 20 amino acid stretch. The peptide Efb68‐87 was able to bind to resting and, to a greater extent, thrombin‐stimulated platelets in the absence of fibrinogen. Dot blots, pull‐down assays and P‐selectin glycoprotein ligand‐1 (PSGL‐1) competitive binding experiments identified P‐selectin as the cellular docking site mediating Efb68‐87 platelet binding. Accordingly, Efb68‐87 did not bind to other blood cells and captured platelets from human whole blood under low shear stress conditions. Efb68‐87 did not affect platelet activation as tested by aggregometry, flow cytometry and immunoblotting, but inhibited the formation of platelet‐leukocyte aggregates (PLAs). Efb68‐87 also interfered with the platelet‐dependent stimulation of neutrophil extracellular traps (NETs) formation in vitro. ConclusionsWe have identified Efb68‐87 as a novel selective platelet‐binding peptide. Efb68‐87 binds directly to P‐selectin and inhibits interactions of platelets with leukocytes that lead to PLA and NET formation. As PLAs and NETs play a key role in thromboinflammation, Efb68‐87 is an exciting candidate for the development of novel selective inhibitors of the proinflammatory activity of platelets.

INVESTIGATION OF 3D CULTURE OF HUMAN ADIPOSE TISSUE-DERIVED MESENCHYMAL STEM CELLS IN A MICROFLUIDIC PLATFORM

Mesenchymal stem cells (MSCs) are multipotent stem cells that can support various tissues including bone marrow, adipose tissue, and synovial fluids, from which they can be readily isolated. The objective of this study is to harness the advantages of microfluidic systems for controlling and enhancing the maintenance and viability, and regenerative properties of MSCs by providing a 3D culture microenvironment with gelatin methacrylate (GelMA) hydrogel and exposing the cells to a slow fluid flow and low shear stress conditions. GelMA has methacryloyl groups and can be crosslinked by a photocuring process using biocompatible photoinitiators. The most common used photoinitiator for cellular encapsulation within hydrogels is the ultraviolet (UV) initiator 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959 or I2959), but due to its low water solubility and the necessity of using a shorter wavelength light (365 nm), it can lead to cellular phototoxic and genotoxic effects. To overcome these limitations, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) have recently been used with GelMA as an alternative photoinitiator. Because LAP is highly water soluble and has a 10 times faster polymerization rate, and it requires a visible light (λ = 405 nm) which makes it much safer for the cells, we use 10% GelMA together with 0.05% LAP photoinitiator for bioprinting human adipose tissue derived MSCs (hAT-MSCs) onto a membrane that has a 40 µm mesh size. To demonstrate a microfluidic culture advancement for improving the biological activities and regenerative capacity of the cells including cell adhesion, growth, viability and proliferation capacity as ultimate goals of this study, the membrane carrying the bioprinted construct was placed in a PDMS microchannel and exposed to the fluid to obtain dynamic microenvironments found in the human body. As a result, the cells were successfully maintained in the microfluidic 3D cell culture for two days, with a high cell viability of 99%.

Melatonin attenuates low shear stress-induced pyroptosis and endothelial cell dysfunction via the RORα/miR-223/STAT-3 signalling pathway.

Endothelial cells sense changes in blood flow shear stress and affect the progression of atherosclerotic plaques. Pyroptosis is an inflammatory form of cell death and has been implicated in cardiovascular diseases. Melatonin and its nuclear receptor retinoid-related orphan receptor α (RORα) have protective effects on the development of atherosclerosis. To date, whether melatonin can prevent endothelial cell pyroptosis and dysfunction in pathological shear stress remains unclear. In the present study, human umbilical vein endothelial cells (ECs) were cultured under low shear stress conditions (5 dyne/cm2) for 24 h and treated with or without melatonin (2 µmol/l). The binding sites of the microRNA (miR)-223 promoter and RORα were predicted using the JASPAR website. Expression of pyroptosis-related proteins, including cleaved N-terminal gasdermin D, caspase-1, intercellular adhesion molecule 1 (ICAM-1) and nitric oxide (NO) were assessed. The results indicated that low shear stress increased pyroptosis and ICAM-1 expression, whereas it decreased NO levels. Melatonin alleviated pyroptosis and ICAM-1 expression and increased the production of NO in ECs. Further assessment revealed that low-level shear stress decreased RORα protein and mRNA expression, whereas melatonin would bind to RORα and thereby promoted miR-223 transcription in ECs. The present study also identified signal transducer and activator of transcription 3 (STAT-3) as a potential target gene of miR-223-3p. When transfected with miR-223 inhibitor, ECs up-regulated the expression of pyroptosis-related proteins and ICAM-1, and down-regulated NO levels. By contrast, silencing STAT-3 expression diminished the protective effect of miR-223. These results indicated that melatonin prevented ECs from undergoing pyroptosis and alleviated dysfunction via the RORα/miR-223/STAT-3 signalling pathway. This information could aid in the development of novel therapeutic approaches and provide new insights into atherosclerosis.

Generalized Bed-Load Function Based on Empirical Data

There exist many bed-load functions in the literature to calculate bed-load transport rates, but none of them fit data from low to high shear stress conditions. This research presents a generalized bed-load function based on empirical data. Specifically, the classic power law in high shear stress conditions is extended to low shear stress conditions by applying a complimentary error function (or logistic function) and using Coles’ mathematical idea for the wake law in turbulent boundary layer velocity distribution. The resulting generalized bed-load function agrees well with the classic data sets; it reduces to Huang’s 5/3 power law in the very low and the high shear stress conditions, and it is numerically close to Paintal’s 16th power law in the transitional regime. It is found that the maximum turbulence-induced lift force and the minimum critical shear stress in the Shields diagram correspond to the inflection point (in terms of logarithmic scale) in the Einstein bed-load diagram, resulting in the most efficient bed-load transport rate. After that, this paper discusses the effects of turbulence-induced lift force, critical shear stress, viscosity, nonlinearity, and uncertainty on bed-load transport. Finally, an example with uncertainty analysis is illustrated for applications.

Restoration of endothelial autophagic flux in vascular endothelial cells exposed to low shear stress reduces atherosclerotic lesions

Atherosclerotic lesions preferentially develop in arterial areas exposed to low shear stress (SS), such as the aortic arch. Here, endothelial cells express a pro-inflammatory and pro-senescent phenotype. Our team has shown that this atheroprone phenotype is in part due to an inhibition of endothelial autophagy under low SS conditions. Previous studies have stated that acetylated microtubules are critical for functional autophagy. The aim of the study is to reduce endothelial inflammation by restoring autophagic flux in low SS conditions, via the inhibition of the α-tubulin deacetylase, Histone Deacetylase 6 (HDAC6). Confluent HUVECs, passage 2–4, were exposed to either low SS (2 dyn/cm 2 ) or high SS (20 dyn/cm 2 ) for 24 hours. Localization and levels of the different proteins involved were assessed by immunostaining and Western blot respectively. Autophagic flux was analyzed using tandem fluorescently tagged LC3. To evaluate atherosclerosis development in vivo, we used hypercholesteremic HDAC6 −/− /ApoE −/− mice. We found that blocking HDAC6 activity, by pharmacological (Tubastatin A) or genetic (shHDAC6) approaches, raised levels of acetylated α-tubulin, as well as autophagic flux. This resulted in a reduced expression of inflammatory markers (ICAM-1, VCAM-1 and MCP-1) in TNF-α-stimulated cells. We observed an increase in the colocalization of LC3 and lysosomal marker, LAMP2, in murine aorta of HDAC6 −/− /ApoE −/− mice, compared to control mice. Finally, atherosclerotic plaque size significantly decreased in the atheroprone areas of chimeric HDAC6 −/− /ApoE −/− mice, transplanted with HDAC6 +/+ /ApoE -/− bone marrow, when compared to HDAC6 +/+ /ApoE −/− littermate controls. These results indicate that targeting α-tubulin acetylation, via HDAC6-inhibition, may be an interesting strategy to restore endothelial autophagy and to promote an atheroprotective endothelial phenotype despite unfavorable shear stress conditions.

Exploring the crosstalk between endothelial autophagy and extracellular vesicle biology: Potential role in atherosclerosis

Atherosclerotic lesions mainly form in arterial areas exposed to low shear stress (LSS), where endothelial cells express an inflammatory phenotype. Our team demonstrated that endothelial autophagy is protective, stimulated by high shear stress (HSS) conditions when compared to LSS, hampering the development of these lesions. Endothelial EVs have been shown to regulate inflammation and senescence, thus playing a crucial role in vascular homeostasis. While previous studies found links between autophagy and EV formation, the exact role of autophagy in the release and uptake of EVs remains elusive. Thus, our aim is to decipher the interplay between these processes in endothelial cells. Confluent HUVECs were exposed to LSS (2 dyn/cm 2 ) or HSS (20 dyn/cm 2 ) for 24 hours. Large (> 200 nm) and small EVs were isolated from conditioned medium by sequential centrifugation and size exclusion chromatography. They were characterized by Western blot, TRPS, flow cytometry and proteomics. Uptake experiments were performed using fluorescently labelled EVs. Large and small EV levels were 50 and 5 times higher in HSS than in LSS conditions, respectively. In vivo and in vitro assays revealed more EV uptake by cells exposed to LSS compared to HSS. Interestingly, endothelial LSS-EVs had greater affinity for HUVECs than HSS-EVs or EVs derived from other cell types. Proteomics showed that LSS-EVs were enriched in adhesion proteins PECAM1 and MCAM. Blocking these proteins on EVs reduced their uptake by HUVECs. Finally, inhibiting autophagy in parent cells raised adhesion protein levels in HSS-EVs and caused them to be more readily taken up. These findings suggest that autophagy modulation, by shear stress, plays a crucial role during EV formation. Given the importance of EVs and autophagy in vascular health, deciphering the relation between these processes may yield new strategies for early detection and treatment of atherosclerosis.

Antithrombotic Effects of Combined PAR (Protease-Activated Receptor)-4 Antagonism and Factor Xa Inhibition.

PAR (protease-activated receptor)-4 antagonism has antiplatelet effects under conditions of high shear stress. We aimed to establish whether PAR4 antagonism had additive antithrombotic activity in the presence of factor Xa inhibition in an ex vivo model of acute arterial injury. Approach and Results: Fifteen healthy volunteers (29±6 years, 7 women) completed a phase zero double-blind randomized controlled crossover trial. Ex vivo platelet activation, platelet aggregation, and thrombus formation were measured following blood perfusion of low shear and high shear stress chambers. Upstream of the chambers, extracorporeal blood was admixed with (1) vehicle, (2) low-dose apixaban (20 ng/mL), (3) high-dose apixaban (80 ng/mL), (4) BMS-986141 (400 ng/mL), (5) BMS-968141 and low-dose apixaban, or (6) BMS-968141 and high-dose apixaban in 6 sequential studies performed in random order. Compared with vehicle, BMS-986141 demonstrated selective inhibition of PAR4-AP (agonist peptide)-stimulated platelet aggregation, platelet-monocyte aggregates, and P-selectin expression (P≤0.01 for all). Total thrombus area was reduced under both low shear and high shear stress conditions for all drug infusions (P<0.0001 for all versus vehicle). BMS-968141 reduced total (≤44.4%) and platelet-rich (≤39.3%) thrombus area, whereas apixaban reduced total (≤42.9%) and fibrin-rich (≤31.6%) thrombus area. Combination of BMS-986141 with apixaban caused a further modest reduction in total thrombus area (9.6%-12.4%), especially under conditions of high shear stress (P≤0.027). In the presence of factor Xa inhibition, PAR4 antagonism with BMS-986141 further reduces thrombus formation, especially under conditions of high shear stress. This suggests the potential for additive efficacy of combination PAR4 antagonism and factor Xa inhibition in the prevention of atherothrombotic events.

A Genome-Scale Insight into the Effect of Shear Stress During the Fed-Batch Production of Clavulanic Acid by Streptomyces Clavuligerus.

Streptomyces clavuligerus is a filamentous Gram-positive bacterial producer of the β-lactamase inhibitor clavulanic acid. Antibiotics biosynthesis in the Streptomyces genus is usually triggered by nutritional and environmental perturbations. In this work, a new genome scale metabolic network of Streptomyces clavuligerus was reconstructed and used to study the experimentally observed effect of oxygen and phosphate concentrations on clavulanic acid biosynthesis under high and low shear stress. A flux balance analysis based on experimental evidence revealed that clavulanic acid biosynthetic reaction fluxes are favored in conditions of phosphate limitation, and this is correlated with enhanced activity of central and amino acid metabolism, as well as with enhanced oxygen uptake. In silico and experimental results show a possible slowing down of tricarboxylic acid (TCA) due to reduced oxygen availability in low shear stress conditions. In contrast, high shear stress conditions are connected with high intracellular oxygen availability favoring TCA activity, precursors availability and clavulanic acid (CA) production.