176 results found
Weinberg P, Ghim M, Warboys C, et al., 2022, NO synthesis but not apoptosis, mitosis or inflammation can explain correlations between flow directionality and paracellular permeability of cultured endothelium, International Journal of Molecular Sciences, ISSN: 1422-0067
Haemodynamic wall shear stress varies from site to site within the arterial system and is thought to cause local variation in endothelial permeability to macromolecules. Our aim was to investigate mechanisms underlying the changes in paracellular permeability caused by different patterns of shear stress in long-term culture. We used the swirling well system and a substrate-binding tracer that permits visualisation of transport at the cellular level. Permeability increased in the centre of swirled wells, where flow is highly multidirectional, and decreased towards the edge, where flow is more uniaxial, compared to static controls. Overall, there was a reduction in permeability. There were also decreases in early- and late-stage apoptosis, proliferation and mitosis, and there were significant correlations between the first three and permeability when considering variation from the centre to the edge under flow. However, data from static controls did not fit the same relation and a cell-by-cell analysis showed that <5% of uptake under shear was associated with each of these events. Nuclear translocation of NF-κB p65 increased and then decreased with the duration of applied shear, as did permeability, but the spatial correlation between them was not significant. Application of an NO synthase inhibitor abolished the overall decrease in permeability caused by chronic shear and the difference in permeability between the centre and edge of the well. Hence shear and paracellular permeability appear to be linked by NO synthesis and not by apoptosis, mitosis or inflammation. The effect was mediated by an increase in transport through tricellular junctions.
Weinberg P, Kandangwa P, Torii R, et al., 2022, Influence of right coronary artery motion, flow pulsatility and non-Newtonian rheology on wall shear stress metrics, Fontiers in Bioengineering and Biotechnology, ISSN: 2296-4185
The patchy distribution of atherosclerosis within the arterial system is consistent with a controlling influence of hemodynamic wall shear stress (WSS). Patterns of low, oscillatory and transverse WSS have been invoked to explain the distribution of disease in the aorta. Disease of coronary arteries has greater clinical importance but blood flow in these vessels may be complicated by their movement during the cardiac cycle. Previous studies have shown that time average WSS is little affected by the dynamic geometry, and that oscillatory shear is influenced more. Here we additionally investigate effects on transverse WSS. We also investigate the influence of non-Newtonian blood rheologyas it can influence vortical structure, on which transverse WSS depends; Carreau-Yasuda models were used. WSS metrics were derived from numerical simulations of blood flow in a model of a moving right coronary artery which, together with a subject-specific inflow waveform, was obtained by MR imaging of a healthy human subject in a previous study. The results confirmed that time average WSS was little affected by dynamic motion, and that oscillatory WSS was more affected. They additionally showed that transverse WSS and its non-dimensional analogue, the Cross Flow Index, were affected still further. This appeared to reflect time-varying vortical structures caused by the changes in curvature. The influence of non-Newtonian rheology was significant with some physiologically realistic parameter values, and hence may be important in certain subjects. Dynamic geometry and non-Newtonian rheology should be incorporated into models designed to produce maps of transverse WSS in coronary arteries.
Shih K-CJ, Peiffer V, Rowland EM, et al., 2022, Non-linear shrinkage of Batson's #17 resin during vascular corrosion casting, JOURNAL OF ANATOMY, ISSN: 0021-8782
Weinberg P, 2022, Haemodynamic wall shear stress, endothelial permeability and atherosclerosis – a triad of controversy, Frontiers in Bioengineering and Biotechnology, Vol: 10, Pages: 1-29, ISSN: 2296-4185
A striking feature of atherosclerosis is its patchy distribution with the vascular system; certain arteries and certain locations within each artery are preferentially affected. Identifying the local risk factors underlying this phenomenon may lead to new therapeutic strategies. The large variation in lesion prevalence in areas of curvature and branching has motivated a search for haemodynamic triggers, particular those related to wall shear stress (WSS). The fact that lesions are rich in blood-derived lipids has motivated studies of local endothelial permeability. However, the location of lesions, the underlying haemodynamic triggers, the role of permeability, the routes by which lipids cross the endothelium, and the mechanisms by which WSS affects permeability have all been areas of controversy. This review presents evidence for and against the current consensus that lesions are triggered by low and/or oscillatory WSS and that this type of shear profile leads to elevated entry of low density lipoprotein (LDL) into the wall via widened intercellular junctions; it also evaluates more recent evidence that lesion location changes with age, that multidirectional shear stress plays a key role, that LDL dominantly crosses the endothelium by transcytosis, and that the link between flow and permeability results from hitherto unrecognised shear-sensitive mediators.
Weinberg P, Riemer K, Rowland E, et al., 2022, Contrast agent free assessment of blood flow and wall shear stress in the rabbit aorta using ultrasound image velocimetry, Ultrasound in Medicine and Biology, Vol: 48, Pages: 437-449, ISSN: 0301-5629
Blood flow velocity and wall shear stress (WSS) influence and are influencedby vascular disease. Their measurement is consequently useful in the laboratory and clinic. Contrast enhanced ultrasound image velocimetry (UIV) canestimate them accurately but the need to inject contrast agents limits utility. Singular value decomposition and high frame rate imaging may rendercontrast agents dispensable. Here we determined whether contrast agent freeUIV can measure flow and WSS. In simulation, accurate measurements wereachieved with a signal-to-noise ratio of 13.5 dB or higher. Signal intensity inthe rabbit aorta increased monotonically with mechanical index and was lowest during stagnant flow and uneven across the vessel. In vivo measurementswith contrast free and contrast enhanced UIV differed by 4.4 % and 1.9 % forvelocity magnitude and angle and by 9.47 % for WSS. Bland–Altman analysis of waveforms showed good agreement between contrast free and contrast enhanced UIV. In five rabbits the root-mean-square error was as low as 0.022m/s (0.81 %) and 0.11 Pa (1.7 %). This study demonstrates that with anoptimised protocol, UIV can assess flow and WSS without contrast agents.Unlike contrast enhanced UIV, it could be routinely employed.
Reavette RM, Sherwin SJ, Tang M-X, et al., 2021, Wave intensity analysis combined with machine learning can detect impaired stroke volume in simulations of heart failure, Frontiers in Bioengineering and Biotechnology, Vol: 9, Pages: 1-13, ISSN: 2296-4185
Heart failure is treatable, but in the United Kingdom, the 1-, 5- and 10-year mortality rates are 24.1, 54.5 and 75.5%, respectively. The poor prognosis reflects, in part, the lack of specific, simple and affordable diagnostic techniques; the disease is often advanced by the time a diagnosis is made. Previous studies have demonstrated that certain metrics derived from pressure-velocity-based wave intensity analysis are significantly altered in the presence of impaired heart performance when averaged over groups, but to date, no study has examined the diagnostic potential of wave intensity on an individual basis, and, additionally, the pressure waveform can only be obtained accurately using invasive methods, which has inhibited clinical adoption. Here, we investigate whether a new form of wave intensity based on noninvasive measurements of arterial diameter and velocity can detect impaired heart performance in an individual. To do so, we have generated a virtual population of two-thousand elderly subjects, modelling half as healthy controls and half with an impaired stroke volume. All metrics derived from the diameter-velocity-based wave intensity waveforms in the carotid, brachial and radial arteries showed significant crossover between groups-no one metric in any artery could reliably indicate whether a subject's stroke volume was normal or impaired. However, after applying machine learning to the metrics, we found that a support vector classifier could simultaneously achieve up to 99% recall and 95% precision. We conclude that noninvasive wave intensity analysis has significant potential to improve heart failure screening and diagnosis.
Arshad M, Rowland EM, Riemer K, et al., 2021, Improvement and validation of a computational model of flow in the swirling well cell culture model, Biotechnology and Bioengineering, ISSN: 0006-3592
Effects of fluid dynamics on cells are often studied by growing the cells on the base of cylindrical wells or dishes that are swirled on the horizontal platform of an orbital shaker. The swirling culture medium applies a shear stress to the cells that varies in magnitude and directionality from the centre to the edge of the vessel. Computational fluid dynamics methods are used to simulate the flow and hence calculate shear stresses at the base of the well. The shear characteristics at each radial location are then compared with cell behaviour at the same position. Previous simulations have generally ignored effects of surface tension and wetting, and results have only occasionally been experimentally validated. We investigated whether such idealized simulations are sufficiently accurate, examining a commonly-used swirling well configuration. The breaking wave predicted by earlier simulations was not seen, and the edge-to-centre difference in shear magnitude (but not directionality) almost disappeared, when surface tension and wetting were included. Optical measurements of fluid height and velocity agreed well only with the computational model that incorporated surface tension and wetting. These results demonstrate the importance of including accurate fluid properties in computational models of the swirling well method.
Weinberg P, Warboys CM, 2021, S1P in the development of atherosclerosis: roles of haemodynamic wall shear stress and endothelial permeability, Tissue Barriers, Vol: 9, Pages: 1-18, ISSN: 2168-8362
Atherosclerosis is characterised by focal accumulations of lipid within the arterial wall, thought to arise from effects of haemodynamic wall shear stress (WSS) on endothelial permeability. Identifying pathways that mediate effects of shear on permeability could therefore provide new therapeutic opportunities. Here we consider whether the sphingosine-1-phosphate (S1P) pathway could constitute such a route. We review effects of S1P in endothelial barrier function, the influence of WSS on S1P production and signalling, the results of trials investigating S1P in experimental atherosclerosis in mice, and associations between S1P levels and cardiovascular disease in humans. Although it seems clear that S1P reduces endothelial permeability and responds to WSS, the evidence that it influences atherosclerosis is equivocal. The effects of specifically pro- and anti-atherosclerotic WSS profiles on the S1P pathway require investigation, as do influences of S1P on the vesicular pathways likely to dominate low density lipoprotein transport across endothelium.
Ghim M, Pang KT, Burnap SA, et al., 2021, Endothelial cells exposed to atheroprotective flow secrete follistatin-like 1 protein which reduces transcytosis and inflammation, Atherosclerosis, Vol: 333, Pages: 56-66, ISSN: 0021-9150
Background and aimsWhen endothelium is cultured in wells swirled on an orbital shaker, cells at the well centre experience putatively pro-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 reported for pro-inflammatory changes. Here we identify the mediator and the flow characteristics that stimulate its release.Methods and resultsMedium 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 vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) expression, activation of nuclear factor κB (NF-κB), and monocyte adhesion. An inhibitor of transcytosis similarly decreased tracer transport. Mass spectrometry identified follistatin-like 1 (FSTL1) as a candidate mediator. Cells from the swirled edge produced significantly more FSTL1 than cells from the swirled centre or from static wells. Exogenous FSTL1 reduced transendothelial transport of the LDL-sized tracer and of LDL itself, as well as TNF-α-induced VCAM-1 and ICAM-1 expression. Bone morphogenetic protein (4BMP4) increased transendothelial transport of the LDL-sized tracer and expression of VCAM-1 and ICAM-1; these effects were abolished by FSTL1.ConclusionsPutatively atheroprotective flow stimulates production of FSTL1 from 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.
Pang KT, Ghim M, Liu C, et al., 2021, Leucine-Rich alpha-2-Glycoprotein 1 Suppresses Endothelial Cell Activation Through ADAM10-Mediated Shedding of TNF-alpha Receptor, FRONTIERS IN CELL AND DEVELOPMENTAL BIOLOGY, Vol: 9, ISSN: 2296-634X
Weinberg P, Pang K, Ghim M, et al., 2021, Segmenting growth of endothelial cells in 6-well plates on an orbital shaker for mechanobiological studies, Journal of Visualized Experiments, Vol: 2021, ISSN: 1940-087X
Shear stress induced on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells. One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype. Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cells seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere i
Weinberg P, Arshad M, Ghim M, et al., 2021, Endothelial cells do not align with the mean wall shear stress vector, Journal of the Royal Society Interface, Vol: 18, Pages: 1-10, ISSN: 1742-5662
Alignment of arterial endothelial cells with the mean wall shear stress (WSS) vector is the prototypical example of their responsiveness to flow. However, evidence for this behaviour rests on experiments where many WSS metrics had the same orientation or where they were incompletely characterised. In the present study, we tested the phenomenon more rigorously. Aortic endothelial cells were cultured in cylindrical wells on the platform of an orbital shaker. In this system, orientation would differ depending on the WSS metric to which the cells aligned. Variation in flow features and hence in possible orientations was further enhanced by altering the viscosity of the medium. Orientation of endothelial nuclei was compared to WSS characteristics obtained by computational fluid dynamics. At low mean WSS magnitudes, endothelial cells aligned with the modal WSS vector whilst at high mean WSS magnitudes they aligned so as to minimise the shear acting across their long axis (“transverse WSS”). Their failure to align with the mean WSS vector implies that other aspects of endothelial behaviour attributed to this metric require re-examination. The evolution of a mechanism for minimising transverse WSS is consistent with it having detrimental effects on the cells and with its putative role in atherogenesis.
Dumas R, Riemer K, Toulemonde M, et al., 2021, 4D ultrafast blood flow imaging comparison: Vector Doppler, transverse oscillation and speckle tracking, ISSN: 1948-5719
Quantitative measurement of volume flow rate is crucial in the assessment of cardiovascular performance and consequently useful in diagnosis e.g. of ischemic events or kidney failure. Recently, a number of methods have been developed to estimate spatiotemporally varying 3D blood flow. Here, we investigate three of these techniques, namely vector Doppler, transverse oscillation and ultrasound speckle tracking to measure three dimensional blood flow using an identical imaging sequence and microbubble contrast agents. We measured the flow in a straight PVA tube phantom at a constant flow rate with known mass flow rate. All three methods underestimated flow rate with the highest accuracy for ultrasound speckle tracking (-0.5%), followed by transverse oscillation (-13.6%) and vector Doppler (-14.6%). Qualitative assessment of flow profiles showed good resemblance between transverse oscillation and ultrasound speckle tracking. Intra-method comparison illustrated that the lowest magnitude bias between measurements was obtained for transverse oscillation and vector Doppler (1.95 cm/s), The lowest angle bias was calculated between ultrasound speckle tracking and transverse oscillation (6.47°). All methods are capable of measuring volumetric flow and mass flow rate with a low to moderate error. Further work is required to evaluate their performance in more complex settings for clinical applications.
Reavette RM, Sherwin SJ, Tang M, et al., 2020, Comparison of arterial wave intensity analysis by pressure-velocity and diameter-velocity methods in a virtual population of adult subjects., Proceedings of the Institution of Mechanical Engineers Part H: Journal of Engineering in Medicine, Vol: 234, Pages: 1260-1276, ISSN: 0954-4119
Pressure-velocity-based analysis of arterial wave intensity gives clinically relevant information about the performance of the heart and vessels, but its utility is limited because accurate pressure measurements can only be obtained invasively. Diameter-velocity-based wave intensity can be obtained noninvasively using ultrasound; however, due to the nonlinear relationship between blood pressure and arterial diameter, the two wave intensities might give disparate clinical indications. To test the magnitude of the disagreement, we have generated an age-stratified virtual population to investigate how the two dominant nonlinearities 'viscoelasticity and strain-stiffening' cause the two formulations to differ. We found strong agreement between the pressure-velocity and diameter-velocity methods, particularly for the systolic wave energy, the ratio between systolic and diastolic wave heights, and older subjects. The results are promising regarding the introduction of noninvasive wave intensities in the clinic.
Weinberg P, Dazzi M, Rowland E, et al., 2020, 3D confocal microscope imaging of macromolecule uptake in the intact brachiocephalic artery, Atherosclerosis, Vol: 310, Pages: 93-101, ISSN: 0021-9150
Background and aimsElevated uptake of plasma macromolecules by the arterial wall is an early event in atherogenesis. Existing optical techniques for detecting macromolecular tracers in the wall have poor depth penetration and hence require en face imaging of flattened arterial segments. Imaging uptake in undistorted curved and branched vessels would be useful in understanding disease development.MethodsDepth penetration was increased by applying optical clearing techniques. The rat aorto-brachiocephalic junction was imaged intact by confocal microscopy after it had been exposed to circulating rhodamine-labelled albumin in vivo, fixed in situ, excised and then cleared with benzyl alcohol/benzyl benzoate. Tracer uptake was mapped onto a 3D surface mesh of the arterial geometry.ResultsTracer fluorescence was detectable throughout the wall closest to the objective lens and, despite a vessel diameter of c. 1 mm, in the wall on the other side of the artery, across the lumen. By tile scanning, tracer concentrations were mapped in the aorta, the brachiocephalic artery and their junction without opening or flattening either vessel. Optical clearing was also shown to be compatible with immunofluorescent staining and imaging of experimental atherosclerosis.ConclusionsThe technique obviates the need for labour-intensive sample preparation associated with standard en face imaging. More importantly, it preserves arterial geometry, facilitating co-localisation of uptake maps with maps of biomechanical factors, which typically exist on 3D surface meshes. It will permit the correlation of haemodynamic wall shear stress with macromolecule permeability more accurately in regions of high curvature or branching, such as in the coronary arteries.
Rowland EM, Bailey EL, Weinberg PD, 2020, Estimating Arterial Cyclic Strain from the Spacing of Endothelial Nuclei, EXPERIMENTAL MECHANICS, Vol: 61, Pages: 171-190, ISSN: 0014-4851
Ghim M, Mohamied Y, Weinberg PD, 2020, The role of tricellular junctions in the transport of macromolecules across endothelium, Cardiovascular Engineering and Technology, Vol: 12, Pages: 101-113, ISSN: 1869-408X
PurposeTransport of water and solutes across vascular endothelium is important in normal physiology and critical in the development of various diseases, including atherosclerosis. However, there is debate about the routes for such transport. We recently showed that an albumin-sized tracer crossed endothelium at bicellular and tricellular junctions, a tracer having the size of high density lipoprotein crossed only through tricellular junctions, and a tracer with the size of low density lipoprotein was unable to cross by either route and instead traversed the cells themselves. Here we review previous work on the structure and function of tricellular junctions. We then describe a study in which we assessed the role of such junctions in the transport of an albumin-sized tracer.MethodsWe examined normal endothelial monolayers, the effect of agonists that modify their permeability, and the influence of different patterns of shear stress.ResultsUnder normal conditions, approximately 85% of transendothelial transport occurred through tricellular junctions. This fraction was unchanged when permeability was reduced by sphingosine-1-phosphate or increased by thrombin, and also did not differ between endothelium exposed to multidirectional as opposed to uniaxial shear stress despite a > 50% difference in permeability.ConclusionThese data show that tricellular junctions dominate normal transport of this tracer and largely determine influences of agonists and shear. The effects were attributable to changes in both the number and conductivity of the junctions. Further investigation of these structures will lead to increased understanding of endothelial barrier function and may suggest new therapeutic strategies in disease.
Riemer K, Rowland EM, Leow CH, et al., 2020, Determining haemodynamic wall shear stress in the rabbit aorta in vivo using contrast-enhanced ultrasound image velocimetry, Annals of Biomedical Engineering, Vol: 48, Pages: 1728-1739, ISSN: 0090-6964
Abnormal blood flow and wall shear stress (WSS) can cause and be caused by cardiovascular disease. To date, however, no standard method has been established for mapping WSS in vivo. Here we demonstrate wide-field assessment of WSS in the rabbit abdominal aorta using contrast-enhanced ultrasound image velocimetry (UIV). Flow and WSS measurements were made independent of beam angle, curvature or branching. Measurements were validated in an in silico model of the rabbit thoracic aorta with moving walls and pulsatile flow. Mean errors over a cardiac cycle for velocity and WSS were 0.34 and 1.69%, respectively. In vivo time average WSS in a straight segment of the suprarenal aorta correlated highly with simulations (PC = 0.99) with a mean deviation of 0.29 Pa or 5.16%. To assess fundamental plausibility of the measurement, UIV WSS was compared to an analytic approximation derived from the Poiseuille equation; the discrepancy was 17%. Mapping of WSS was also demonstrated in regions of arterial branching. High time average WSS (TAWSSxz = 3.4 Pa) and oscillatory flow (OSIxz = 0.3) were observed near the origin of conduit arteries. In conclusion, we have demonstrated that contrast-enhanced UIV is capable of measuring spatiotemporal variation in flow velocity, arterial wall location and hence WSS in vivo with high accuracy over a large field of view.
Ghim M, Weinberg PD, Warboys CM, 2020, beta-CATENIN TRANSCRIPTIONAL ACTIVITY REGULATES THE PERMEABILITY OF ENDOTHELIAL CELLS EXPOSED TO DISTURBED FLOW, Publisher: SPRINGER, Pages: 284-284, ISSN: 0920-3206
Riemer K, Toulemonde M, Rowland EM, et al., 2020, 4D Blood Flow and Wall Shear Stress measured using Volumetric Ultrasound Image Velocimetry, IEEE International Ultrasonics Symposium (IEEE IUS), Publisher: IEEE, ISSN: 1948-5719
Zhang G, Toulemonde M, Riemer K, et al., 2020, Effects of Mechanical Index on Repeated Sparse Activation of Nanodroplets In Vivo, IEEE International Ultrasonics Symposium (IEEE IUS), Publisher: IEEE, ISSN: 1948-5719
Zhang G, Harput S, Toulemonde M, et al., 2019, Acoustic wave sparsely-activated localization microscopy (AWSALM): in vivo fast ultrasound super-resolution imaging using nanodroplets, IEEE International Ultrasonics Symposium (IUS), Publisher: IEEE, Pages: 1930-1933, ISSN: 1948-5719
Current localization-based super-resolution ultrasound imaging requires a low concentration of flowing microbubbles to visualize microvasculature beyond the diffraction limit and acquisition is slow. Nanodroplets offer a promising solution as they can be sparsely activated and deactivated on-demand. In this study, acoustic wave sparsely-activated localization microscopy (AWSALM) using activation and deactivation of nanodroplets, an acoustic counterpart of photo-activated localization microscopy (PALM) which is less dependent on agent concentration and the presence of flow, is demonstrated for super-resolution imaging in deep tissues in vivo. An in vivo super-resolution image of a rabbit kidney is obtained in 1.1 seconds using AWSALM, where micro-vessels with apparent sizes far below the half-wavelength of 220 μm were visualized. This preliminary result demonstrates the feasibility of applying AWSALM for in vivo super-resolution imaging.
Wen Y, Ahmad F, Mohri Z, et al., 2019, Cysteamine inhibits lysosomal oxidation of low density lipoprotein in human macrophages and reduces atherosclerosis in mice, Atherosclerosis, Vol: 291, Pages: 9-18, ISSN: 0021-9150
BACKGROUND AND AIMS: We have shown previously that low density lipoprotein (LDL) aggregated by vortexing is internalised by macrophages and oxidised by iron in lysosomes to form the advanced lipid/protein oxidation product ceroid. We have now used sphingomyelinase-aggregated LDL, a more pathophysiological form of aggregated LDL, to study lysosomal oxidation of LDL and its inhibition by antioxidants, including cysteamine (2-aminoethanethiol), which concentrates in lysosomes by several orders of magnitude. We have also investigated the effect of cysteamine on atherosclerosis in mice. METHODS: LDL was incubated with sphingomyelinase, which increased its average particle diameter from 26 to 170 nm, and was then incubated for up to 7 days with human monocyte-derived macrophages. LDL receptor-deficient mice were fed a Western diet (19-22 per group) and some given cysteamine in their drinking water at a dose equivalent to that used in cystinosis patients. The extent of atherosclerosis in the aortic root and the rest of the aorta was measured. RESULTS: Confocal microscopy revealed lipid accumulation in lysosomes in the cultured macrophages. Large amounts of ceroid were produced, which colocalised with the lysosomal marker LAMP2. The antioxidants cysteamine, butylated hydroxytoluene, amifostine and its active metabolite WR-1065, inhibited the production of ceroid. Cysteamine at concentrations well below those expected to be present in lysosomes inhibited the oxidation of LDL by iron ions at lysosomal pH (pH 4.5) for prolonged periods. Finally, we showed that the extent of atherosclerotic lesions in the aortic root and arch of mice was significantly reduced by cysteamine. CONCLUSIONS: These results support our hypothesis that lysosomal oxidation of LDL is important in atherosclerosis and hence antioxidant drugs that concentrate in lysosomes might provide a novel therapy for this disease.
Zhu J, Lin S, Leow CH, et al., 2019, High Frame Rate Contrast-Enhanced Ultrasound Imaging for Slow Lymphatic Flow: Influence of Ultrasound Pressure and Flow Rate on Bubble Disruption and Image Persistence, Ultrasound in Medicine and Biology, Vol: 45, Pages: 2456-2470, ISSN: 0301-5629
Contrast enhanced ultrasound (CEUS) utilising microbubbles shows great potential for visualising lymphatic vessels and identifying sentinel lymph nodes (SLN) which is valuable for axillary staging in breast cancer patients. However, current CEUS imaging techniques have limitations that affect the accurate visualisation and tracking of lymphatic vessels and SLN. (1) Tissue artefacts and bubble disruption can reduce the image contrast. (2) Limited spatial and temporal resolution diminishes the amount of information that can be captured by CEUS. (3) The slow lymph flow makes Doppler based approaches less effective. This work evaluates on a lymphatic vessel phantom the use of high frame-rate (HFR) CEUS for the detection of lymphatic vessels where flow is slow. Specifically the work particularly investigates the impact of key factors in lymphatic imaging, including ultrasound pressure and flow velocity as well as probe motion during vessel tracking, on bubble disruption and image contrast. A trail was also conducted to apply HFR CEUS imaging on vasculature in a rabbit popliteal lymph node (LN). Our results show that (1) HFR imaging and SVD filtering can significantly reduce tissue artefacts in the phantom; (2) the slow flow rate within the phantom makes image contrast and signal persistence more susceptible to changes in ultrasound amplitude/MI, and an MI value can be chosen to reach a compromise between images contrast and bubble disruption under slow flow condition; (3) probe motion significantly decreases image contrast of the vessel, which can be improved by applying motion correction prior to SVD filtering; (4) the optical observation of the impact of ultrasound pressure in HFR CEUS further confirm the importance of optimising ultrasound amplitude MI; (5) Vessels inside rabbit LN with blood flow less than 3 mm/s are clearly visualised.
Zhu J, Rowland E, Harput S, et al., 2019, 3D super-resolution ultrasound imaging of rabbit lymph node vasculature in vivo using microbubbles, Radiology, Vol: 291, Pages: 642-650, ISSN: 0033-8419
Background: Variations in lymph node (LN) microcirculation can be indicative of metastasis. Identifying and quantifying metastatic LNs remains essential for prognosis and treatment planning but a reliable non-invasive imaging technique is lacking. 3D super-resolution (SR) ultrasound has shown potential to noninvasively visualize microvascular networks in vivo.Purpose: To study the feasibility of 3D SR ultrasound imaging of rabbit lymph node (LN) microvascular structure and blood flow using microbubbles.Materials and Methods: In vivo studies were carried out to image popliteal LNs of two healthy male New Zealand White rabbits aged 6-8 weeks. 3D high frame rate contrast enhanced ultrasound was achieved by mechanically scanning a linear imaging probe. Individual microbubbles were identified, localized, and tracked to form 3D SR images and super-resolved velocity maps. Acoustic sub-aperture processing (ASAP)was used to improve image contrast and generateenhanced power Doppler (PD) and color Doppler (CD) images. Vessel size and blood flow velocity distributions were evaluated and assessed by Student’s paired t-test. Results:SR images revealed micro-vessels in the rabbitLN, with branches clearly resolved when separated by 30 μm, which is less than half of the acoustic wavelength and not resolvable by power or color Doppler. The apparent size distribution of most vessels in the SR images was below 80 μm and agrees with micro-CT data whereas most of those detected by Doppler techniques were larger than 80 μm. The blood flow velocity distribution indicated that most of the blood flow in the rabbit popliteal LN was at velocities lower than 5mm/s. Conclusion: 3D super-resolution ultrasound imaging using microbubbles allows non-invasive and non-ionizing visualization and quantification of rabbit lymph node microvascular structures and blood flow dynamics with resolution below the wave diffraction limit.
Warboys CM, Ghim M, Weinberg PD, 2019, Understanding mechanobiology in cultured endothelium: A review of the orbital shaker method, Atherosclerosis, Vol: 285, Pages: 170-177, ISSN: 0021-9150
A striking feature of atherosclerosis is its highly non-uniform distribution within the arterial tree. This has been attributed to variation in the haemodynamic wall shear stress (WSS) experienced by endothelial cells, but the WSS characteristics that are important and the mechanisms by which they lead to disease remain subjects of intensive investigation despite decades of research. In vivo evidence suggests that multidirectional WSS is highly atherogenic. This possibility is increasingly being studied by culturing endothelial cells in wells that are swirled on an orbital shaker. The method is simple and cost effective, has high throughput and permits chronic exposure, but interpretation of the results can be difficult because the fluid mechanics are complex; hitherto, their description has largely been restricted to the engineering literature. Here we review the findings of such studies, which indicate that putatively atherogenic flow characteristics occur at the centre of the well whilst atheroprotective ones occur towards the edge, and we describe simple mathematical methods for choosing experimental variables that avoid resonance, wave breaking and uncovering of the cells. We additionally summarise a large number of studies showing that endothelium cultured at the centre of the well expresses more pro-inflammatory and fewer homeostatic genes, has higher permeability, proliferation, apoptosis and senescence, and shows more endothelial-to-mesenchymal transition than endothelium at the edge. This simple method, when correctly interpreted, has the potential to greatly increase our understanding of the homeostatic and pathogenic mechanobiology of endothelial cells and may help identify new therapeutic targets in vascular disease.
Zhang G, Pang KT, Ghim M, et al., 2018, Investigation of Nanodroplet Adhesion to Endothelial Cells under Atheroprone Flow Conditions, IEEE International Ultrasonics Symposium (IUS), Publisher: IEEE, ISSN: 1948-5719
Zhou X, Leow CH, Rowland E, et al., 2018, 3D velocity and volume flow measurement in vivo using speckle decorrelation and 2D high frame rate contrast-enhanced ultrasound, IEEE Transactions on Ultrasonics, Ferroelectrics and Frequency Control, Vol: 65, Pages: 2233-2244, ISSN: 0885-3010
Being able to measure 3D flow velocity and volumetric flow rate effectively in the cardiovascular system is valuable but remains a significant challenge in both clinical practice and research. Currently there has not been an effective and practical solution to the measurement of volume flow using ultrasound imaging systems due to challenges in existing 3D imaging techniques and high system cost. In this study, a new technique for quantifying volumetric flow rate from the cross-sectional imaging plane of the blood vessel was developed by using speckle decorrelation, 2D high frame rate imaging with a standard 1D array transducer, microbubble contrast agents, and ultrasound imaging velocimetry (UIV). Through speckle decorrelation analysis of microbubble signals acquired with a very high frame rate and by using UIV to estimate the two in-plane flow velocity components, the third and out-of-plane velocity component can be obtained over time and integrated to estimate volume flow. The proposed technique was evaluated on a wall-less flow phantom in both steady and pulsatile flow. UIV in the longitudinal direction was conducted as a reference. The influences of frame rate, mechanical index, orientation of imaging plane, and compounding on velocity estimation were also studied. In addition, an in vivo trial on the abdominal aorta of a rabbit was conducted. The results show that the new system can estimate volume flow with an averaged error of 3.65±2.37% at a flow rate of 360 ml/min and a peak velocity of 0.45 m/s, and an error of 5.03±2.73% at a flow rate of 723 ml/min and a peak velocity of 0.8 m/s. The accuracy of the flow velocity and volumetric flow rate estimation directly depend on the imaging frame rate. With a frame rate of 6000 Hz, a velocity up to 0.8 m/s can be correctly estimated. A higher mechanical index (MI=0.42) is shown to produce greater errors (up to 21.78±0.49%, compared to 3.65±2.37% at MI=0.19). An in vivo trial, where velo
van Ooij P, Cibis M, Rowland EM, et al., 2018, Spatial correlations between MRI-derived wall shear stress and vessel wall thickness in the carotid bifurcation, European Radiology Experimental, Vol: 2, ISSN: 2509-9280
BACKGROUND: To explore the possibility of creating three-dimensional (3D) estimation models for patient-specific wall thickness (WT) maps using patient-specific and cohort-averaged WT, wall shear stress (WSS), and vessel diameter maps in asymptomatic atherosclerotic carotid bifurcations. METHODS: Twenty subjects (aged 75 ± 6 years [mean ± standard deviation], eight women) underwent a 1.5-T MRI examination. Non-gated 3D phase-contrast gradient-echo images and proton density-weighted echo-planar images were retrospectively assessed for WSS, diameter estimation, and WT measurements. Spearman's ρ and scatter plots were used to determine correlations between individual WT, WSS, and diameter maps. A bootstrapping technique was used to determine correlations between 3D cohort-averaged WT, WSS, and diameter maps. Linear regression between the cohort-averaged WT, WSS, and diameter maps was used to predict individual 3D WT. RESULTS: Spearman's ρ averaged over the subjects was - 0.24 ± 0.18 (p < 0.001) and 0.07 ± 0.28 (p = 0.413) for WT versus WSS and for WT versus diameter relations, respectively. Cohort-averaged ρ, averaged over 1000 bootstraps, was - 0.56 (95% confidence interval [- 0.74,- 0.38]) for WT versus WSS and 0.23 (95% confidence interval [- 0.06, 0.52]) for WT versus diameter. Scatter plots did not reveal relationships between individual WT and WSS or between WT and diameter data. Linear relationships between these parameters became apparent after averaging over the cohort. Spearman's ρ between the original and predicted WT maps was 0.21 ± 0.22 (p < 0.001). CONCLUSIONS: With a combination of bootstrapping and cohort-averaging methods, 3D WT maps can be predicted from the individual 3D WSS and diameter maps. The methodology may help to elucidate pathological p
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