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The focal nature of atherosclerotic lesions suggests an important role of local hemodynamic environment. Recent studies have demonstrated significant roles of Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in mediating mechanotransduction and vascular homeostasis. The objective of this study is to investigate the functional role of YAP/TAZ in the flow regulation of atheroprone endothelial phenotypes and the consequential development of atherosclerotic lesions. We found that exposure of cultured endothelial cells (ECs) to the atheroprone disturbed flow resulted in YAP/TAZ activation and translocation into EC nucleus to up-regulate the target genes, including cysteine-rich angiogenic inducer 61 (CYR61), connective tissue growth factor (CTGF), and ankyrin repeat domain 1 (ANKRD1). In contrast, the athero-protective laminar flow suppressed YAP/TAZ activities. En face analysis of mouse arteries demonstrated an increased nuclear localization of YAP/TAZ and elevated levels of the target genes in the endothelium in atheroprone areas compared with athero-protective areas. YAP/TAZ knockdown significantly attenuated the disturbed flow induction of EC proliferative and proinflammatory phenotypes, whereas overexpression of constitutively active YAP was sufficient to promote EC proliferation and inflammation. In addition, treatment with statin, an antiatherosclerotic drug, inhibited YAP/TAZ activities to diminish the disturbed flow-induced proliferation and inflammation. In vivo blockade of YAP/TAZ translation by morpholino oligos significantly reduced endothelial inflammation and the size of atherosclerotic lesions. Our results demonstrate a critical role of the activation of YAP/TAZ by disturbed flow in promoting atheroprone phenotypes and atherosclerotic lesion development. Therefore, inhibition of YAP/TAZ activation is a promising athero-protective therapeutic strategy., Competing Interests: The authors declare no conflict of interest.

Arteriovenous fistula (AVF) is the first choice for providing vascular access for hemodialysis patients, but maintaining its patency is challenging. AVF failure is primarily due to development of neointimal hyperplasia (NH) and subsequent stenosis. Using idealized models of AVF we previously suggested that reciprocating hemodynamic wall shear is implicated in vessel stenosis. The aim of the present study was to investigate local hemodynamics in patient-specific side-to-end AVF. We reconstructed realistic geometrical models of four AVFs from magnetic resonance images acquired in a previous clinical study. High-resolution computational fluid dynamics simulations using patient-specific blood rheology and flow boundary conditions were performed. We then characterized the flow field and categorized disturbed flow areas by means of established hemodynamic wall parameters. In all AVF, either in upper or lower arm location, we consistently observed transitional laminar to turbulent-like flow developing in the juxta-anastomotic vein and damping towards the venous outflow, but not in the proximal artery. High-frequency fluctuations of the velocity vectors in these areas result in eddies that induce similar oscillations of wall shear stress vector. This condition may importantly impair the physiological response of endothelial cells to blood flow and be responsible for NH formation in newly created AVF.

The blood flow pathway within a device, together with the biomaterial surfaces and status of the patient's blood, are well-recognized factors in the development of thrombotic deposition and subsequent embolization. Blood flow patterns are of particular concern for devices such as blood pumps (i.e. ventricular assist devices, VADs) where shearing forces can be high, volumes are relatively large, and the flow fields can be complex. However, few studies have examined the effect of geometric irregularities on thrombus formation on clinically relevant opaque materials under flow. The objective of this study was to quantify human platelet deposition onto Ti6Al4V alloys, as well as positive and negative control surfaces, in the region of defined crevices (∼50-150 μm in width) that might be encountered in many VADs or other cardiovascular devices. To achieve this, reconstituted fresh human blood with hemoglobin-depleted red blood cells (to achieve optical clarity while maintaining relevant rheology), long working optics, and a custom designed parallel plate flow chamber were employed. The results showed that the least amount of platelet deposition occurred in the largest crevice size examined, which was counterintuitive. The greatest levels of deposition occurred in the 90 μm and 53 μm crevices at the lower wall shear rate. The results suggest that while crevices may be unavoidable in device manufacturing, the crevice size might be tailored, depending on the flow conditions, to reduce the risk of thromboembolic events. Further, these data might be used to improve the accuracy of predictive models of thrombotic deposition in cardiovascular devices to help optimize the blood flow path and reduce device thrombogenicity., (Copyright © 2016. Published by Elsevier Ltd.)

Thrombus in a femoral artery may form under stagnant flow conditions which vary depending on the local arterial waveform. Four different physiological flow waveforms - poor (blunt) monophasic, sharp monophasic, biphasic and triphasic - can exist in the femoral artery as a result of different levels of peripheral arterial disease progression. This study aims to examine the effect of different physiological waveforms on femoral artery haemodynamics. In this regard, a fluid-structure interaction analysis was carried out in idealised models of bifurcated common femoral artery. The results showed that recirculation zones occur in almost all flow waveforms; however, the sites at where these vortices are initiated, the size and structure of vortices are highly dependent on the type of flow waveform being used. It was shown that the reverse diastolic flow in biphasic and triphasic waveforms leads to the occurrence of a retrograde flow which aids in 'washout' of the disturbed flow regions. This may limit the likelihood of thrombus formation, indicating the antithrombotic role of retrograde flow in femoral arteries. Furthermore, our data revealed that the flow particles experience considerably higher residence time under blunt and sharp monophasic waveforms than under biphasic and triphasic waveforms. This confirms that the risk of atherothrombotic plaque initiation and development in femoral arteries is higher under blunt and sharp monophasic waveforms than under biphasic and triphasic flow waveforms.

Disturbed blood flow with low-oscillatory shear stress (OSS) is a predominant atherogenic factor leading to dysfunctional endothelial cells (ECs). Recently, it was found that disturbed flow can directly induce endoplasmic reticulum (ER) stress in ECs, thereby playing a critical role in the development and progression of atherosclerosis. Ursodeoxycholic acid (UDCA), a naturally occurring bile acid, has long been used to treat chronic cholestatic liver disease and is known to alleviate endoplasmic reticulum (ER) stress at the cellular level. However, its role in atherosclerosis remains unexplored. In this study, we demonstrated the anti-atherogenic activity of UDCA via inhibition of disturbed flow-induced ER stress in atherosclerosis. UDCA effectively reduced ER stress, resulting in a reduction in expression of X-box binding protein-1 (XBP-1) and CEBP-homologous protein (CHOP) in ECs. UDCA also inhibits the disturbed flow-induced inflammatory responses such as increases in adhesion molecules, monocyte adhesion to ECs, and apoptosis of ECs. In a mouse model of disturbed flow-induced atherosclerosis, UDCA inhibits atheromatous plaque formation through the alleviation of ER stress and a decrease in adhesion molecules. Taken together, our results revealed that UDCA exerts anti-atherogenic activity in disturbed flow-induced atherosclerosis by inhibiting ER stress and the inflammatory response. This study suggests that UDCA may be a therapeutic agent for prevention or treatment of atherosclerosis.

The distribution of arterial lesions is attributed by the prevalent mechanistic theory to the proatherogenic role played by low and oscillatory wall shear stress (WSS). However, discrepancies observed when comparing WSS distribution with location of regions with lesion prevalence challenge this theory and have recently stimulated the idea that a role in endothelial mechanosensing is played by WSS multidirectionality, which could contribute to explain the observed discrepancies. Here an approach is presented for analyzing the multidirectional nature of WSS in complex vascular geometries. Using an essential geometric attribute of the vessel (its centerline), the local WSS vector is projected along an "axial" direction (aligned with the tangent to the vessel׳s centerline), and "secondary" direction (orthogonal to centerline׳s tangent), which is related to secondary flow. The WSS projection scheme is applied: (1) to a realistic computational hemodynamic model of human aorta, with the aim to come up with a plausibility checking regarding its consistency; and (2) to investigate if an aortic hemodynamics characterized by different amount and topology of helical flow (HF) could influence WSS directionality. The projection scheme confirmed its consistency and plausibility in realistic arterial geometries and allowed to get insight into the relationship between aortic intravascular fluid structures and WSS directionality. The findings of this study clearly show the potential of the projection scheme as quantitative tool for an in depth investigation of the WSS multidirectional nature. The proposed approach enriches the arsenal of tools available to study and exploit the role played by local hemodynamics in vascular disease., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Laminar shear stress (SS) induces an antiproliferative and anti-inflammatory endothelial phenotype and increases Klf2 expression. We altered the diameter of an arteriovenous fistula (AVF) in the mouse model to determine whether increased fistula diameter produces disturbed SS in vivo and if acutely increased disturbed SS results in decreased Klf2 expression. The mouse aortocaval fistula model was performed with 22, 25, or 28 gauge needles to puncture the aorta and the inferior vena cava. Duplex ultrasound was used to examine the AVF and its arterial inflow and venous outflow, and SS was calculated. Arterial samples were examined with western blot, immunohistochemistry, and immunofluorescence analysis for proteins and qPCR for RNA. Mice with larger diameter fistulae had diminished survival but increased AVF patency. Increased SS magnitudes and range of frequencies were directly proportional to the needle diameter in the arterial limb proximal to the fistula but not in the venous limb distal to the fistula, with 22-gauge needles producing the most disturbed SS in vivo. Klf2 mRNA and protein expression was diminished in the artery proximal to the fistula in proportion to increasing SS. Increased fistula diameter produces increased SS magnitude and frequency, consistent with disturbed SS in vivo. Disturbed SS is associated with decreased mRNA and protein expression of Klf2. Disturbed SS and reduced Klf2 expression near the fistula are potential therapeutic targets to improve AVF maturation., (© 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)

Histone deacetylases (HDACs) play a crucial role in transcriptional regulation through modulation of chromatin structure. The class I HDAC, HDAC3, is involved in maintaining endothelial cell integrity. Sustained activation of the x-box binding protein 1 (XBP1), an endoplasmic reticulum stress response transcription factor, results in the development of atherosclerosis in apoE-/- mice. HDAC3 and XBP1 are similarly expressed in the bifurcation regions of the aorta. In this study, we investigated whether cross-talk existed between HDAC3 and XBP1, and its role in the maintenance of endothelial cell integrity. Our study demonstrated that disturbed flow upregulated HDAC3 and unspliced XBP1 (XBP1u) protein production through the KDR/PI3K/Akt pathway. Knockdown of XBP1 by shRNA lentiviral transfection ablated disturbed flow-induced HDAC3 upregulation. Similarly to HDAC3, overexpression of XBP1u by adenoviral gene transfer increased Akt phosphorylation at Serine473 and haem oxygenase 1 gene transcription, which showed a protective role in hydrogen peroxide-induced apoptosis of endothelial cells. Co-immunoprecipitation assays demonstrated that HDAC3 physically associates with XBP1u and this could be enhanced by disturbed flow and VEGF treatment. The use of truncated HDAC3 constructs demonstrated that XBP1 binds to the central section of HDAC3. Further experiments indicated that overexpression of XBP1u increased the binding of HDAC3 to IRE1α, Akt and PI3K, especially after VEGF treatment. In contrast, sustained activation of spliced XBP1 decreased HDAC3 protein production through transcriptional suppression, leading to endothelial apoptosis. These results suggest that XBP1u protects endothelial cells from oxidative stress by interacting with HDAC3. Targeting this interaction may provide novel therapeutic strategies for vascular disease via maintaining endothelial integrity. [ABSTRACT FROM AUTHOR]

Extracellular signal-regulated kinase 5 (ERK5) has been reported to regulate endothelial cell integrity and protect from vascular dysfunction under continuous laminar flow. However, the effect of flow on ERK5 levels has not been determined. Confluent human umbilical vein endothelial cells (HUVECs) were seeded on fibronectin coated glass slides and serum starved for 2 hours with 1% fetal bovine serum (FBS). HUVECs were then exposed to to and fro flow (TFF), pulsatile forward flow (PFF), or continuous laminar flow (CLF) in a parallel plate flow chamber for up to 2 hours. At the end of experiment, cell lysates were prepared and immunoblotted with antibodies to total ERK5. Both CLF and TFF exhibited a decrease in ERK5 after levels after 2-hour exposure. However, the level of ERK5 for PFF remained the same. Disturbed, but not uniform pulsatile, flow decreases ERK5 levels in HUVECs.

The anatomy of the sinuses of Valsalva has not been considered from the viewpoint of a converging nozzle. Converging nozzles reduce turbulence. We reviewed computed tomographic images of the left and right sinuses of Valsalva in 20 consecutive patients. The sinuses of Valsalva were shown to have a shape in the axial projection that approximates a cubic equation nozzle, although the sinuses of Valsalva are not axisymmetric. The ratios of the cross-sectional area of the inlet to cross-sectional areas of the outlet, assuming the sinuses are axisymmetric, were 14 and 17 in the left and right sinuses, respectively. Calculations by others show that turbulent kinetic energy at the exit (at the coronary ostia) of such axisymmetric nozzles would be reduced by 97%. We conclude that the sinuses of Valsalva have the configuration of a converging nozzle and prevent or reduce turbulent flow in the proximal portions of the coronary arteries., (© 2014 Anatomical Society.)

Rationale: Fluid shear stress differentially regulates endothelial cell stress fiber formation with decreased stress fibers in areas of disturbed flow compared with steady flow areas. Importantly, stress fibers are critical for several endothelial cell functions including cell shape, mechano-signal transduction, and endothelial cell-cell junction integrity. A key mediator of steady flow-induced stress fiber formation is Src that regulates downstream signaling mediators such as phosphorylation of cortactin, activity of focal adhesion kinase, and small GTPases., Objective: Previously, we showed that thioredoxin-interacting protein (TXNIP, also VDUP1 [vitamin D upregulated protein 1] and TBP-2 [thioredoxin binding protein 2]) was regulated by fluid shear stress; TXNIP expression was increased in disturbed flow compared with steady flow areas. Although TXNIP was originally characterized for its role in redox and metabolic cellular functions, recent reports show important scaffold functions related to its α-arrestin structure. Based on these findings, we hypothesized that TXNIP acts as a biomechanical sensor that regulates Src kinase activity and stress fiber formation., Methods and Results: Using en face immunohistochemistry of the aorta and cultured endothelial cells, we show inverse relationship between TXNIP expression and Src activity. Specifically, steady flow increased Src activity and stress fiber formation, whereas it decreased TXNIP expression. In contrast, disturbed flow had opposite effects. We studied the role of TXNIP in regulating Src homology phosphatase-2 plasma membrane localization and vascular endothelial cadherin binding because Src homology phosphatase-2 indirectly regulates dephosphorylation of Src tyrosine 527 that inhibits Src activity. Using immunohistochemistry and immunoprecipitation, we found that TXNIP prevented Src homology phosphatase-2-vascular endothelial cadherin interaction., Conclusions: In summary, these data characterize a fluid shear stress-mediated mechanism for stress fiber formation that involves a TXNIP-dependent vascular endothelial cadherin-Src homology phosphatase-2-Src pathway.

Spatial variation of the haemodynamic stresses acting on the arterial wall is commonly assumed to explain the focal development of atherosclerosis. Disturbed flow in particular is thought to play a key role. However, widely-used metrics developed to quantify its extent are unable to distinguish between uniaxial and multidirectional flows. We analysed pulsatile flow fields obtained in idealised and anatomically-realistic arterial geometries using computational fluid dynamics techniques, and in particular investigated the multidirectionality of the flow fields, capturing this aspect of near-wall blood flow with a new metric - the transverse wall shear stress (transWSS) - calculated as the time-average of wall shear stress components perpendicular to the mean flow direction. In the idealised branching geometry, multidirectional flow was observed downstream of the branch ostium, a region of flow stagnation, and to the sides of the ostium. The distribution of the transWSS was different from the pattern of traditional haemodynamic metrics and more dependent on the velocity waveform imposed at the branch outlet. In rabbit aortas, transWSS patterns were again different from patterns of traditional metrics. The near-branch pattern varied between intercostal ostia, as is the case for lesion distribution; for some branches there were striking resemblances to the age-dependent patterns of disease seen in rabbit and human aortas. The new metric may lead to improved understanding of atherogenesis., (© 2013 The Authors. Published by Elsevier Ltd. All rights reserved.)