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Clinical failure of arteriovenous neointimal hyperplasia (NIH) fistulae (AVF) is frequently due to juxta-anastomotic NIH (JANIH). Although the mouse AVF model recapitulates human AVF maturation, previous studies focused on the outflow vein distal to the anastomosis. We hypothesized that the juxta-anastomotic area (JAA) has increased NIH compared with the outflow vein. AVF was created in C57BL/6 mice without or with chronic kidney disease (CKD). Temporal and spatial changes of the JAA were examined using histology and immunofluorescence. Computational techniques were used to model the AVF. RNA-seq and bioinformatic analyses were performed to compare the JAA with the outflow vein. The jugular vein to carotid artery AVF model was created in Wistar rats. The neointima in the JAA shows increased volume compared with the outflow vein. Computational modeling shows an increased volume of disturbed flow at the JAA compared with the outflow vein. Endothelial cells are immediately lost from the wall contralateral to the fistula exit, followed by thrombus formation and JANIH. Gene Ontology (GO) enrichment analysis of the 1,862 differentially expressed genes (DEG) between the JANIH and the outflow vein identified 525 overexpressed genes. The rat jugular vein to carotid artery AVF showed changes similar to the mouse AVF. Disturbed flow through the JAA correlates with rapid endothelial cell loss, thrombus formation, and JANIH; late endothelialization of the JAA channel correlates with late AVF patency. Early thrombus formation in the JAA may influence the later development of JANIH. NEW & NOTEWORTHY: Disturbed flow and focal endothelial cell loss in the juxta-anastomotic area of the mouse AVF colocalizes with acute thrombus formation followed by late neointimal hyperplasia. Differential flow patterns between the juxta-anastomotic area and the outflow vein correlate with differential expression of genes regulating coagulation, proliferation, collagen metabolism, and the immune response. The rat jugular vein to carotid artery AVF model shows changes similar to the mouse AVF model. [ABSTRACT FROM AUTHOR]

Redox processes can modulate vascular pathophysiology. The endoplasmic reticulum redox chaperone protein disulfide isomerase A1 (PDIA1) is overexpressed during vascular proliferative diseases, regulating thrombus formation, endoplasmic reticulum stress adaptation, and structural remodeling. However, both protective and deleterious vascular effects have been reported for PDIA1, depending on the cell type and underlying vascular condition. Further understanding of this question is hampered by the poorly studied mechanisms underlying PDIA1 expression regulation. Here, we showed that PDIA1 mRNA and protein levels were upregulated (average 5-fold) in the intima and media/adventitia following partial carotid ligation (PCL). Our search identified that miR-204-5p and miR-211-5p (miR-204/211), two broadly conserved miRNAs, share PDIA1 as a potential target. MiR-204/211 was downregulated in vascular layers following PCL. In isolated endothelial cells, gain-of-function experiments of miR-204 with miR mimic decreased PDIA1 mRNA while having negligible effects on markers of endothelial activation/stress response. Similar effects were observed in vascular smooth muscle cells (VSMCs). Furthermore, PDIA1 downregulation by miR-204 decreased levels of the VSMC contractile differentiation markers. In addition, PDIA1 overexpression prevented VSMC dedifferentiation by miR-204. Collectively, we report a new mechanism for PDIA1 regulation through miR-204 and identify its relevance in a model of vascular disease playing a role in VSMC differentiation. This mechanism may be regulated in distinct stages of atherosclerosis and provide a potential therapeutic target. [ABSTRACT FROM AUTHOR]

Endothelial cells in steady laminar flow assume a healthy, quiescent phenotype, while endothelial cells in oscillating disturbed flow become dysfunctional. Since endothelial dysfunction leads to atherosclerosis and cardiovascular disease, it is important to understand the mechanisms by which endothelial cells change their function in varied flow environments. Endothelial metabolism has recently been proven a powerful tool to regulate vascular function. Endothelial cells generate most of their energy from glycolysis, and steady laminar flow may reduce endothelial glycolytic flux. We hypothesized that steady laminar but not oscillating disturbed flow would reduce glycolytic flux and alter glycolytic side branch pathways. In this study, we exposed human umbilical vein endothelial cells to static culture, steady laminar flow (20 dynes/cm2 shear stress), or oscillating disturbed flow (4 ± 6 dynes/cm2 shear stress) for 24 h using a cone-and-plate device. We then measured glucose and lactate uptake and secretion, respectively, and glycolytic metabolites. Finally, we explored changes in the expression and protein levels of endothelial glycolytic enzymes. Our data show that endothelial cells in steady laminar flow had decreased glucose uptake and 13C labeling of glycolytic metabolites while cells in oscillating disturbed flow did not. Steady laminar flow did not significantly change glycolytic enzyme gene or protein expression, suggesting that glycolysis may be altered through enzyme activity. Flow also modulated glycolytic side branch pathways involved in proteoglycan and glycosaminoglycan synthesis, as well as oxidative stress. These flow-induced changes in endothelial glucose metabolism may impact the atheroprone endothelial phenotype in oscillating disturbed flow. [ABSTRACT FROM AUTHOR]

Disturbed flow is one of the pathological initiators of endothelial dysfunction in intimal hyperplasia (IH) which is commonly seen in vascular bypass grafts, and arteriovenous fistulas. Various in vitro disease models have been designed to simulate the hemodynamic conditions found in the vasculature. Nonetheless, prior investigations have encountered challenges in establishing a robust disturbed flow model, primarily attributed to the complex bifurcated geometries and distinctive flow dynamics. In the present study, we aim to address this gap by introducing an in vitro bypass flow model capable of inducing disturbed flow and other hemodynamics patterns through a pulsatile flow in the same model. To assess the model's validity, we employed computational fluid dynamics (CFD) to simulate hemodynamics and compared the morphology and functions of human umbilical venous endothelial cells (HUVECs) under disturbed flow conditions to those in physiological flow or stagnant conditions. CFD analysis revealed the generation of disturbed flow within the model, pinpointing the specific location in the channel where the effects of disturbed flow were observed.High-content screening, a single-cell morphological profile assessment, demonstrated that HUVECs in the disturbed flow area exhibited random orientation, and morphological features were significantly distinct compared to cells in the physiological flow or stagnant condition after a two days of flow exposure. Furthermore, HUVECs exposed to disturbed flow underwent extensive remodeling of the adherens junctions and expressed higher levels of endothelial cell activation markers compared to other hemodynamic conditions.In conclusion, our in vitro bypass flow model provides a robust platform for investigating the associations between disturbed flow pattern and vascular diseases.

Several compounds have been used for atherosclerosis treatment, including clinical trials; however, no anti-atherosclerotic drugs based on hemodynamic force-mediated atherogenesis have been discovered. Our previous studies demonstrated that "small mothers against decapentaplegic homolog 1/5" (Smad1/5) is a convergent signaling molecule for chemical [e.g., bone morphogenetic proteins (BMPs)] and mechanical (e.g., disturbed flow) stimulations and hence may serve as a promising hemodynamic-based target for anti-atherosclerosis drug development. The goal of this study was to develop a high-throughput screening (HTS) platform to identify potential compounds that can inhibit disturbed flow-and BMP-induced Smad1/5 activation and atherosclerosis. Through HTS using a Smad1/5 downstream target inhibitor of DNA binding 1 (Id-1) as a luciferase reporter, we demonstrated that KU-55933 and Apicidin suppressed Id-1 expression in AD-293 cells. KU-55933 (10 µM), Apicidin (10 µM), and the combination of half doses of each [1/2(K + A)] inhibited disturbed flow-and BMP4-induced Smad1/5 activation in human vascular endothelial cells (ECs). KU-55933, Apicidin, and 1/2(K + A) treatments caused 50.6%, 47.4%, and 73.3% inhibitions of EC proliferation induced by disturbed flow, respectively, whereas EC inflammation was only suppressed by KU-55933 and 1/2(K + A), but not Apicidin alone. Administrations of KU-55933 and 1/2(K + A) to apolipoprotein E-deficient mice inhibited Smad1/5 activation in ECs in athero-susceptible regions, thereby suppressing endothelial proliferation and inflammation, with the attenuation of atherosclerotic lesions in these mice. A unique drug screening platform has been developed to demonstrate that KU-55933 and its combination with Apicidin are promising therapeutic compounds for atherosclerosis based on hemodynamic considerations. [ABSTRACT FROM AUTHOR]

Endothelial cells (ECs) experience two different blood flow patterns: laminar and disturbed flow. Their responses to laminar flow contribute to vascular homeostasis, whereas their responses to disturbed flow result in EC dysfunction and vascular diseases. However, it remains unclear how ECs differentially sense laminar and disturbed flow and trigger signaling that elicits different responses. Here, we showed that ECs differentially sense laminar and disturbed flows by altering the lipid order of their plasma and mitochondrial membranes in opposite directions. This results in distinct changes in mitochondrial function, namely, increased adenosine triphosphate (ATP) production for laminar flow and increased hydrogen peroxide (H2O2) release for disturbed flow, leading to ATP- and H2O2-mediated signaling, respectively. When cultured human aortic ECs were subjected to laminar or disturbed flow in flow-loading devices, the lipid order of their plasma membranes immediately decreased in response to laminar flow and increased in response to disturbed flow. Laminar flow also decreased the lipid order of mitochondrial membranes and increased mitochondrial ATP production. In contrast, disturbed flow increased the lipid order of mitochondrial membranes and increased the release of H2O2 from the mitochondria. The addition of cholesterol to the cells increased the lipid order of both membranes and abrogated laminar flow-induced ATP production, while treatment of the cells with a cholesterol-depleting reagent, methyl-β cyclodextrin, decreased the lipid order of both membranes and abolished disturbed flow-induced H2O2 release, indicating that changes in the membrane lipid order and/or cholesterol content are closely linked to flow-induced changes in mitochondrial functions. [ABSTRACT FROM AUTHOR]

Disturbed flow promotes progression of atherosclerosis at particular regions of arteries where the recent studies show the arterial wall becomes stiffer. Objective of this study is to show how mechanotransduction in subcellular organelles of endothelial cells (ECs) will alter with changes in blood flow profiles applied on ECs surface and mechanical properties of arterial wall where ECs are attached to. We will examine the exposure of ECs to atherogenic flow profiles (disturbed flow) and non-atherogenic flow profiles (purely forward flow), while stiffness and viscoelasticity of arterial wall will change. A multicomponent model of endothelial cell monolayer was applied to quantify the response of subcellular organelles to the changes in their microenvironment. Our results show that arterial stiffening alters mechanotransduction in intra/inter-cellular organelles of ECs by slight increase in the transmitted stresses, particularly over central stress fibers (SFs). We also observed that degradation of glycocalyx and exposure to non-atherogenic flow profiles result in significantly higher stresses in subcellular organelles, while degradation of glycocalyx and exposure to atherogenic flow profiles result in dramatically lower stresses in the organelles. Moreover, we show that increasing the arterial wall viscoelasticity leads to slight increase in the stresses transmitted to subcellular organelles. FAs are particularly influenced with the changes in the arterial wall properties and viscoelasticity. Our study suggests that changes in viscoelasticity of arterial wall and degradation state of glycocalyx have to be considered along with arterial stiffening in designing more efficient treatment strategies for atherosclerosis. Our study provides insight into significant role of mechanotransduction in the localization of atherosclerosis by quantifying the role of ECs mechanosensors and suggests that mechanotransduction may play a key role in design of more efficient and precision therapeutics to slow down or block the progression of atherosclerosis. [ABSTRACT FROM AUTHOR]

Redox processes can modulate vascular pathophysiology. The endoplasmic reticulum redox chaperone protein disulfide isomerase A1 (PDIA1) is overexpressed during vascular proliferative diseases, regulating thrombus formation, endoplasmic reticulum stress adaptation, and structural remodeling. However, both protective and deleterious vascular effects have been reported for PDIA1, depending on the cell type and underlying vascular condition. Further understanding of this question is hampered by the poorly studied mechanisms underlying PDIA1 expression regulation. Here, we showed that PDIA1 mRNA and protein levels were upregulated (average 5-fold) in the intima and media/adventitia following partial carotid ligation (PCL). Our search identified that miR-204-5p and miR-211-5p (miR-204/211), two broadly conserved miRNAs, share PDIA1 as a potential target. MiR-204/211 was downregulated in vascular layers following PCL. In isolated endothelial cells, gain-of-function experiments of miR-204 with miR mimic decreased PDIA1 mRNA while having negligible effects on markers of endothelial activation/stress response. Similar effects were observed in vascular smooth muscle cells (VSMCs). Furthermore, PDIA1 downregulation by miR-204 decreased levels of the VSMC contractile differentiation markers. In addition, PDIA1 overexpression prevented VSMC dedifferentiation by miR-204. Collectively, we report a new mechanism for PDIA1 regulation through miR-204 and identify its relevance in a model of vascular disease playing a role in VSMC differentiation. This mechanism may be regulated in distinct stages of atherosclerosis and provide a potential therapeutic target.

Atherosclerosis is a chronic inflammatory disease, occurring preferentially in bifurcation, branching, and bending of blood vessels exposed to disturbed flow. Disturbed flow in atheroprone areas activates elevated proteases, degrading elastin lamellae and collagenous matrix, resulting in endothelial dysfunction and vascular remodeling. As a mediator for extracellular matrix protein degradation, cathepsin K (CTSK) was directly regulated by hemodynamics and contributed to atherosclerosis. The mechanism of CTSK responding to disturbed flow and contributing to disturbed flow-induced atherosclerosis is unclear. In this study, the partial carotid ligation model of mice and in vitro disturbed shear stress model were constructed to explore the contribution and potential mechanism of CTSK in atherosclerosis. Our results indicated that CTSK elevated in the disturbed flow area in vivo and in vitro along with endothelial inflammation and atherogenesis. Additionally, the expression of integrin αvβ3 was upregulated in these atheroprone areas. We found that inhibition of the integrin αvβ3-cytoskeleton pathway could significantly block the activation of NF-κB and the expression of CTSK. Collectively, our findings unraveled that disturbed flow induces increased CTSK expression, and contributes to endothelial inflammation and vascular remodeling, leading to atherogenesis eventually. This study is helpful to provide new enlightenment for the therapy of atherosclerosis.

IntroductionAtherosclerosis is a progressive disease that develops in areas of disturbed flow (d-flow). Progressive atherosclerosis is characterized by bulky plaques rich in mesenchymal cells and high-grade inflammation that can rupture leading to sudden cardiac death or acute myocardial infarction. In response to d-flow, endothelial cells acquire a mesenchymal phenotype through endothelial-to-mesenchymal transition (EndMT). However, the signaling intermediaries that link d-flow to EndMT are incompletely understood.Methods and ResultsIn this study we found that in human atherosclerosis, cells expressing SNAI1 (Snail 1, EndMT transcription factor) were highly expressed within the endothelial cell (EC) layer and in the pre-necrotic areas in unstable lesions, whereas stable lesions did not show any SNAI1 positive cells, suggesting a role for EndMT in lesion instability. The interleukin-1 (IL-1), which signals through the type-I IL-1 receptor (IL-1R1), has been implicated in plaque instability and linked to EndMT formation in vitro. Interestingly, we observed an association between SNAI1 and IL-1R1 within ECs in the unstable lesions. To establish the causal relationship between EndMT and IL-1R1 expression, we next examined IL-1R1 levels in our Cre-lox endothelial-specific lineage tracing mice. IL-1R1 and Snail1 were highly expressed in ECs under atheroprone compared to athero-protective areas, and oscillatory shear stress (OSS) increased IL-1R1 protein and mRNA levels in vitro. Exposure of ECs to OSS resulted in loss of their EC markers and higher induction of EndMT markers. By contrast, genetic silencing of IL-1R1 significantly reduced the expression of EndMT markers and Snail1 nuclear translocation, suggesting a direct role for IL-1R1 in d-flow-induced EndMT. In vivo, re-analysis of scRNA-seq datasets in carotid artery exposed to d-flow confirmed the IL-1R1 upregulation among EndMT population, and in our partial carotid ligation model of d-flow, endothelial cell specific IL-1R1 KO significantly reduced SNAI1 expression.DiscussionGlobal inhibition of IL-1 signaling in atherosclerosis as a therapeutic target has recently been tested in the completed CANTOS trial, with promising results. However, the data on IL-1R1 signaling in different vascular cell-types are inconsistent. Herein, we show endothelial IL-1R1 as a novel mechanosensitive receptor that couples d-flow to IL-1 signaling in EndMT.

Atherosclerosis preferentially develops at bifurcations exposed to disturbed flow. Plexin D1 (PLXND1) responds to mechanical forces and drives macrophage accumulation in atherosclerosis. Here, multiple strategies were used to identify the role of PLXND1 in site-specific atherosclerosis. Using computational fluid dynamics and three-dimensional light-sheet fluorescence-microscopy, the elevated PLXND1 in M1 macrophages was mainly distributed in disturbed flow area of ApoE−/− carotid bifurcation lesions, and visualization of atherosclerosis in vivo was achieved by targeting PLXND1. Subsequently, to simulate the microenvironment of bifurcation lesions in vitro, we co-cultured oxidized low-density lipoprotein (oxLDL)-treated THP-1-derived macrophages with shear-treated human umbilical vein endothelial cells (HUVECs). We found that oscillatory shear induced the increase of PLXND1 in M1 macrophages, and knocking down PLXND1 inhibited M1 polarization. Semaphorin 3E, the ligand of PLXND1 which was highly expressed in plaques, strongly enhanced M1 macrophage polarization via PLXND1 in vitro. Our findings provide insights into pathogenesis in site-specific atherosclerosis that PLXND1 mediates disturbed flow-induced M1 macrophage polarization.

Cancer survivors undergone treatment face an increased risk of developing atherosclerotic cardiovascular disease (CVD), yet the underlying mechanisms remain elusive. Recent studies have revealed that chemotherapy can drive senescent cancer cells to acquire a proliferative phenotype known as senescence-associated stemness (SAS). These SAS cells exhibit enhanced growth and resistance to cancer treatment, thereby contributing to disease progression. Endothelial cell (EC) senescence has been implicated in atherosclerosis and cancer, including among cancer survivors. Treatment modalities for cancer can induce EC senescence, leading to the development of SAS phenotype and subsequent atherosclerosis in cancer survivors. Consequently, targeting senescent ECs displaying the SAS phenotype hold promise as a therapeutic approach for managing atherosclerotic CVD in this population. This review aims to provide a mechanistic understanding of SAS induction in ECs and its contribution to atherosclerosis among cancer survivors. We delve into the mechanisms underlying EC senescence in response to disturbed flow and ionizing radiation, which play pivotal role in atherosclerosis and cancer. Key pathways, including p90RSK/TERF2IP, TGFβR1/SMAD, and BH4 signaling are explored as potential targets for cancer treatment. By comprehending the similarities and distinctions between different types of senescence and the associated pathways, we can pave the way for targeted interventions aim at enhancing the cardiovascular health of this vulnerable population. The insights gained from this review may facilitate the development of novel therapeutic strategies for managing atherosclerotic CVD in cancer survivors.

Background and aims Atherosclerosis preferentially develops in arterial branches and curvatures where vascular endothelium is exposed to disturbed flow. In this study, the effects of disturbed flow on the regulation of vascular endothelial phosphoproteins and their contribution to therapeutic application in atherogenesis were elucidated. Methods Porcine models, large-scale phosphoproteomics, transgenic mice, and clinical specimens were used to discover novel site-specific phosphorylation alterations induced by disturbed flow in endothelial cells (ECs). Results A large-scale phosphoproteomics analysis of native endothelium from disturbed (athero-susceptible) vs. pulsatile flow (athero-resistant) regions of porcine aortas led to the identification of a novel atherosclerosis-related phosphoprotein vinculin (VCL) with disturbed flow-induced phosphorylation at serine 721 (VCLS721p). The induction of VCLS721p was mediated by G-protein-coupled receptor kinase 2 (GRK2)S29p and resulted in an inactive form of VCL with a closed conformation, leading to the VE-cadherin/catenin complex disruption to enhance endothelial permeability and atherogenesis. The generation of novel apolipoprotein E-deficient (ApoE−/−) mice overexpressing S721-non-phosphorylatable VCL mutant in ECs confirmed the critical role of VCLS721p in promoting atherosclerosis. The administration of a GRK2 inhibitor to ApoE−/− mice suppressed plaque formation by inhibiting endothelial VCLS721p. Studies on clinical specimens from patients with coronary artery disease (CAD) revealed that endothelial VCLS721p is a critical clinicopathological biomarker for atherosclerosis progression and that serum VCLS721p level is a promising biomarker for CAD diagnosis. Conclusions The findings of this study indicate that endothelial VCLS721p is a valuable hemodynamic-based target for clinical assessment and treatment of vascular disorders resulting from atherosclerosis. [ABSTRACT FROM AUTHOR]

Although the hemodynamics of cerebral aneurysms have been extensively studied using patient-specific computational fluid dynamics techniques, no specific hemodynamic factors characteristic of cerebral aneurysm development have yet been identified. We believe that one problem with previous hemodynamic studies of cerebral aneurysms has been the manner in which control groups were created for comparison with experimental groups. The purpose of this study was to determine hemodynamic factors that correlated with the development of cerebral aneurysms. The control group was established in a manner that differed from those of previous works. This allowed us to demonstrate the effectiveness of our method. We artificially removed aneurysms in the middle cerebral artery bifurcations of nine patients and reconstructed the vessel geometries before the aneurysms had occurred. Pulsatile blood flow simulations were performed using the vessel geometries ipsilateral and contralateral to the sites of aneurysm removal, and hemodynamic metrics were calculated. Use of the ipsilateral and contralateral sides as the experimental and control sites, respectively, allowed us to evaluate statistically the hemodynamic metrics between the two corresponding sites/groups. The results showed that only the normalized transverse wall shear stress (NtransWSS) was significantly higher at the MCA bifurcation ipsilateral to the site of aneurysm removal than at the contralateral bifurcation (p = 0.01). There were no significant differences in the other hemodynamic metrics between the bilateral bifurcations. Our findings imply that multi-directional disturbed wall shear stress, which is detected by the NtransWSS metric, may be one hemodynamic risk factor for the development of cerebral aneurysms. [ABSTRACT FROM AUTHOR]

Endothelial cells are highly sensitive to hemodynamic shear stresses, which act in the blood flow’s direction on the blood vessel’s luminal surface. Thus, endothelial cells on that surface are exposed to various physiological and pathological stimuli, such as disturbed flow-induced shear stress, which may exert effects on adaptive vascular diameter or structural wall remodeling. Here we showed that plasma thioredoxin-interactive protein (TXNIP) and malondialdehyde levels were significantly increased in patients with slow coronary flow. In addition, human endothelial cells exposed to disturbed flow exhibited increased levels of TXNIP in vitro. On the other hand, deletion of human endothelial TXNIP increased capillary formation, nitric oxide production and mitochondrial function, as well as lessened oxidative stress response and endothelial cell inflammation. Additional beneficial impacts from TXNIP deletion were also seen in a glucose utilization study, as reflected by augmented glucose uptake, lactate secretion and extracellular acidification rate. Taken together, our results suggested that TXNIP is a key component involved in mediating shear stress-induced inflammation, energy homeostasis, and glucose utilization, and that TXNIP may serve as a potentially novel endothelial dysfunction regulator.

Abstracts PURPOSE Accumulation of senescent endothelial cells (ECs) and development of vascular aging have been implicated in the etiology of vascular dysfunction and disease. Aerobic exercise has been recognized as the single most effective non-pharmacological anti-aging intervention via increased laminar shear stress (LSS). This study aimed to determine the protective effects of LSS against premature senescence and the underlying mechanism. METHODS Carotid artery partial ligation surgery was performed on the left carotid arteries (LCAs) of C57BL/6J male mice to determine the effect of disturbed flow on the development of endothelial senescence. Senescence-associated β-galactosidase (SA-β-gal) staining was performed to measure cellular senescence. Expression levels of protein markers for cell senescence including p21, p16, and p53 were measured by western blotting. RESULTS En face SA-β-gal staining was identified only in the partially ligated LCAs of voluntary wheel-running mice, suggesting a direct relevance of LSS on the prevention of vascular senescence. In the in vitro H2 O2-induced premature senescence model, preconditioning of high-flow LSS (20 dyne/cm2, 36 hours) induced significant reduction in the percentage of SA-β-gal positive ECs. Expression of the molecular markers of cellular senescence such as p21, p16, and p53 was significantly decreased by LSS pretreatment. However, the protective effects of LSS against premature senescence were completely abolished by SIRT1 inhibition. CONCLUSIONS The results suggest that high-flow LSS has protective effects against oxidative stress-induced premature endothelial senescence through a SIRT1-dependent mechanism.

Navigation of nanoparticles to target sites of blood flow disturbance markedly upgrades the diagnostic paradigm in vascular medicine. The theranostic treatment of pre‐stenotic vessels can prevent the irreversible occlusion process effectively. Here, these nanotheranostic functions are established by displaying CDK9(cyclin‐dependent kinase 9)‐targeting peptide (P.) onto nanovesicles (NV) and liposomes using the navigation function and subsequent binding‐on signaling of P. as a game‐changer. When rabbit vessels are allografted with injecting contrast‐loaded P. liposomes, the case‐dependent stenotic degree after 2–6 weeks can be diagnosed accurately within 2–4 days via computed tomography imaging with cross‐validation in a mouse model of partial carotid ligation. Furthermore, the anti‐CDK9 signaling of P. NV is activated post‐targeting and effectively prevents vascular stenosis by suppressing inflammation and lipotoxicity in the vessels, serum, and/or liver. CDK9 targeting is confirmed using computer, in vitro, and in vivo models. This study demonstrates an unprecedented nanotheranostic function for future clinical applications. [ABSTRACT FROM AUTHOR]

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.

Aneurysms are malformations within the arterial vasculature brought on by the structural breakdown of the microarchitecture of the vessel wall, with aneurysms posing serious health risks in the event of their rupture. Blood flow within vessels is generally laminar with high, unidirectional wall shear stressors that modulate vascular endothelial cell functionality and regulate vascular smooth muscle cells. However, altered vascular geometry induced by bifurcations, significant curvature, stenosis, or clinical interventions can alter the flow, generating low stressor disturbed flow patterns. Disturbed flow is associated with altered cellular morphology, upregulated expression of proteins modulating inflammation, decreased regulation of vascular permeability, degraded extracellular matrix, and heightened cellular apoptosis. The understanding of the effects disturbed flow has on the cellular cascades which initiate aneurysms and promote their subsequent growth can further elucidate the nature of this complex pathology. This review summarizes the current knowledge about the disturbed flow and its relation to aneurysm pathology, the methods used to investigate these relations, as well as how such knowledge has impacted clinical treatment methodologies. This information can contribute to the understanding of the development, growth, and rupture of aneurysms and help develop novel research and aneurysmal treatment techniques. [ABSTRACT FROM AUTHOR]

Atherosclerosis, the chronic accumulation of cholesterol-rich plaque within arteries, is associated with a broad spectrum of cardiovascular diseases including myocardial infarction, aortic aneurysm, peripheral vascular disease, and stroke. Atherosclerotic cardiovascular disease remains a leading cause of mortality in high-income countries and recent years have witnessed a notable increase in prevalence within low- and middle-income regions of the world. Considering this prominent and evolving global burden, there is a need to identify the cellular mechanisms that underlie the pathogenesis of atherosclerosis to discover novel therapeutic targets for preventing or mitigating its clinical sequelae. Despite decades of research, we still do not fully understand the complex cell-cell interactions that drive atherosclerosis, but new investigative approaches are rapidly shedding light on these essential mechanisms. The vascular endothelium resides at the interface of systemic circulation and the underlying vessel wall and plays an essential role in governing pathophysiological processes during atherogenesis. In this review, we present emerging evidence that implicates the activated endothelium as a driver of atherosclerosis by directing site-specificity of plaque formation and by promoting plaque development through intracellular processes, which regulate endothelial cell proliferation and turnover, metabolism, permeability, and plasticity. Moreover, we highlight novel mechanisms of intercellular communication by which endothelial cells modulate the activity of key vascular cell populations involved in atherogenesis, and discuss how endothelial cells contribute to resolution biology – a process that is dysregulated in advanced plaques. Finally, we describe important future directions for preclinical atherosclerosis research, including epigenetic and targeted therapies, to limit the progression of atherosclerosis in at-risk or affected patients. [ABSTRACT FROM AUTHOR]

Focal adhesion kinase (FAK) activation plays a crucial role in vascular diseases. In endothelial cells, FAK activation is involved in the activation of pro-inflammatory signaling and the progression of atherosclerosis. Disturbed flow (D-flow) induces endothelial activation and senescence, but the exact role of FAK in D-flow-induced endothelial activation and senescence remains unclear. The objective of this study is to investigate the role of FAK SUMOylation in D-flow-induced endothelial activation and senescence. The results showed that D-flow induced reactive oxygen species (ROS) production via NADPH oxidase activation and activated a redox-sensitive kinase p90RSK, leading to FAK activation by upregulating FAK K152 SUMOylation and the subsequent Vav2 phosphorylation, which in turn formed a positive feedback loop by upregulating ROS production. This feedback loop played a crucial role in regulating endothelial activation and senescence. D-flow-induced endothelial activation and senescence were significantly inhibited by mutating a FAK SUMOylation site lysine152 to arginine. Collectively, we concluded that FAK K152 SUMOylation plays a key role in D-flow-induced endothelial activation and senescence by forming a positive feedback loop through ROS production. • Disturbed flow increased FAK K152 SUMOylation regulating p90RSK and SENP2 function. • FAK K152 SUMOylation upregulates FAK kinase activity and increases Vav2 tyrosine phosphorylation and subsequently upregulates NOX2 activation. • Vav2 -induced ROS production via NOX2 activation forms a positive feedback loop by activating redox-sensitive kinase of p90RSK, and consequently induces endothelial activation. • This positive feedback loop consisting with p90RSK, SENP2, FAK, and Vav2-NOX2 maintains ROS production and promotes endothelial inflammation and senescence. [ABSTRACT FROM AUTHOR]

Objective: Central venous stenosis (CVS) is a major cause of arteriovenous fistula (AVF) failure. However, central veins are relatively inaccessible to study with conventional Doppler ultrasound methods. To understand mechanisms underlying AVF failure owing to CVS, an animal model was established that creates a stenosis distal to an AVF. We hypothesized that this mouse model will show comparable morphology and physiology to human CVS. Methods: An aortocaval fistula was created between the distal aorta and inferior vena cava (IVC); a stenosis was then created distal to the fistula by partial IVC ligation. Sham-operated animals, AVF without venous stenosis, and venous stenosis without AVF were used as controls. Physiologic properties of the IVC, both upstream and downstream of the stenosis, or the corresponding sites in models without stenosis, were assessed with ultrasound examination on days 0 to 21. The spectral broadening index was measured to assess the degree of disturbed shear stress. The IVC was harvested at day 21 and specimens were analyzed with immunofluorescence. Results: The IVC diameter of mice with an AVF and stenosis showed increased upstream (P = .013), but decreased downstream diameter (P = .001) compared with mice with an AVF but without a stenosis, at all postoperative times (days 3-21). IVC wall thickness increased in mice with an AVF, compared with IVC without an AVF (upstream of stenosis: 13.9 μm vs 11.0 μm vs 4.5 μm vs 3.9 μm; P = .020; downstream of stenosis: 6.0 μm vs 6.6 μm vs μm 4.5 μm vs 3.8 μm; P = .002; AVF with stenosis, AVF, stenosis, sham, respectively). AVF patency significantly decreased in mice with an AVF and stenosis by day 21 (50% vs 90%; P = .048). The IVC of mice with AVF and stenosis showed a venous waveform with pulsatility as well as enhanced velocity at and downstream of the stenosis; similar waveforms were observed in a human case of CVS. Downstream to the stenosis, the spectral broadening index was significantly higher compared with mice with AVF alone (1.06 vs 0.78; P = .011; day 21), and there was a trend towards less immunoreactivity of both Krüppel-like factor 2 and phosphorylated-endothelial nitric oxide synthase compared with mice with an AVF alone. Conclusions: Partial IVC ligation distal to a mouse aortocaval fistula alters the fistula diameter and wall thickness, decreases patency, and increases distal disturbed flow compared with fistulae without a distal stenosis. Our mouse model of stenosis distal to an AVF may be a faithful representation of human CVS that shows similar morphology and physiology, including disturbed shear stress. : Clinical Relevance: A mouse model of venous stenosis distal to an arteriovenous fistula shows similar Doppler waveforms as those observed in a human case of central venous stenosis. These mice retain disturbed shear stress in the vein distal to the fistula, characterized by a sustained increase of the spectral broadening index and diminished expression of proteins upregulated by laminar shear stress. This novel mouse model will enable investigation of the physiology and downstream molecular pathways involved in central venous stenosis in humans.

Atherosclerosis, the chronic accumulation of cholesterol-rich plaque within arteries, is associated with a broad spectrum of cardiovascular diseases including myocardial infarction, aortic aneurysm, peripheral vascular disease, and stroke. Atherosclerotic cardiovascular disease remains a leading cause of mortality in high-income countries and recent years have witnessed a notable increase in prevalence within low- and middle-income regions of the world. Considering this prominent and evolving global burden, there is a need to identify the cellular mechanisms that underlie the pathogenesis of atherosclerosis to discover novel therapeutic targets for preventing or mitigating its clinical sequelae. Despite decades of research, we still do not fully understand the complex cell-cell interactions that drive atherosclerosis, but new investigative approaches are rapidly shedding light on these essential mechanisms. The vascular endothelium resides at the interface of systemic circulation and the underlying vessel wall and plays an essential role in governing pathophysiological processes during atherogenesis. In this review, we present emerging evidence that implicates the activated endothelium as a driver of atherosclerosis by directing site-specificity of plaque formation and by promoting plaque development through intracellular processes, which regulate endothelial cell proliferation and turnover, metabolism, permeability, and plasticity. Moreover, we highlight novel mechanisms of intercellular communication by which endothelial cells modulate the activity of key vascular cell populations involved in atherogenesis, and discuss how endothelial cells contribute to resolution biology – a process that is dysregulated in advanced plaques. Finally, we describe important future directions for preclinical atherosclerosis research, including epigenetic and targeted therapies, to limit the progression of atherosclerosis in at-risk or affected patients.

Background: Anticoagulation and antiplatelet therapy effectively inhibit neointimal hyperplasia (NIH) in both arterial and venous systems but not in arteriovenous fistulae (AVF). The main site of AVF failure is the juxta-anastomotic area that is characterized by disturbed flow compared with laminar flow in the arterial inflow and the venous outflow., Objectives: We hypothesized that early thrombus formation is required for eccentric and heterogeneous NIH in the presence of disturbed flow., Methods: Needle puncture and sutured AVF were created in C57BL/6 mice, in PF4-Cre × mT/mG reporter mice, and in Wistar rats. Human AVF samples were second-stage basilic vein transpositions. The tissues were examined by histology, immunofluorescence, immunohistochemistry, and en face staining., Results: In the presence of disturbed flow, both mouse and human AVF showed eccentric and heterogeneous NIH. Maladapted vein wall was characterized by eccentric and heterogeneous neointima that was composed of a different abundance of thrombus and smooth muscle cells. PF4-cre × mT/mG reporter mice AVF showed that green fluorescent protein-labeled platelets deposit on the wall directly facing the fistula exit with endothelial cell loss and continue to accumulate in the presence of disturbed flow. Neither disturbed flow with limited endothelial cell loss nor nondisturbed flow induced heterogeneous neointima in different animal models., Conclusion: Early thrombus contributes to late heterogeneous NIH in the presence of disturbed flow. Disturbed flow, large area of endothelial cell loss, and thrombus formation are critical to form eccentric and heterogeneous NIH. Categorization of adapted or maladapted walls may be helpful for therapy targeting heterogeneous NIH., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Published by Elsevier Inc.)

Disturbed flow is one of the pathological initiators of endothelial dysfunction in intimal hyperplasia (IH) which is commonly seen in vascular bypass grafts, and arteriovenous fistulas. Various in vitro disease models have been designed to simulate the hemodynamic conditions found in the vasculature. Nonetheless, prior investigations have encountered challenges in establishing a robust disturbed flow model, primarily attributed to the complex bifurcated geometries and distinctive flow dynamics. In the present study, we aim to address this gap by introducing an in vitro bypass flow model capable of inducing disturbed flow and other hemodynamics patterns through a pulsatile flow in the same model. To assess the model's validity, we employed computational fluid dynamics (CFD) to simulate hemodynamics and compared the morphology and functions of human umbilical venous endothelial cells (HUVECs) under disturbed flow conditions to those in physiological flow or stagnant conditions. CFD analysis revealed the generation of disturbed flow within the model, pinpointing the specific location in the channel where the effects of disturbed flow were observed. High-content screening, a single-cell morphological profile assessment, demonstrated that HUVECs in the disturbed flow area exhibited random orientation, and morphological features were significantly distinct compared to cells in the physiological flow or stagnant condition after a two days of flow exposure. Furthermore, HUVECs exposed to disturbed flow underwent extensive remodeling of the adherens junctions and expressed higher levels of endothelial cell activation markers compared to other hemodynamic conditions. In conclusion, our in vitro bypass flow model provides a robust platform for investigating the associations between disturbed flow pattern and vascular diseases., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Disturbed fluid flow is well understood to have significant ramifications on endothelial function, but the impact disturbed flow has on endothelial biomechanics is not well understood. In this study, we measured tractions, intercellular stresses, and cell velocity of endothelial cells exposed to disturbed flow using a custom-fabricated flow chamber. Our flow chamber exposed cells to disturbed fluid flow within the following spatial zones: zone 1 (inlet; length 0.676–2.027 cm): 0.0037 ± 0.0001 Pa; zone 2 (middle; length 2.027–3.716 cm): 0.0059 ± 0.0005 Pa; and zone 3 (outlet; length 3.716–5.405 cm): 0.0051 ± 0.0025 Pa. Tractions and intercellular stresses were observed to be highest in the middle of the chamber (zone 2) and lowest at the chamber outlet (zone 3), while cell velocity was highest near the chamber inlet (zone 1), and lowest near the middle of the chamber (zone 2). Our findings suggest endothelial biomechanical response to disturbed fluid flow to be dependent on not only shear stress magnitude, but the spatial shear stress gradient as well. We believe our results will be useful to a host of fields including endothelial cell biology, the cardiovascular field, and cellular biomechanics in general. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRs) and bone morphogenetic protein receptor–specific Smads are mechano-responsive molecules that play vital roles in modulating endothelial cell (EC) functions in response to blood flow. However, the roles of interplay between these molecules in modulating EC functions under flows remain unclear. We elucidated the regulatory roles of the interplay between miR-487a and Smad5 in EC proliferation in response to different flow patterns. Microarray and quantitative RT-PCR showed that disturbed flow with low and oscillatory shear stress (OS, 0.5 ± 4 dynes/cm2) upregulates EC miR-487a in comparison to static controls and pulsatile shear stress (12 ± 4 dynes/cm2). MiR-487a expression was higher in ECs in the inner curvature (OS region) than the outer curvature of the rat aortic arch and thoracic aorta and also elevated in diseased human coronary arteries. MiR-487a expression was promoted by nuclear phospho-Smad5, which bound to primary-miR-487a to facilitate miR-487a processing. Algorithm prediction and luciferase reporter and argonaute 2-immunoprecipitation assays demonstrated that miR-487a binds to 3′UTR of CREB binding protein (CBP) and p53. Knockdown and overexpression of miR-487a decreased and increased, respectively, phospho-Rb and cyclin A expressions through CBP and p53. A BrdU incorporation assay showed that miR-487a enhanced EC proliferation under OS in vitro and in disturbed flow regions of experimentally stenosed rat abdominal aorta in vivo. These results demonstrate that disturbed flow with OS induces EC expression of miR-487a through its enhanced processing by activated-Smad5. MiR-487 inhibits its direct targets CBP and p53 to induce EC cycle progression and proliferation. Our findings suggest that EC miR-487 may serve as an important molecular target for intervention against disturbed flow–associated vascular disorders resulting from atherosclerosis.

Pulsatile non-Newtonian fluid flow was simulated in an artery bifurcation model, with three different inlet flow waveform shapes and an identical inlet mean velocity steady-state condition, to quantify the impact of three different waveform shapes on hemodynamics such as flow patterns, wall shear stress, and oscillatory shear index during a cardiac cycle. It was found that the degree of flow separation is insensitive to changes in flow waveform shape. There is remarkable similarity in the position and magnitude of the maximum wall shear stress at peak systole and the maximum time-averaged wall shear stress for all examined cases. The oscillatory shear index distributions are broadly similar except that the local maximum oscillatory shear index increases proportionally with the pulsatility index of the waveform shape. The maximum oscillatory shear index values on the planar branch are within 8.7% in all examined cases, while these oscillatory shear index values on the nonplanar branch are identical due to the effects of its curvature. The negligible hemodynamic differences between simplified and characteristic waveform cases suggest that changes in waveform shape play a minimal role in the progression and development of atherosclerosis. Although the computed hemodynamics is almost consistent for the three different waveform shapes, several slight differences were observed. [ABSTRACT FROM AUTHOR]

Background: MER proto-oncogene tyrosine kinase (MerTK) is a key receptor for efferocytosis, a process for the clearance of apoptotic cells. MerTK is mainly expressed in macrophages and immature dendritic cells. There are very limited reports focused on MerTK biology in aortic endothelial cells (ECs). It remains unclear for the role of blood flow patterns in regulating MerTK-mediated efferocytosis in aortic ECs. This study was designed to investigate whether endothelial MerTK and EC efferocytosis respond to blood flow patterns during atherosclerosis. Methods: Big data analytics, RNA-seq and proteomics combined with our in vitro and in vivo studies were applied to reveal the potential molecular mechanisms. Partial carotid artery ligation combined with AAV-PCSK9 and high fat diet were used to set up acute atherosclerosis in 4 weeks. Results: Our data showed that MerTK is sensitive to blood flow patterns and is inhibited by disturbed flow and oscillatory shear stress in primary human aortic ECs (HAECs). The RNA-seq data in HAECs incubated with apoptotic cells showed that d-flow promotes pro-inflammatory pathway and senescence pathway. Our in vivo data of proteomics and immunostaining showed that, compared with WT group, MerTK -/- aggravates atherosclerosis in d-flow areas through upregulation of endothelial dysfunction markers (e.g. IL-1β, NF-κB, TLR4, MAPK signaling, vWF, VCAM-1 and p22 phox ) and mitochondrial dysfunction. Interestingly, MerTK -/- induces obvious abnormal endothelial thickening accompanied with decreased endothelial efferocytosis, promoting the development of atherosclerosis. Conclusions: Our data suggests that blood flow patterns play an important role in regulating MerTK-mediated efferocytosis in aortic ECs, revealing a new promising therapeutic strategy with EC efferocytosis restoration to against atherosclerosis., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Disturbed blood flow has been recognized to promote platelet aggregation and thrombosis via increasing accumulation of von Willebrand factor (VWF) at the arterial post-stenotic sites. The mechanism underlying the disturbed-flow regulated endothelial VWF production remains elusive. Here we described a mouse model, in which the left external carotid artery (LECA) is ligated to generate disturbed flow in the common carotid artery. Ligation of LECA increased VWF accumulation in the plasma. Carotid arterial thrombosis was induced by ferric chloride (FeCl3) application and the time to occlusion in the ligated vessels was reduced in comparison with the unligated vessels. In vitro, endothelial cells were subjected to oscillatory shear (OS, 0.5 ± 4 dynes/cm2) or pulsatile shear (PS, 12 ± 4 dynes/cm2). OS promoted VWF secretion as well as the cell conditioned media-induced platelet aggregation by regulating the intracellular localization of vesicle-associated membrane protein 3 (VAMP3) and synaptosomal-associated protein 23 (SNAP23). Disruption of vimentin intermediate filaments abolished the OS-induced translocation of SNAP23 to the cell membrane. Knockdown of VAMP3 and SNAP23 reduced the endothelial secretion of VWF. Systemic inhibition of VAMP3 and SNAP23 by treatment of mice with rapamycin significantly ameliorated the FeCl3-induced thrombogenesis, whereas intraluminal overexpression of VAMP3 and SNAP23 aggravated it. Our findings demonstrate VAMP3 and SNAP23 as potential targets for preventing the disturbed flow-accelerated thrombus formation.

Vascular access required to perform a dialysis treatment need to construct an arteriovenous fistula, though it has a problem of stenosis. It is considered to be caused by the influence of wall shear stress on the blood vessel wall. Recently, the influence of flow disturbance is also focused on as one of the causes, and is usually evaluated by the indexes such as TAWSS, transWSS, OSI and RRT. However, the correspondence of these indexes to the characteristic of flow disturbance is not clear. In the present study, these indexes are investigated with two different turbulence intensities, one is composed of fluctuation from the time averaged velocity, and the other is from the phase (ensemble) averaged velocity. As a result, it was found that TAWSS could estimate the extent of pulsatile flow unsteadiness, but the flow disturbance could not. TransWSS could evaluate the intensity of flow disturbance, only under the condition of high correlation between fluctuating velocity perpendicular to the main-flow and that parallel to the main-flow. Therefore, we formulated a new index capable of evaluating the disturbance with any characteristics. The new index shows reasonable performance for the purpose, althought it requires further improvement such as a formulation without post-processing using historical data.

The juxta-anastomotic stenosis of an arteriovenous fistula (AVF) is a significant clinical problem in hemodialysis patients with no effective treatment. Previous studies of AV anastomotic angles on hemodynamics and vascular wall injury were based on computational fluid dynamics (CFD) simulations using standardized AVF geometry, not the real-world patient images. The present study is the first CFD study to use angiographic images with patient-specific outcome information, i.e., the exact location of the AVF stenotic lesion. We conducted the CFD analysis utilizing patient-specific AVF geometric models to investigate hemodynamic parameters at different locations of an AVF, and the association between hemodynamic parameters and the anastomotic angle, particularly at the stenotic location. We analyzed 27 patients who used radio-cephalic AVF for hemodialysis and received an angiographic examination for juxta-anastomotic stenosis. The three-dimensional geometrical model of each patient’s AVF was built using the angiographic images, in which the shape and the anastomotic angle of the AVF were depicted. CFD simulations of AVF hemodynamics were conducted to obtain blood flow parameters at different locations of an AVF. We found that at the location of the stenotic lesion, the AV angle was significantly correlated with access flow disturbance (r = 0.739; p < 0.001) and flow velocity (r = 0.563; p = 0.002). Furthermore, the receiver operating characteristic (ROC) curve analysis revealed that the AV angle determines the lesion’s flow disturbance with a high area under the curve value of 0.878. The ROC analysis also identified a cut-off value of the AV angle as 46.5°, above or below which the access flow disturbance was significantly different. By applying CFD analysis to real-world patient images, the present study provides evidence that an anastomotic angle wider than 46.5° might lead to disturbed flow generation, demonstrating a reference angle to adopt during the anastomosis surgery.

Therapies for atherosclerotic cardiovascular disease should target early disease stages and specific vascular sites where disease occurs. Endothelial glycocalyx (GCX) degradation compromises endothelial barrier function and increases vascular permeability. This initiates pro-atherosclerotic lipids and inflammatory cells to penetrate vessel walls, and at the same time this can be leveraged for targeted drug delivery. In prior cell culture studies, GCX degradation significantly increased endothelial cell uptake of nanoparticle vehicles that are designed for drug delivery, compared to the effects of intact GCX. The present study assessed if the cell culture findings translate to selective nanoparticle uptake in animal vessels. In mice, the left carotid artery (LCA) was partially ligated to disturb blood flow, which induces GCX degradation, endothelial dysfunction, and atherosclerosis. After ligation, the LCA vessel wall exhibited a loss of continuity of the GCX layer on the intima. 10-nm gold nanospheres (GNS) coated with polyethylene glycol (PEG) were delivered intravenously. GCX degradation in the ligated LCA correlated to increased GNS infiltration of the ligated LCA wall. This suggests that GCX dysfunction, which coincides with atherosclerosis, can indeed be targeted for enhanced drug delivery, offering a new approach in cardiovascular disease therapy. [ABSTRACT FROM AUTHOR]

Cancer metastasis and secondary tumor initiation largely depend on circulating tumor cell (CTC) and vascular endothelial cell (EC) interactions by incompletely understood mechanisms. Endothelial glycocalyx (GCX) dysfunction may play a significant role in this process. GCX structure depends on vascular flow patterns, which are irregular in tumor environments. This work presents evidence that disturbed flow (DF) induces GCX degradation, leading to CTC homing to the endothelium, a first step in secondary tumor formation. A 2‐fold greater attachment of CTCs to human ECs was found to occur under DF conditions, compared to uniform flow (UF) conditions. These results corresponded to an approximately 50% decrease in wheat germ agglutinin (WGA)‐labeled components of the GCX under DF conditions, vs UF conditions, with undifferentiated levels of CTC‐recruiting E‐selectin under DF vs UF conditions. Confirming the role of the GCX, neuraminidase induced the degradation of WGA‐labeled GCX under UF cell culture conditions or in Balb/C mice and led to an over 2‐fold increase in CTC attachment to ECs or Balb/C mouse lungs, respectively, compared to untreated conditions. These experiments confirm that flow‐induced GCX degradation can enable metastatic CTC arrest. This work, therefore, provides new insight into pathways of secondary tumor formation. [ABSTRACT FROM AUTHOR]

Arteriovenous grafts (AVGs) typically lose patency within two years of creation due to venous neointimal hyperplasia, which is initiated by disturbed haemodynamics after AVG surgery. Haemodialysis needle flow can further disturb haemodynamics and thus impact AVG longevity. In this computational study it was assessed how dialysis flow and venous needle positioning impacts flow at the graft-vein anastomosis. Furthermore, it was studied how negative effects of dialysis needle flow could be mitigated. Non-physiological wall shear stress and disturbed blood flow were assessed in an AVG model with and without dialysis needle flow. Needle distance to the venous anastomosis was set to 6.5, 10.0, or 13.5 cm, whereas dialysis needle flow was set to 200, 300 or 400 mL/min. Intraluminal needle tip depth was varied between superficial, central, or deep. The detrimental effects of dialysis needle flow were summarised by a haemodynamic score (HS), ranging from 0 (minimal) to 5 (severe). Dialysis needle flow resulted in increased disturbed flow and/or non-physiological wall shear stress in the venous peri-anastomotic region. Increasing cannulation distance from 6.5 to 13.5 cm reduced the HS by a factor 4.0, whereas a central rather than a deep or superficial needle tip depth reduced the HS by a maximum factor of 1.9. Lowering dialysis flow from 400 to 200 mL/min reduced the HS by a factor 7.4. Haemodialysis needle flow, cannulation location, and needle tip depth considerably increase the amount of disturbed flow and non-physiological wall shear stress in the venous anastomotic region of AVGs. Negative effects of haemodialysis needle flow could be minimised by more upstream cannulation, by lower dialysis flow and by ensuring a central needle tip depth. Since disturbed haemodynamics are associated with neointimal hyperplasia development, optimising dialysis flow and needle positioning during haemodialysis could play an important role in maintaining AVG patency. [ABSTRACT FROM AUTHOR]

Arterial endothelium experience physical stress associated with blood flow and play a central role in maintaining vascular integrity and homeostasis in response to hemodynamic forces. Blood flow within vessels is generally laminar and streamlined. However, abrupt changes in the vessel geometry due to branching, sharp turns or stenosis can disturb the laminar blood flow, causing secondary flows in the form of vortices. Such disturbed flow patterns activate pro-inflammatory phenotypes in endothelial cells, damaging the endothelial layer and can lead to atherosclerosis and thrombosis. Here, we report a microfluidic system with integrated ridge-shaped obstacles for generating controllable disturbed flow patterns. This system is used to study the effect of disturbed flow on the cytoskeleton remodeling and nuclear shape and size of cultured human aortic endothelial cells. Our results demonstrate that the generated disturbed flow changes the orientation angle of actin stress fibers and reduces the nuclear size while increases the nuclear circularity.

Several compounds have been used for atherosclerosis treatment, including clinical trials; however, no anti-atherosclerotic drugs based on hemodynamic force-mediated atherogenesis have been discovered. Our previous studies demonstrated that "small mothers against decapentaplegic homolog 1/5" (Smad1/5) is a convergent signaling molecule for chemical [e.g., bone morphogenetic proteins (BMPs)] and mechanical (e.g., disturbed flow) stimulations and hence may serve as a promising hemodynamic-based target for anti-atherosclerosis drug development. The goal of this study was to develop a high-throughput screening (HTS) platform to identify potential compounds that can inhibit disturbed flow- and BMP-induced Smad1/5 activation and atherosclerosis. Through HTS using a Smad1/5 downstream target inhibitor of DNA binding 1 ( Id-1 ) as a luciferase reporter, we demonstrated that KU-55933 and Apicidin suppressed Id-1 expression in AD-293 cells. KU-55933 (10 μM), Apicidin (10 μM), and the combination of half doses of each [1/2(K + A)] inhibited disturbed flow- and BMP4-induced Smad1/5 activation in human vascular endothelial cells (ECs). KU-55933, Apicidin, and 1/2(K + A) treatments caused 50.6%, 47.4%, and 73.3% inhibitions of EC proliferation induced by disturbed flow, respectively, whereas EC inflammation was only suppressed by KU-55933 and 1/2(K + A), but not Apicidin alone. Administrations of KU-55933 and 1/2(K + A) to apolipoprotein E-deficient mice inhibited Smad1/5 activation in ECs in athero-susceptible regions, thereby suppressing endothelial proliferation and inflammation, with the attenuation of atherosclerotic lesions in these mice. A unique drug screening platform has been developed to demonstrate that KU-55933 and its combination with Apicidin are promising therapeutic compounds for atherosclerosis based on hemodynamic considerations., Competing Interests: Competing interests statement:The authors declare no competing interest.

Background: Arteriovenous fistula (AVF) is the preferred vascular access (VA) for hemodialysis, but it is still affected by high non-maturation and early failure rates due to stenosis development. Increasing evidence suggests that the presence of turbulent-like flow may play a key role, therefore, to stabilize the flow in the venous segment, an external support device (VasQ TM ) has been designed. The aim of this study was to provide preliminary evidence of VasQ TM impact on AVF hemodynamics as compared to AVFs created with conventional surgery., Methods: In this pilot single-center prospective randomized study six patients were enrolled, three in the VasQ group and three in the control group. Contrast-free magnetic resonance imaging (MRI) scans were acquired at 3 days, 3 months and 1 year after AVF surgery and were used to generate 3D patient-specific models. Computational fluid dynamic (CFD) simulations were performed using pimpleFoam, imposing patient-specific flow waveforms derived from ultrasound (US) examinations at the inlet of the proximal and distal artery, and a traction-free condition at the venous outflow. Morphologic and hemodynamic changes occurring over time were compared between VasQ and control AVFs., Results: Our MRI protocol provided high-quality images suitable for reliable segmentation and reconstruction of patient-specific 3D models of AVFs at all three timepoints in four out of six patients. The VasQ TM device maintained the angle between the artery and the vein almost unchanged over time, with a more stable flow in the AVFs supported by the device. In contrast, one of the AVFs of the control group evolved to an extreme dilatation of the vein and highly disturbed flow, while the other developed a stenosis in the juxta-anastomotic region., Conclusions: This study demonstrated the feasibility of characterizing the morphological and hemodynamic changes occurring over time in AVFs created using the VasQ TM device and provided preliminary evidence of the potential hemodynamic benefits of its use., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Endothelial cells (ECs) experience two different blood flow patterns: laminar and disturbed flow. Their responses to laminar flow contribute to vascular homeostasis, whereas their responses to disturbed flow result in EC dysfunction and vascular diseases. However, it remains unclear how ECs differentially sense laminar and disturbed flow and trigger signaling that elicits different responses. Here, we showed that ECs differentially sense laminar and disturbed flows by altering the lipid order of their plasma and mitochondrial membranes in opposite directions. This results in distinct changes in mitochondrial function, namely, increased adenosine triphosphate (ATP) production for laminar flow and increased hydrogen peroxide (H 2 O 2 ) release for disturbed flow, leading to ATP- and H 2 O 2 -mediated signaling, respectively. When cultured human aortic ECs were subjected to laminar or disturbed flow in flow-loading devices, the lipid order of their plasma membranes immediately decreased in response to laminar flow and increased in response to disturbed flow. Laminar flow also decreased the lipid order of mitochondrial membranes and increased mitochondrial ATP production. In contrast, disturbed flow increased the lipid order of mitochondrial membranes and increased the release of H 2 O 2 from the mitochondria. The addition of cholesterol to the cells increased the lipid order of both membranes and abrogated laminar flow-induced ATP production, while treatment of the cells with a cholesterol-depleting reagent, methyl-β cyclodextrin, decreased the lipid order of both membranes and abolished disturbed flow-induced H 2 O 2 release, indicating that changes in the membrane lipid order and/or cholesterol content are closely linked to flow-induced changes in mitochondrial functions. NEW & NOTEWORTHY How vascular endothelial cells (ECs) differentially sense laminar and disturbed flows and trigger intracellular signaling remains unclear. Here, we show that EC plasma membranes act as mechanosensors to discriminate between laminar and disturbed flows by undergoing opposite changes in their lipid order. Similar lipid order changes occur simultaneously in the mitochondrial membranes, which are linked to changes in mitochondrial function, that is, increased ATP production for laminar flow and increased H 2 O 2 release for disturbed flow.

Thoracic aortic aneurysm (TAA) is fatal diseases, which leads to aortic rupture and sudden death. Blood pressure-lowering drugs are ineffective for most of the patients. Our previous study demonstrated the inhibition of endothelial secreted miR-126–3p by rapamycin ameliorate the aneurysmal phenotype of smooth muscle cells (SMCs) in vitro. Hence, this study aimed to evaluate the modulation and mechanism of miR-126–3p in a murine model of TAA (Fbn1C1039G/+). Our results showed that noticeable disturbed flow (DF) was observed in the aorta of Fbn1C1039G/+ mice, and the expression of miR-126–3p was significantly increased under the DF in the cell chamber. This finding was also confirmed by tests in the corresponding DF area of the human aortic aneurysm tissue. Constant rapamycin administration significantly ameliorates the incidence and severity of Fbn1C1039G/+ mice characterized by decreased aortic media degradation, macrophage infiltration and MMP2/9 expression in the aortic wall. Mechanistic studies showed that rapamycin attenuates TAA progression by inhibiting miR-126–3p through ERK1/2 inactivation. • Disturbed flow (DF) induces upregulation of miR-126-3p both in vitro and in vivo. • TAA mice had an increased disturbed flow inside the aneurysm sac. • Rapamycin downregulated miR-126-3p through MAPK/ERK inactivation. • Rapamycin inhibited thoracic aortic aneurysm in a murine model of thoracic aortic aneurysm(TAA). • Inhibition of the flow-induced dysregulated miR-126-3p may be a new mechanism to treat TAA. [ABSTRACT FROM AUTHOR]

Atherosclerosis is the primary underlying cause of cardiovascular disease which preferentially develops at arterial regions exposed to disturbed flow (DF), but much less at regions of unidirectional laminar flow (UF). Recent studies have demonstrated that DF and UF differentially regulate important aspects of endothelial function, such as vascular inflammation, oxidative stress, vascular tone, cell proliferation, senescence, mitochondrial function, and glucose metabolism. DF and UF regulate vascular pathophysiology via differential regulation of mechanosensitive transcription factors (MSTFs) (KLF2, KLF4, NRF2, YAP/TAZ/TEAD, HIF-1α, NF-κB, AP-1, and others). Emerging studies show that MSTFs represent promising therapeutic targets for the prevention and treatment of atherosclerosis. We present here a comprehensive overview of the role of MSTFs in atherosclerosis, and highlight future directions for developing novel therapeutic agents by targeting MSTFs. Highlights UF and DF differentially modulate several MSTFs in endothelial cells. MSTFs regulate multiple aspects of endothelial function, including inflammation, proliferation, thrombosis, oxidative stress, and endothelial cell metabolism. MSTFs crosstalk with each other and functionally regulate endothelial gene expression and function. Targeting MSTFs could be a promising therapeutic strategy for atherosclerosis. [ABSTRACT FROM AUTHOR]

The present experimental study investigates a new configuration of vortex flowmeter to improve the turndown ratio and flow distortion sensitivity. An optimized contraction cone is introduced upstream to the vortex shedder to enhance the strength of the shed vortices and to suppress upstream flow distortion effects. A trapezoidal bluff body with two different blockage ratios (19% and 28%) is studied as a vortex shedder. The performance of this new configuration is studied under fully developed flow condition and also evaluated with various flow disturbances. The flow distortion is introduced with 90° degree in-plane and out-of-plane elbows. Transient wall pressure measurement technique is used to capture the vortex shedding frequency. It is found that 28% blockage ratio with optimized contraction cone is the best combination to improve the turn down ratio of vortex flowmeter. The installation of contraction cone helps in reducing the non-uniformity in the velocity profiles which improves the strength and stability of the shed vortices. Minimum measurable Reynolds number obtained with this technique is 5000 which is significantly lower than conventional vortex flowmeters. The turn down ratio achieved with this technique is 1:80 as opposed to 1:20 for conventional vortex flowmeters. [ABSTRACT FROM AUTHOR]

Background: Arterial stiffness and wall shear stress are powerful determinants of cardiovascular health, and arterial stiffness is associated with increased cardiovascular mortality. Low and oscillatory wall shear stress, termed disturbed flow (d-flow), promotes atherosclerotic arterial remodeling, but the relationship between d-flow and arterial stiffness is not well understood. The objective of this study was to define the role of d-flow on arterial stiffening and discover the relevant signaling pathways by which d-flow stiffens arteries.Methods: D-flow was induced in the carotid arteries of young and old mice of both sexes. Arterial stiffness was quantified ex vivo with cylindrical biaxial mechanical testing and in vivo from duplex ultrasound and compared with unmanipulated carotid arteries from 80-week-old mice. Gene expression and pathway analysis was performed on endothelial cell-enriched RNA and validated by immunohistochemistry. In vitro testing of signaling pathways was performed under oscillatory and laminar wall shear stress conditions. Human arteries from regions of d-flow and stable flow were tested ex vivo to validate critical results from the animal model.Results: D-flow induced arterial stiffening through collagen deposition after partial carotid ligation, and the degree of stiffening was similar to that of unmanipulated carotid arteries from 80-week-old mice. Intimal gene pathway analyses identified transforming growth factor-β pathways as having a prominent role in this stiffened arterial response, but this was attributable to thrombospondin-1 (TSP-1) stimulation of profibrotic genes and not changes to transforming growth factor-β. In vitro and in vivo testing under d-flow conditions identified a possible role for TSP-1 activation of transforming growth factor-β in the upregulation of these genes. TSP-1 knockout animals had significantly less arterial stiffening in response to d-flow than wild-type carotid arteries. Human arteries exposed to d-flow had similar increases TSP-1 and collagen gene expression as seen in our model.Conclusions: TSP-1 has a critical role in shear-mediated arterial stiffening that is mediated in part through TSP-1's activation of the profibrotic signaling pathways of transforming growth factor-β. Molecular targets in this pathway may lead to novel therapies to limit arterial stiffening and the progression of disease in arteries exposed to d-flow. [ABSTRACT FROM AUTHOR]