Identification of KU-55933 as an anti-atherosclerosis compound by using a hemodynamic-based high-throughput drug screening platform.

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.

Disturbed flow in the juxta-anastomotic area of an arteriovenous fistula correlates with endothelial loss, acute thrombus formation, and neointimal hyperplasia.

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.

MST1 Suppresses Disturbed Flow Induced Atherosclerosis.

BACKGROUND: Atherosclerosis occurs mainly at arterial branching points exposed to disturbed blood flow. How MST1 (mammalian sterile 20-like kinase 1), the primary kinase in the mechanosensitive Hippo pathway modulates disturbed flow induced endothelial cells (ECs) activation and atherosclerosis remains unclear. METHODS: To assess the role of MST1 in vivo, mice with EC-specific Mst1 deficiency on ApoE-/- background (Mst1iECKOApoE-/-) were used in an atherosclerosis model generated by carotid artery ligation. Mass spectrometry, immunoprecipitation, proximity ligation assay, and dye uptake assay were used to identify the functional substrate of MST1. Human umbilical vein endothelial cells and human aortic endothelial cells were subjected to oscillatory shear stress that mimic disturbed flow in experiments conducted in vitro. RESULTS: We found that the phosphorylation of endothelial MST1 was significantly inhibited in oscillatory shear stress-exposed regions of human and mouse arteries and ECs. Ectopic lenti-mediated overexpression of wild-type MST1, but not a kinase-deficient mutant of MST1, reversed disturbed flow-caused EC activation and atherosclerosis in EC-specific Mst1 deficiency on ApoE-/- background (Mst1iECKOApoE-/-). Inhibition of MST1 by oscillatory shear stress led to reduced phosphorylation of Cx43 (connexin 43) at Ser255, the Cx43 hemichannel open, EC activation, and atherosclerosis, which were blocked by TAT-GAP19, a Cx43 hemichannel inhibitory peptide. Mass spectrometry studies identified that Filamin B fueled the translocation of Cx43 to lipid rafts for further hemichannel open. Finally, lenti-mediated overexpression of the Cx43S255 mutant into glutamate to mimic phosphorylation blunted disturbed flow-induced EC activation, thereby inhibiting the atherogenesis in both ApoE-/- and Mst1 iECKOApoE-/- mice. CONCLUSIONS: Our study reveals that inhibition of the MST1-Cx43 axis is an essential driver of oscillatory shear stress-induced endothelial dysfunction and atherosclerosis, which provides a new therapeutic target for the treatment of atherosclerosis.

Vinculin phosphorylation impairs vascular endothelial junctions promoting atherosclerosis.

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.

Steady Laminar Flow Decreases Endothelial Glycolytic Flux While Enhancing Proteoglycan Synthesis and Antioxidant Pathways.

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.

Endothelial cells differentially sense laminar and disturbed flows by altering the lipid order of their plasma and mitochondrial membranes.

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-ß 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.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 H2O2 release for disturbed flow.

Osteopontin mediation of disturbed flow-induced endothelial mesenchymal transition through CD44 is a novel mechanism of neointimal hyperplasia in arteriovenous fistulae for hemodialysis access.

In dysfunctional arteriovenous fistulae (AVF) for hemodialysis access, neointimal hyperplasia (NH) is prone to occur in the region exposed to disturbed flow. We hypothesized that disturbed flow contributes to NH in AVF by inducing endothelial mesenchymal transition (EndMT) through activation of the osteopontin/CD44 axis. In rats with aortocaval fistula, a rodent model of AVF, we demonstrated development of EndMT and expression of osteopontin and CD44 specifically in the vicinity of the arteriovenous junction using immunostaining. Duplex scan confirmed this region was exposed to a disturbed flow. A mixed ultrastructural phenotype of endothelium and smooth muscle cells was found in luminal endothelial cells of the arteriovenous junction by electron microscopy ascertaining the presence of EndMT. Endothelial lineage tracing using Cdh5-Cre/ERT2;ROSA26-tdTomato transgenic mice showed that EndMT was involved in NH of AVF since the early stage and that the endothelial-derived cells contributed to 24% of neointimal cells. In human umbilical vein endothelial cells (HUVECs) in culture, osteopontin treatment induced EndMT, which was suppressed by CD44 knockdown. Exposure to low oscillatory wall shear stress using a parallel-plate system induced EndMT in HUVECs, also suppressed by osteopontin or CD44 knockdown. In AVF of CD44 knockout mice, EndMT was mitigated and NH decreased by 35% compared to that in wild-type mice. In dysfunctional AVF of patients with uremia, expressions of osteopontin, CD44, and mesenchymal markers in endothelial cells overlying the neointima was also found by immunostaining. Thus, the osteopontin/CD44 axis regulates disturbed flow-induced EndMT, plays an important role in neointimal hyperplasia of AVF, and may act as a potential therapeutic target to prevent AVF dysfunction.

Thioridazine protects against disturbed flow-induced atherosclerosis by inhibiting RhoA/YAP-mediated endothelial inflammation.

Atherosclerotic diseases remain the leading cause of adult mortality and impose heavy burdens on health systems globally. Our previous study found that disturbed flow enhanced YAP activity to provoke endothelial activation and atherosclerosis, and targeting YAP alleviated endothelial inflammation and atherogenesis. Therefore, we established a luciferase reporter assay-based drug screening platform to seek out new YAP inhibitors for anti-atherosclerotic treatment. By screening the FDA-approved drug library, we identified that an anti-psychotic drug thioridazine markedly suppressed YAP activity in human endothelial cells. Thioridazine inhibited disturbed flow-induced endothelial inflammatory response in vivo and in vitro. We verified that the anti-inflammatory effects of thioridazine were mediated by inhibition of YAP. Thioridazine regulated YAP activity via restraining RhoA. Moreover, administration of thioridazine attenuated partial carotid ligation- and western diet-induced atherosclerosis in two mouse models. Overall, this work opens up the possibility of repurposing thioridazine for intervention of atherosclerotic diseases. This study also shed light on the underlying mechanisms that thioridazine inhibited endothelial activation and atherogenesis via repression of RhoA-YAP axis. As a new YAP inhibitor, thioridazine might need further investigation and development for the treatment of atherosclerotic diseases in clinical practice.

Disturbed flow's impact on cellular changes indicative of vascular aneurysm initiation, expansion, and rupture: A pathological and methodological review.

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.

Flow-regulated endothelial glycocalyx determines metastatic cancer cell activity.

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.

Endothelial-to-mesenchymal transition shapes the atherosclerotic plaque and modulates macrophage function.

Endothelial cells can acquire a mesenchymal phenotype upon irritation [endothelial-to-mesenchymal transition (EndMT)]. Macrophages accumulate in the atherosclerotic plaque. This study addressed whether macrophages modulate EndMT and delineated a reciprocal effect of EndMT on macrophage functions in atherosclerosis. In atherosclerotic murine and human aortas, endothelial cells with mesenchymal markers were elevated by confocal microscopy and flow cytometric analysis. Increased EndMT master transcription factor Snai1 expression and extracellular matrix are consistent with enhanced EndMT in this condition. Hypoxia was detected in individual aortic EndMT cells in vivo and rapidly induced a similar EndMT phenotype in vitro. As a novel inducer of EndMT, macrophages, which are abundant in the atherosclerotic lesions, enhance mesothelial marker expression during coculture in vitro. In the reverse relationship, EndMT altered endothelial colony-stimulating factor expression. Functionally, EndMT cell-conditioned media attenuated macrophage proliferation, antigen-presenting cell marker expression, and TNF-α production in response to oxidized LDL but increased oxidized LDL uptake and scavenger receptor expression. These experiments demonstrate that macrophages promote partial EndMT. In turn, EndMT cells modulate macrophage phenotype and lipid uptake. Our data suggest that EndMT shapes macrophage and endothelial cell phenotypes, thus affecting internal atherosclerotic plaque in addition to surface structure.-Helmke, A., Casper, J., Nordlohne, J., David, S., Haller, H., Zeisberg, E. M., von Vietinghoff, S. Endothelial-to-mesenchymal transition shapes the atherosclerotic plaque and modulates macrophage function.

Computational Modelling Based Recommendation on Optimal Dialysis Needle Positioning and Dialysis Flow in Patients With Arteriovenous Grafts.

OBJECTIVE: 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. METHODS: 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). RESULTS: 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. CONCLUSION: 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.

Targeting Platelet in Atherosclerosis Plaque Formation: Current Knowledge and Future Perspectives.

Besides their role in hemostasis and thrombosis, it has become increasingly clear that platelets are also involved in many other pathological processes of the vascular system, such as atherosclerotic plaque formation. Atherosclerosis is a chronic vascular inflammatory disease, which preferentially develops at sites under disturbed blood flow with low speeds and chaotic directions. Hyperglycemia, hyperlipidemia, and hypertension are all risk factors for atherosclerosis. When the vascular microenvironment changes, platelets can respond quickly to interact with endothelial cells and leukocytes, participating in atherosclerosis. This review discusses the important roles of platelets in the plaque formation under pro-atherogenic factors. Specifically, we discussed the platelet behaviors under disturbed flow, hyperglycemia, and hyperlipidemia conditions. We also summarized the molecular mechanisms involved in vascular inflammation during atherogenesis based on platelet receptors and secretion of inflammatory factors. Finally, we highlighted the studies of platelet migration in atherogenesis. In general, we elaborated an atherogenic role of platelets and the aspects that should be further studied in the future.

Rapamycin attenuates a murine model of thoracic aortic aneurysm by downregulating the miR-126-3p mediated activation of MAPK/ERK signalling pathway.

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.

Perivascular adipose tissue modulates carotid plaque formation induced by disturbed flow in mice.

OBJECTIVE: Emerging evidence shows that perivascular adipose tissue (PVAT) is crucially involved in inflammation and cardiovascular diseases. However, controversial results have been reported regarding the effect of PVAT in atherosclerosis. This study aimed to determine the role of PVAT in disturbed blood flow (d-flow)-induced carotid plaque formation. METHODS: ApoE-/- male mice underwent partial carotid ligation (PCL) to induce d-flow in the left carotid artery (LCA) and were fed a high-fat diet for 2 weeks. Oil Red O and hematoxylin and eosin stains were used to determine adipose tissue. Thoracic PVAT from ApoE-/- or wild-type female mice were transplanted to the LCA of PCL-treated ApoE-/- mice. Carotid arteries were stained with Sudan IV to detect atherosclerotic lesions. Quantitative real-time reverse transcription polymerase chain reaction and immunofluorescence staining were performed to assess macrophage infiltration. RESULTS: By 2 weeks of the high-fat diet after PCL surgery, de novo adipose tissue was formed around the ligated LCA, where atherosclerotic plaques were also observed. Quantitative real-time reverse transcription polymerase chain reaction analysis of the newly formed PVAT revealed a similar transcription profile to native PVAT. Treatment with bisphenol A diglycidyl ether, a peroxisome proliferator-activated receptor γ inhibitor, diminished PVAT formation but increased plaque size and macrophage infiltration. Transplantation of thoracic PVAT from wild-type mice (PVAT-TWT) rather than from ApoE-/- mice (PVAT-TApoE-/-) nearly abrogated LCA plaque macrophage content without affecting plaque size. Mechanistically, PVAT-TApoE-/- showed higher messenger RNA levels of inflammatory cytokines compared with PVAT-TWT. CONCLUSIONS: Our findings suggest that regulated PVAT formation may confer protection against atherosclerosis-prone shear stress, probably through attenuation of focal inflammation.

Flow Perturbation Mediates Neutrophil Recruitment and Potentiates Endothelial Injury via TLR2 in Mice: Implications for Superficial Erosion.

RATIONALE: Superficial erosion currently causes up to a third of acute coronary syndromes; yet, we lack understanding of its mechanisms. Thrombi because of superficial intimal erosion characteristically complicate matrix-rich atheromata in regions of flow perturbation. OBJECTIVE: This study tested in vivo the involvement of disturbed flow and of neutrophils, hyaluronan, and Toll-like receptor 2 ligation in superficial intimal injury, a process implicated in superficial erosion. METHODS AND RESULTS: In mouse carotid arteries with established intimal lesions tailored to resemble the substrate of human eroded plaques, acute flow perturbation promoted downstream endothelial cell activation, neutrophil accumulation, endothelial cell death and desquamation, and mural thrombosis. Neutrophil loss-of-function limited these findings. Toll-like receptor 2 agonism activated luminal endothelial cells, and deficiency of this innate immune receptor decreased intimal neutrophil adherence in regions of local flow disturbance, reducing endothelial cell injury and local thrombosis (P<0.05). CONCLUSIONS: These results implicate flow disturbance, neutrophils, and Toll-like receptor 2 signaling as mechanisms that contribute to superficial erosion, a cause of acute coronary syndrome of likely growing importance in the statin era.

Cystathionine γ-Lyase Modulates Flow-Dependent Vascular Remodeling.

Objective- Flow patterns differentially regulate endothelial cell phenotype, with laminar flow promoting vasodilation and disturbed flow promoting endothelial proinflammatory activation. CSE (cystathionine γ-lyase), a major source of hydrogen sulfide (H2S) in endothelial cells, critically regulates cardiovascular function, by both promoting vasodilation and reducing endothelial activation. Therefore, we sought to investigate the role of CSE in the endothelial response to flow. Approach and Results- Wild-type C57Bl/6J and CSE knockout ( CSE-/-) mice underwent partial carotid ligation to induce disturbed flow in the left carotid. In addition, endothelial cells isolated from wild-type and CSE -/- mice were exposed to either laminar or oscillatory flow, an in vitro model of disturbed flow. Interestingly, laminar flow significantly reduced CSE expression in vitro, and only disturbed flow regions show discernable CSE protein expression in vivo, correlating with enhanced H2S production in wild-type C57BL/6J but not CSE-/- mice. Lack of CSE limited disturbed flow-induced proinflammatory gene expression (ICAM-1[intercellular adhesion molecule 1], VCAM-1 [vascular cell adhesion molecular 1]) and monocyte infiltration and CSE-/- endothelial cells showed reduced NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation and proinflammatory gene expression in response to oscillatory flow in vitro. In addition, CSE-/- mice showed reduced inward remodeling after partial carotid ligation. CSE-/- mice showed elevated vascular nitrite levels (measure of nitric oxide [NO]) in the unligated carotids, suggesting an elevation in baseline NO production, and the NO scavenger 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3-oxide normalized the reduced inward remodeling, but not inflammation, of ligated carotids in CSE-/- mice. Conclusions- CSE expression in disturbed flow regions critically regulates both endothelial activation and flow-dependent vascular remodeling, in part through altered NO availability.

Flow-dependent YAP/TAZ activities regulate endothelial phenotypes and atherosclerosis.

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.

Disturbed Flow Promotes Arterial Stiffening Through Thrombospondin-1.

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-ß 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-ß. In vitro and in vivo testing under d-flow conditions identified a possible role for TSP-1 activation of transforming growth factor-ß 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-ß. 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.

C1q/TNF-related protein 1 promotes endothelial barrier dysfunction under disturbed flow.

Endothelial hyper-permeability is a major determinant factor that contributes to the accelerated development of atherosclerotic lesions at hemodynamically disturbed sites. Previously, we showed that C1q/TNF related protein (CTRP) 1 promotes endothelium-leukocyte interactions and inflammatory responses in vascular cells. Here, we sought to investigate the role of CTRP1 in modulation of endothelial permeability under disturbed flow condition. By using en face staining of mouse aorta, we found CTRP1 expression was significantly increased in vascular endothelial cells under disturbed flow as compared to steady laminar flow. Vascular permeability to Evans blue dye was notably enhanced in CTRP1 knockin mice as compared to wild type animals, whereas aortic hyper-permeability at disturbed sites was remarkably restored after deletion of CTRP1. In cultured endothelial cells, treatment of CTRP1 led to increased permeability to fluorescent-labelled dextran and apparent formation of paracellular holes as observed after disturbed flow exposure, which was evidently reduced in the presence of a CTRP1-specific neutralizing antibody. Mechanistically, we found activation of VEGFR2 by CTRP1 might be involved in vascular hyper-permeability under disturbed flow condition. Taken together, this study suggests that CTRP1 is a mechano-sensitive proinflammatory factor that mediates disturbed flow-induced vascular barrier dysfunction. Inhibition of CTRP1 may inhibit the pathogenesis of atherosclerosis at early stage.