High glucose induced NF-κB DNA-binding activity in HAEC is maintained under low shear stress but inhibited under high shear stress: role of nitric oxide

We have investigated the role of inhibitor κBα (IκBα) in the activation of nuclear factor κB (NF-κB) observed in human aortic endothelial cells (HAEC) undergoing a low shear stress of 2 dynes/cm2. Low shear for 6 h resulted in a reduction of IκBα levels, an activation of NF-κB, and an increase in κB-dependent vascular cell adhesion molecule 1 (VCAM-1) mRNA expression and endothelial-monocyte adhesion. Overexpression of IκBα in HAEC attenuated all of these shear-induced responses. These results suggest that downregulation of IκBα is the major factor in the low shear-induced activation of NF-κB in HAEC. We then investigated the role of nitric oxide (NO) in the regulation of IκBα/NF-κB. Overexpression of endothelial nitric oxide synthase (eNOS) inhibited NF-κB activation in HAEC exposed to 6 h of low shear stress. Addition of the structurally unrelated NO donors S-nitrosoglutathione (300 μM) or sodium nitroprusside (1 mM) before low shear stress significantly increased cytoplasmic IκBα and concomitantly reduced NF-κB binding activity and κB-dependent VCAM-1 promoter activity. Together, these data suggest that NO may play a major role in the regulation of IκBα levels in HAEC and that the application of low shear flow increases NF-κB activity by attenuating NO generation and thus IκBα levels.

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Albumin Uptake Into Endothelial Cells in Separated Flow

10.1115/imece2000-2526 ◽

2000 ◽

Author(s):

Susumu Kudo ◽

Ryuhei Yamaguchi ◽

Masashi Sato ◽

Kotaro Oka ◽

Kazuo Tanishita

Keyword(s):

Endothelial Cells ◽

Shear Stress ◽

Cell Morphology ◽

Stress Gradient ◽

Separated Flow ◽

High Shear ◽

High Shear Stress ◽

Low Shear Stress ◽

Shear Stress Gradient ◽

Low Shear

Abstract The purpose of this study is to reveal the albumin uptake into endothelial cells in the separated flow area. After 24 hr of exposure to flow induced in a back step flow channel, the endothelial cells were incubated in 37°C for 60 minutes in PBS containing tetramethylrhodamine isothiocyanate conjugated albumin (TRITC-albumin). Thereafter, the cell morphology and the albumin uptake were observed by a confocal laser scanning microscope (CLSM). In a low shear stress area (stagnant and reattachment areas), the cells aligned randomly. In a high shear stress area (reversal and fully developed areas), the cells were elongated and aligned to flow direction. In low-shear-stress and high-shear-stress gradient areas (reattachment areas), the amount of albumin uptake into the cells was the largest in all areas. These data indicate that shear stress and shear stress gradient affect the endothelial cell morphology and the albumin uptake into endothelial cells.

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Endothelial Cell Morphometry of Atherosclerotic Lesions and Flow Profiles at Aortic Bifurcations in Cholesterol Fed Rabbits

Journal of Biomechanical Engineering ◽

10.1115/1.2891387 ◽

1992 ◽

Vol 114 (3) ◽

pp. 301-308 ◽

Cited By ~ 28

Author(s):

Mitsuji Okano ◽

Yoji Yoshida

Keyword(s):

Endothelial Cells ◽

Shear Stress ◽

Lipid Deposition ◽

High Shear ◽

High Shear Stress ◽

Atherosclerotic Lesions ◽

Flow Profiles ◽

Flow Divider ◽

Low Shear Stress ◽

Low Shear

Observations on shapes of endothelial cells both in sudanophilic and nonsudanophilic regions at bifurcations of the brachiocephalic (BC) and left subclavian (SA) arteries in hyperlipidemic rabbits were performed under a SEM. The stagnation point of flow and leading edges of flow dividers were nonsudanophilic and covered by round and long fusiform endothelial cells, respectively. The hips of flow dividers of both branchings, proven to be relatively low shear stress regions, by movement of microspheres in steady flow, were sudanophilic and covered by ellipsoidal cells. Similar studies were carried out in normolipidemic rabbits. It might be concluded that lipid deposition in hyperlipidemic rabbits occurs in relatively low shear stress regions, where endothelial cells are functionally activated, rather than in laminar high shear stress regions at the flow divider.

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Low Shear Stress Increases Recombinant Protein Production and High Shear Stress Increases Apoptosis in Human Cells

iScience ◽

10.1016/j.isci.2020.101653 ◽

2020 ◽

Vol 23 (11) ◽

pp. 101653

Author(s):

Caijuan Zhan ◽

Gholamreza Bidkhori ◽

Hubert Schwarz ◽

Magdalena Malm ◽

Aman Mebrahtu ◽

...

Keyword(s):

Shear Stress ◽

Recombinant Protein ◽

Recombinant Protein Production ◽

Protein Production ◽

Human Cells ◽

High Shear ◽

High Shear Stress ◽

Low Shear Stress ◽

Low Shear

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Nitric Oxide–Dependent and Nitric Oxide–Independent Transcriptional Responses to High Shear Stress in Endothelial Cells

Hypertension ◽

10.1161/01.hyp.0000154683.33414.94 ◽

2005 ◽

Vol 45 (4) ◽

pp. 672-680 ◽

Cited By ~ 20

Author(s):

Branko Braam ◽

Remmert de Roos ◽

Hans Bluyssen ◽

Patrick Kemmeren ◽

Frank Holstege ◽

...

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Shear Stress ◽

High Shear ◽

High Shear Stress ◽

Transcriptional Responses

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Role of cFOS in mechanosensitive transcriptional regulation in diabetes associated atherosclerosis

European Heart Journal ◽

10.1093/eurheartj/ehab724.3403 ◽

2021 ◽

Vol 42 (Supplement_1) ◽

Author(s):

A Khan ◽

M Lee ◽

A Watson ◽

S Maxwell ◽

M Cooper ◽

...

Keyword(s):

Endothelial Cells ◽

Shear Stress ◽

Myocardial Dysfunction ◽

Transcriptional Profile ◽

Gene Set Enrichment Analysis ◽

Regulation Of Transcription ◽

Gene Markers ◽

Low Shear Stress ◽

Low Shear

Abstract Atherosclerosis, as manifested clinically by myocardial infarction, stroke and peripheral vascular disease, is a major contributor to cardiovascular disease, the leading cause of death in patients with diabetes. The lipid-laden plaque development within the arterial vessel wall is a progressive process initiated with endothelial cell activation and monocyte adhesion. These cellular events occur primarily at the regions of blood vessels exposed to turbulent blood flow (TBF) and low shear stress such as vascular bends and bifurcations. Exposure of the vascular cells to chronic hyperglycaemia and TBF induces a proatherogenic transcriptional profile. Studies have shown that shear stress regulates vascular pathophysiology via differential regulation of transcription factors (TFs) such as KLF4, EGR1 and AP-1, hence named as mechanosensitive TFs. AP-1 is a heterodimer composed of FOS, Jun and ATF family of TFs. Studies have shown that it is activated by low shear stress in cultured endothelial cells. Increasing evidence supports the vital role of AP-1 family members in inflammation and diabetes-induced myocardial dysfunction. However, gene targets and the mechanisms underlying hyperglycemia-induced activation of AP-1 transcription factor cFOS in vascular regions exposed to TBF are not known.Although a novel approach not previously studied in diabetes associated atherosclerosis, we used a single cell RNA sequencing (scRNA-seq) approach to identify endothelial cells from TBF regions of aorta. Diabetes was induced with streptozotocin (STZ) in Apoe−/− mice and followed for 10 weeks. Cells from digested aortae of control and diabetic mice were subjected to scRNA-seq using 10X Genomics system and Illumina Nova-seq 6000. Unsupervised graph based clustering grouped cells into fourteen cell clusters with similar gene expression profile. We applied a list of mechanosensitive gene markers including EGR1, cFOS, Junb and ICAM1 in scRNA-seq analysis to identify endothelial cells from TBF regions of aorta. This approach identified atheroprone endothelial cells exposed to persistent TBF that showed a distinct transcriptional profile with more than six hundred genes differentially expressed. Importantly, cFOS was the most significantly upregulated gene in endothelial cells exposed to TBF. We next generated adiabetes associated transcriptional signature unique to endothelial cells exposed to TBF as compared to all other cell types in the aorta. We identified several genes in endothelial cells exposed to TBF and hyperglycaemia uniquely dysregulated in diabetic Apoe−/− mice as compared to control mice (cut off = FDR<0.05, fold change at least 2-fold). Gene set enrichment analysis identified “fluid shear stress and atherosclerosis” as most significantly dysregulated pathway in endothelial cells. These novel findings indicate that AP-1 TF subunit cFOS is a potential therapeutic target in diabetes associated atherosclerosis that warrant further experimental exploration. FUNDunding Acknowledgement Type of funding sources: Foundation. Main funding source(s): National Heart Foundation of Australia

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Low shear stress preferentially enhances IKK activity through selective sources of ROS for persistent activation of NF-κB in endothelial cells

AJP Cell Physiology ◽

10.1152/ajpcell.00535.2005 ◽

2007 ◽

Vol 292 (1) ◽

pp. C362-C371 ◽

Cited By ~ 49

Author(s):

Sumathy Mohan ◽

Koichi Koyoma ◽

Amalraj Thangasamy ◽

Hiroyasu Nakano ◽

Randolph D. Glickman ◽

...

Keyword(s):

Endothelial Cells ◽

Shear Stress ◽

Nadph Oxidase ◽

Significant Proportion ◽

Time Frame ◽

Oxidase Inhibitor ◽

Ros Generation ◽

High Shear ◽

Low Shear Stress ◽

Low Shear

NF-κB signaling pathway has been known to play a major role in the pathological process of atherogenesis. Unlike high shear stress, in which the NF-κB activity is transient, our earlier studies have demonstrated a persistent activation of NF-κB in response to low shear stress in human aortic endothelial cells. These findings partially explained why low shear regions that exist at bifurcations of arteries are prone to atherosclerosis, unlike the relatively atheroprotective high shear regions. In the present study, we further investigated 1) the role of NF-κB signaling kinases (IKKα and β) that may be responsible for the sustained activation of NF-κB in low shear stress and 2) the regulation of these kinases by reactive oxygen species (ROS). Our results demonstrate that not only is a significant proportion of low shear-induced-kinase activity is contributed by IKKβ, but it is also persistently induced for a prolonged time frame. The IKK activity (both α and β) is blocked by apocynin (400 μM), a specific NADPH oxidase inhibitor, and diphenyleneiodonium chloride (DPI; 10 μM), an inhibitor of flavin-containing oxidases like NADPH oxidases. Determination of ROS also demonstrated an increased generation in low shear stress that could be blocked by DPI. These results suggest that the source of ROS generation in endothelial cells in response to low shear stress is NADPH oxidase. The DPI-inhibitable component of ROS is the primary regulator of specific upstream kinases that determine the persistent NF-κB activation selectively in low shear-induced endothelial cells.

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Calin from Hirudo medicinalis, an inhibitor of von Willebrand factor binding to collagen under static and flow conditions

Blood ◽

10.1182/blood.v85.3.705.bloodjournal853705 ◽

1995 ◽

Vol 85 (3) ◽

pp. 705-711 ◽

Cited By ~ 37

Author(s):

J Harsfalvi ◽

JM Stassen ◽

MF Hoylaerts ◽

E Van Houtte ◽

RT Sawyer ◽

...

Keyword(s):

Shear Stress ◽

Platelet Adhesion ◽

Thrombus Formation ◽

High Shear ◽

High Shear Stress ◽

Flow Conditions ◽

Shear Rates ◽

Von Willebrand ◽

Willebrand Factor ◽

Low Shear

Calin from the saliva of the medicinal leech, Hirudo medicinalis, is a potent inhibitor of collagen mediated platelet adhesion and activation. In addition to inhibition of the direct platelet-collagen interaction, we presently demonstrate that binding of von Willebrand to coated collagen can be prevented by Calin, both under static and flow conditions in agreement with the occurrence of binding of Calin to collagen, confirmed by Biospecific Interaction Analysis. To define whether Calin acted by inhibiting the platelet-collagen or the platelet- von Willebrand factor (vWF)-collagen-mediated thrombus formation, platelet adhesion to different types of collagens was studied in a parallel-plate flow chamber perfused with whole blood at different shear rates. Calin dose-dependently prevented platelet adhesion to the different collagens tested both at high- and low-shear stress. The concentration of Calin needed to cause 50% inhibition of platelet adhesion at high-shear stress was some fivefold lower than that needed for inhibition of vWF-binding under similar conditions, implying that at high-shear stress, the effect of Calin on the direct platelet- collagen interactions, suffices to prevent thrombus formation. Platelet adhesion to extracellular matrix (ECM) of cultured human umbilical vein endothelial cells was only partially prevented by Calin, and even less so at a high-shear rather than a low-shear rate, whereas the platelet binding to coated vWF and fibrinogen were minimally affected at both shear rates. Thus, Calin interferes with both the direct platelet- collagen interaction and the vWF-collagen binding. Both effects may contribute to the inhibition of platelet adhesion in flowing conditions, although the former seems to predominate.

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Plaque-prone hemodynamics impair endothelial function in pig carotid arteries

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00486.2005 ◽

2006 ◽

Vol 290 (6) ◽

pp. H2320-H2328 ◽

Cited By ~ 69

Author(s):

Veronica Gambillara ◽

Céline Chambaz ◽

Gabriela Montorzi ◽

Sylvain Roy ◽

Nikos Stergiopulos ◽

...

Keyword(s):

Gene Expression ◽

Nitric Oxide ◽

Endothelial Cells ◽

Shear Stress ◽

Endothelial Dysfunction ◽

Oscillatory Shear ◽

Enos Gene ◽

Low Shear Stress ◽

Oscillatory Shear Stress ◽

Low Shear

Hemodynamic forces play an active role in vascular pathologies, particularly in relation to the localization of atherosclerotic lesions. It has been established that low shear stress combined with cyclic reversal of flow direction (oscillatory shear stress) affects the endothelial cells and may lead to an initiation of plaque development. The aim of the study was to analyze the effect of hemodynamic conditions in arterial segments perfused in vitro in the absence of other stimuli. Left common porcine carotid segments were mounted into an ex vivo arterial support system and perfused for 3 days under unidirectional high and low shear stress (6 ± 3 and 0.3 ± 0.1 dyn/cm2) and oscillatory shear stress (0.3 ± 3 dyn/cm2). Bradykinin-induced vasorelaxation was drastically decreased in arteries exposed to oscillatory shear stress compared with unidirectional shear stress. Impaired nitric oxide-mediated vasodilation was correlated to changes in both endothelial nitric oxide synthase (eNOS) gene expression and activation in response to bradykinin treatment. This study determined the flow-mediated effects on native tissue perfused with physiologically relevant flows and supports the hypothesis that oscillatory shear stress is a determinant factor in early stages of atherosclerosis. Indeed, oscillatory shear stress induces an endothelial dysfunction, whereas unidirectional shear stress preserves the function of endothelial cells. Endothelial dysfunction is directly mediated by a downregulation of eNOS gene expression and activation; consequently, a decrease of nitric oxide production and/or bioavailability occurs.

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

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvbaha.120.314960 ◽

2020 ◽

Vol 40 (11) ◽

pp. 2678-2685

Author(s):

Mohammed N. Meah ◽

Jennifer Raftis ◽

Simon J. Wilson ◽

Vidya Perera ◽

Samira M. Garonzik ◽

...

Keyword(s):

Shear Stress ◽

Low Dose ◽

Ex Vivo ◽

Thrombus Formation ◽

Factor Xa ◽

High Dose ◽

High Shear ◽

High Shear Stress ◽

Factor Xa Inhibition ◽

Low Shear

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

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