Construction of a liver sinusoid based on the laminar flow on chip and self-assembly of endothelial cells

Rationale: In diabetic animals as well as high glucose cell culture conditions, endothelial nitric oxide synthase (eNOS) is heavily O-GlcNAcylated, which inhibits its phosphorylation and nitric oxide (NO) production. It is unknown, however, whether varied blood flow conditions, which affect eNOS phosphorylation, modulate eNOS activity via O-GlcNAcylation-dependent mechanisms. Objective: The goal of this study was to test if steady laminar flow, but not oscillating disturbed flow, decreases eNOS O-GlcNAcylation, thereby elevating eNOS phosphorylation and NO production. Methods and Results: Human umbilical vein endothelial cells (HUVEC) were exposed to either laminar flow (20 dynes/cm2 shear stress) or oscillating disturbed flow (4{plus minus}6 dynes/cm2 shear stress) for 24 hours in a cone-and-plate device. eNOS O-GlcNAcylation was almost completely abolished in cells exposed to steady laminar but not oscillating disturbed flow. Interestingly, there was no change in protein level or activity of key O-GlcNAcylation enzymes (OGT, OGA, or GFAT). Instead, metabolomics data suggest that steady laminar flow decreases glycolysis and hexosamine biosynthetic pathway (HBP) activity, thereby reducing UDP-GlcNAc pool size and consequent O-GlcNAcylation. Inhibition of glycolysis via 2-deoxy-2-glucose (2-DG) in cells exposed to disturbed flow efficiently decreased eNOS O-GlcNAcylation, thereby increasing eNOS phosphorylation and NO production. Finally, we detected significantly higher O-GlcNAcylated proteins in endothelium of the inner aortic arch in mice, suggesting that disturbed flow increases protein O-GlcNAcylation in vivo. Conclusions: Our data demonstrate that steady laminar but not oscillating disturbed flow decreases eNOS O-GlcNAcylation by limiting glycolysis and UDP-GlcNAc substrate availability, thus enhancing eNOS phosphorylation and NO production. This research shows for the first time that O-GlcNAcylation is regulated by mechanical stimuli, relates flow-induced glycolytic reductions to macrovascular disease, and highlights targeting HBP metabolic enzymes in endothelial cells as a novel therapeutic strategy to restore eNOS activity and prevent EC dysfunction in cardiovascular disease.

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Abstract 660: Local Hemodynamic Forces and Aqueous Cigarette Smoke Extract Affect Development of Endothelial Dysfunction

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.34.suppl_1.660 ◽

2014 ◽

Vol 34 (suppl_1) ◽

Author(s):

Sindy Giebe ◽

Coy Brunssen ◽

Melanie Brux ◽

Natalia Cockcroft ◽

Katherine Hewitt ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Dysfunction ◽

Laminar Flow ◽

Tobacco Smoking ◽

Molecular Mechanisms ◽

Low Flow ◽

Dependent Manner ◽

Flow Conditions ◽

Hemodynamic Forces ◽

Plaque Phenotype

Endothelial dysfunction is one of the first steps in the development of atherosclerosis. This proinflammatory phenotype is associated with decreased bioavailability of nitric oxide and a corresponding expression profile in the endothelial cells. Tobacco smoking promotes development of atherosclerotic plaques and local hemodynamic forces are key stimuli in this process. Low laminar flow is involved in the development of an unstable plaque phenotype, while high laminar flow has atheroprotective role. The molecular mechanisms controlling plaque stability in response to tobacco smoking remain largely unknown so far. Therefore, we exposed human endothelial cells to cigarette smoke extract (CSEaq) under disturbed flow conditions. Primary human endothelial cells were stimulated with increasing dosages of CSEaq for 24h. Cell viability was reduced by CSEaq in a dose-dependent manner. The impact of specific flow conditions and different doses of CSEaq on the expression of atherosclerosis-related genes was investigated using a cone-and-plate viscometer. High laminar flow induced elongation of endothelial cells in the direction of flow, increased eNOS expression and NO release in a time-dependent manner. This increase was inhibited by CSEaq. Low laminar flow showed no effect on eNOS expression and NO release. The NRF2 antioxidative defense system was also induced by high laminar flow. NRF2 and NRF2 target genes HMOX1 and NQO1 were strongly activated by CSEaq. Furthermore, we monitored the expression of proinflammatory genes. CSEaq strongly induced adhesion molecule ICAM-1. Interestingly, VCAM-1 was unaffected by CSEaq. Induction of endothelial NADPH oxidase isoform 4 by CSEaq was prevented by high laminar flow. Catalase expression was not affected by flow and CSEaq, whereas CSEaq transiently increased SOD1 expression. Endothelial wound healing was improved by atheroprotective high laminar flow. Low flow did not affect wound healing. Furthermore, high laminar flow decreased adhesion of monocytes to endothelial cells, compared to low flow. We suggest novel molecular mechanisms how tobacco smoking promotes the development of endothelial dysfunction. This can contribute to the formation of an unstable atherosclerotic plaque phenotype.

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Pleiotropic effects of laminar flow and statins depend on the Krüppel-like factor-induced lncRNA MANTIS

European Heart Journal ◽

10.1093/eurheartj/ehz393 ◽

2019 ◽

Vol 40 (30) ◽

pp. 2523-2533 ◽

Cited By ~ 22

Author(s):

Matthias S Leisegang ◽

Sofia-Iris Bibli ◽

Stefan Günther ◽

Beatrice Pflüger-Müller ◽

James A Oo ◽

...

Keyword(s):

Endothelial Cells ◽

Transcription Factors ◽

Laminar Flow ◽

Therapeutic Potential ◽

Statin Treatment ◽

Telomerase Activity ◽

Dependent Manner ◽

Protective Effects ◽

Monocyte Adhesion ◽

Beneficial Effects

Abstract Aims To assess the functional relevance and therapeutic potential of the pro-angiogenic long non-coding RNA MANTIS in vascular disease development. Methods and results RNA sequencing, CRISPR activation, overexpression, and RNAi demonstrated that MANTIS, especially its Alu-element, limits endothelial ICAM-1 expression in different types of endothelial cells. Loss of MANTIS increased endothelial monocyte adhesion in an ICAM-1-dependent manner. MANTIS reduced the binding of the SWI/SNF chromatin remodelling factor BRG1 at the ICAM-1 promoter. The expression of MANTIS was induced by laminar flow and HMG-CoA-reductase inhibitors (statins) through mechanisms involving epigenetic rearrangements and the transcription factors KLF2 and KLF4. Mutation of the KLF binding motifs in the MANTIS promoter blocked the flow-induced MANTIS expression. Importantly, the expression of MANTIS in human carotid artery endarterectomy material was lower compared with healthy vessels and this effect was prevented by statin therapy. Interestingly, the protective effects of statins were mediated in part through MANTIS, which was required to facilitate the atorvastatin-induced changes in endothelial gene expression. Moreover, the beneficial endothelial effects of statins in culture models (spheroid outgrowth, proliferation, telomerase activity, and vascular organ culture) were lost upon knockdown of MANTIS. Conclusion MANTIS is tightly regulated by the transcription factors KLF2 and KLF4 and limits the ICAM-1 mediated monocyte adhesion to endothelial cells and thus potentially atherosclerosis development in humans. The beneficial effects of statin treatment and laminar flow are dependent on MANTIS.

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Fluid shear stress modulates surface expression of adhesion molecules by endothelial cells

Blood ◽

10.1182/blood.v85.7.1696.bloodjournal8571696 ◽

1995 ◽

Vol 85 (7) ◽

pp. 1696-1703 ◽

Cited By ~ 138

Author(s):

M Morigi ◽

C Zoja ◽

M Figliuzzi ◽

M Foppolo ◽

G Micheletti ◽

...

Keyword(s):

Endothelial Cells ◽

Shear Stress ◽

Laminar Flow ◽

Fluid Shear Stress ◽

Leukocyte Adhesion ◽

Interleukin 1 ◽

Static Condition ◽

Surface Expression ◽

Cell Surface Expression ◽

Interleukin 1 Beta

We investigated the effect of hemodynamic shear forces on the expression of adhesive molecules, E-selectin, and intercellular adhesion molecule-1 (ICAM-1) on human umbilical vein endothelial cells (HUVEC) exposed to laminar (8 dynes/cm2) or turbulent shear stress (8.6 dynes/cm2 average), or to a static condition. Laminar flow induced a significant time-dependent increase in the surface expression of ICAM-1, as documented by flow cytometry studies. Endothelial cell surface expression of ICAM-1 in supernatants of HUVEC exposed to laminar flow was not modified, excluding the possibility that HUVEC exposed to laminar flow synthetize factors that upregulate ICAM-1. The effect of laminar flow was specific for ICAM-1, while E-selectin expression was not modulated by the flow condition. Turbulent flow did not affect surface expression of either E-selectin or ICAM-1. To evaluate the functional significance of the laminar-flow-induced increase in ICAM-1 expression, we studied the dynamic interaction of total leukocyte suspension with HUVEC exposed to laminar flow (8 dynes/cm2 for 6 hours) in a parallel-plate flow chamber or to static condition. Leukocyte adhesion to HUVEC pre-exposed to flow was significantly enhanced, compared with HUVEC maintained in static condition (233 +/- 67 v 43 +/- 16 leukocytes/mm2, respectively), and comparable with that of interleukin-1 beta treated HUVEC. Mouse monoclonal antibody anti-ICAM-1 completely blocked flow-induced upregulation of leukocyte adhesion. Interleukin-1 beta, which upregulated E-selectin expression, caused leukocyte rolling on HUVEC that was significantly lower on flow- conditioned HUVEC and almost absent on untreated static endothelial cells. Thus, laminar flow directly and selectively upregulates ICAM-1 expression on the surface of endothelial cells and promotes leukocyte adhesion. These data are relevant to the current understanding of basic mechanisms that govern local inflammatory reactions and tissue injury.

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On-chip integrated differential optical microring refractive index sensing platform based on a laminar flow scheme

Optics Letters ◽

10.1364/ol.40.004106 ◽

2015 ◽

Vol 40 (17) ◽

pp. 4106 ◽

Cited By ~ 13

Author(s):

Dongwan Kim ◽

Paula Popescu ◽

Mark Harfouche ◽

Jacob Sendowski ◽

Maria-Eleni Dimotsantou ◽

...

Keyword(s):

Refractive Index ◽

Laminar Flow ◽

Flow Scheme ◽

Refractive Index Sensing ◽

Sensing Platform ◽

On Chip

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HDAC3 is crucial in shear- and VEGF-induced stem cell differentiation toward endothelial cells

The Journal of Cell Biology ◽

10.1083/jcb.200605113 ◽

2006 ◽

Vol 174 (7) ◽

pp. 1059-1069 ◽

Cited By ~ 175

Author(s):

Lingfang Zeng ◽

Qingzhong Xiao ◽

Andriana Margariti ◽

Zhongyi Zhang ◽

Anna Zampetaki ◽

...

Keyword(s):

Endothelial Cells ◽

Shear Stress ◽

Stem Cell ◽

Cell Differentiation ◽

Laminar Flow ◽

Progenitor Cell ◽

Stem Cell Differentiation ◽

Es Cell ◽

Local Flow ◽

Proliferation And Differentiation

Reendothelialization involves endothelial progenitor cell (EPC) homing, proliferation, and differentiation, which may be influenced by fluid shear stress and local flow pattern. This study aims to elucidate the role of laminar flow on embryonic stem (ES) cell differentiation and the underlying mechanism. We demonstrated that laminar flow enhanced ES cell–derived progenitor cell proliferation and differentiation into endothelial cells (ECs). Laminar flow stabilized and activated histone deacetylase 3 (HDAC3) through the Flk-1–PI3K–Akt pathway, which in turn deacetylated p53, leading to p21 activation. A similar signal pathway was detected in vascular endothelial growth factor–induced EC differentiation. HDAC3 and p21 were detected in blood vessels during embryogenesis. Local transfer of ES cell–derived EPC incorporated into injured femoral artery and reduced neointima formation in a mouse model. These data suggest that shear stress is a key regulator for stem cell differentiation into EC, especially in EPC differentiation, which can be used for vascular repair, and that the Flk-1–PI3K–Akt–HDAC3–p53–p21 pathway is crucial in such a process.

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Structure of artificial and natural VE-cadherinbased adherens junctions

Biochemical Society Transactions ◽

10.1042/bst0360189 ◽

2008 ◽

Vol 36 (2) ◽

pp. 189-193 ◽

Cited By ~ 24

Author(s):

Jean-Christophe Taveau ◽

Mathilde Dubois ◽

Olivier Le Bihan ◽

Sylvain Trépout ◽

Sébastien Almagro ◽

...

Keyword(s):

Endothelial Cells ◽

Actin Cytoskeleton ◽

Self Assembly ◽

Adherens Junctions ◽

Actin Binding ◽

Vascular Endothelial ◽

Vascular Integrity ◽

Trans Orientation ◽

Annexin 2

In vascular endothelium, adherens junctions between endothelial cells are composed of VE-cadherin (vascular endothelial cadherin), an adhesive receptor that is crucial for the proper assembly of vascular structures and the maintenance of vascular integrity. As a classical cadherin, VE-cadherin links endothelial cells together by homophilic interactions mediated by its extracellular part and associates intracellularly with the actin cytoskeleton via catenins. Although, from structural crystallographic data, a dimeric structure arranged in a trans orientation has emerged as a potential mechanism of cell–cell adhesion, the cadherin organization within adherens junctions remains controversial. Concerning VE-cadherin, its extracellular part possesses the capacity to self-associate in solution as hexamers consisting of three antiparallel cadherin dimers. VE-cadherin-based adherens junctions were reconstituted in vitro by assembly of a VE-cadherin EC (extracellular repeat) 1–EC4 hexamer at the surfaces of liposomes. The artificial adherens junctions revealed by cryoelectron microscopy appear as a two-dimensional self-assembly of hexameric structures. This cadherin organization is reminiscent of that found in native desmosomal junctions. Further structural studies performed on native VE-cadherin junctions would provide a better understanding of the cadherin organization within adherens junctions. Homophilic interactions between cadherins are strengthened intracellularly by connection to the actin cytoskeleton. Recently, we have discovered that annexin 2, an actin-binding protein connects the VE-cadherin–catenin complex to the actin cytoskeleton. This novel link is labile and promotes the endothelial cell switch from a quiescent to an angiogenic state.

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