Abstract 15187: Shear Stress-regulated Mir-27b Controls Pericyte Recruitment
            in vitro
            and
            in vivo
            and Contributes to Vessel Maturation

Vessel maturation involves recruitment of mural cells. Laminar shear stress is a major trigger for vessel maturation. However, the molecular mechanisms by which shear stress affects recruitment of pericytes are unclear. MicroRNAs are small non-coding RNAs, which post-transcriptionally control gene expression. Since shear stress regulates various miRs, we hypothesize that flow-induced miRs inhibit repulsive cues and facilitate mural cell coverage. Laminar shear stress for 72h induces the up-regulation of miR-27b (2.8±0.24-fold vs static, p<0.05) in cultured endothelial cells (ECs) and in mouse femoral artery segments that were exposed to physiological shear stress ex vivo (1.5±0.14-fold vs no flow, p<0.05). Predicted targets for miR-27b include members of the semaphorin (SEMA) family (known to regulate repulsive signaling) and angiopoietin-2 (Ang2), which causes vessel destabilization. MiR-27b overexpression reduces SEMA6A (63.5±13.5%), SEMA6D (58±26%), and Ang2 (51.5±11%) expression. To determine whether miR-27b controls pericyte recruitment, we tested the effect of endothelial miR-27b on adhesion of pericytes. Overexpression of miR-27b increased the adhesion of pericytes (p<0.05) and inhibition of miR-27b reduced pericyte adhesion in vitro (p<0.05). In an in vitro matrigel assay, overexpression of miR-27b increased pericyte coverage of endothelial cell tubes (155±45%, p<0.05), whereas inhibition of miR-27b reduced pericyte coverage (81±15%). In addition, overexpression of miR-27b in vitro increases barrier function (p<0.05) in endothelial cell-pericyte co-culture assays whereas inhibition of miR-27b decreases barrier function (p<0.05). Silencing of SEMA6A and SEMA6D rescued the reduced pericyte adhesion that is caused by miR-27 inhibition (p<0.05). Furthermore, inhibition of SEMA6D increases barrier function. In vivo inhibition of miR-27a/b significantly increased edema (p<0.05) in the uterus which was associated with a significantly reduced pericyte coverage of the vessels (p<0.05). Taken together, our data demonstrate that shear stress regulate miR-27b, which targets Ang2 and semaphorins, thereby controlling pericyte adhesion and coverage in vitro and in vivo.

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Endothelial albumin permeability is shear dependent, time dependent, and reversible

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1991.260.6.h1992 ◽

1991 ◽

Vol 260 (6) ◽

pp. H1992-H1996 ◽

Cited By ~ 32

Author(s):

H. Jo ◽

R. O. Dull ◽

T. M. Hollis ◽

J. M. Tarbell

Keyword(s):

Shear Stress ◽

Endothelial Cell ◽

Fluorescein Isothiocyanate ◽

Vascular Wall ◽

Laminar Shear Stress ◽

Aortic Endothelial Cells ◽

Transendothelial Permeability ◽

Steady Laminar Shear Stress ◽

Laminar Shear

Altered permeability of vascular endothelium to macromolecules may play a role in vascular disease as well as vascular homeostasis. Because the shear stress of flowing blood on the vascular wall is known to influence many endothelial cell properties, an in vitro system to measure transendothelial permeability (Pe) to fluorescein isothiocyanate conjugated bovine serum albumin under defined physiological levels of steady laminar shear stress was developed. Bovine aortic endothelial cells grown on polycarbonate filters pretreated with gelatin and fibronectin constituted the model system. Onset of 1 dyn/cm2 shear stress resulted in a Pe rise from 5.1 +/- 1.3 x 10(-6) cm/s to 21.9 +/- 4.6 X 10(-6) cm/s at 60 min (n = 6); while 10 dyn/cm2 shear stress increased Pe from 4.8 +/- 1.5 X 10(-6) cm/s to 50.2 +/- 6.8 X 10(-6) cm/s at 30 min and 49.6 +/- 8.9 X 10(-6) cm/s at 60 (n = 9). Pe returned to preshear values within 120 and 60 min after removal of 1 and 10 dyn/cm2 shear stress, respectively. The data show that endothelial cell Pe in vitro is acutely sensitive to shear stress.

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Resveratrol promotes endothelial cell wound healing under laminar shear stress through an estrogen receptor-α-dependent pathway

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00892.2013 ◽

2014 ◽

Vol 306 (6) ◽

pp. H797-H806 ◽

Cited By ~ 17

Author(s):

Arif Yurdagul ◽

James J. Kleinedler ◽

Marshall C. McInnis ◽

Alok R. Khandelwal ◽

Allyson L. Spence ◽

...

Keyword(s):

Cell Proliferation ◽

Wound Healing ◽

Shear Stress ◽

Estrogen Receptor ◽

Endothelial Cell ◽

Endothelial Cell Proliferation ◽

Laminar Shear Stress ◽

Endothelial Cell Growth ◽

Laminar Shear

Restenosis is an adverse outcome of angioplasty, characterized by vascular smooth muscle cell (VSMC) hyperplasia. However, therapies targeting VSMC proliferation delay reendothelialization, increasing the risk of thrombosis. Resveratrol (RESV) inhibits restenosis and promotes reendothelialization after arterial injury, but in vitro studies assessing RESV-mediated effects on endothelial cell growth contradict these findings. We thus hypothesized that fluid shear stress, mimicking physiological blood flow, would recapitulate RESV-dependent endothelial cell wound healing. Since RESV is an estrogen receptor (ER) agonist, we tested whether RESV promotes reendothelialization through an ER-α-dependent mechanism. Mice fed a high-fat diet or a diet supplemented with RESV were subjected to carotid artery injury. At 7 days after injury, RESV significantly accelerated reendothelialization compared with vehicle. In vitro wound healing assays demonstrated that RESV exhibits cell-type selectivity, inhibiting VSMC, but not endothelial cell growth. Under laminar shear stress (LSS), RESV dramatically enhanced endothelial cell wound healing and increased both the activation of extracellular signal-regulated kinase (ERK) and endothelial cell proliferation. Under LSS, small interfering RNA against ER-α, but not endothelial nitric oxide synthase, abolished RESV-induced ERK activation, endothelial cell proliferation, and wound healing. Thus these studies suggest that the EC phenotype induced by LSS better models the prohealing effects of RESV and that RESV and LSS interact to promote an ER-α-dependent mitogenic effect in endothelial cells.

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Effects of In Vitro Laminar Shear Stress as an Exercise Mimetic on Endothelial Cell Health

Molecular and Translational Medicine - Effects of Exercise on Hypertension ◽

10.1007/978-3-319-17076-3_7 ◽

2015 ◽

pp. 157-184

Author(s):

Michael D. Brown ◽

Joon-Young Park

Keyword(s):

Shear Stress ◽

Endothelial Cell ◽

Laminar Shear Stress ◽

Laminar Shear

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Shear Stress Regulation of Endothelial Glycocalyx Structure Is Determined by Glucobiosynthesis

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvbaha.119.313399 ◽

2020 ◽

Vol 40 (2) ◽

pp. 350-364 ◽

Cited By ~ 8

Author(s):

Gangqi Wang ◽

Sarantos Kostidis ◽

Gesa L. Tiemeier ◽

Wendy M.P.J. Sol ◽

Margreet R. de Vries ◽

...

Keyword(s):

Shear Stress ◽

Glucuronic Acid ◽

Functional Adaptation ◽

Endothelial Glycocalyx ◽

Nuclear Magnetic Resonance Analysis ◽

Laminar Shear Stress ◽

Major Structural Component ◽

Laminar Shear

Objective: Endothelial cells exposed to laminar shear stress express a thick glycocalyx on their surface that plays an important role in reducing vascular permeability and endothelial anti-inflammatory, antithrombotic, and antiangiogenic properties. Production and maintenance of this glycocalyx layer is dependent on cellular carbohydrate synthesis, but its regulation is still unknown. Approach and Results: Here, we show that biosynthesis of the major structural component of the endothelial glycocalyx, hyaluronan, is regulated by shear. Both in vitro as well as in in vivo, hyaluronan expression on the endothelial surface is increased on laminar shear and reduced when exposed to oscillatory flow, which is regulated by KLF2 (Krüppel-like Factor 2). Using a CRISPR-CAS9 edited small tetracysteine tag to endogenous HAS2 (hyaluronan synthase 2), we demonstrated increased translocation of HAS2 to the endothelial cell membrane during laminar shear. Hyaluronan production by HAS2 was shown to be further driven by availability of the hyaluronan substrates UDP-glucosamine and UDP-glucuronic acid. KLF2 inhibits endothelial glycolysis and allows for glucose intermediates to shuttle into the hexosamine- and glucuronic acid biosynthesis pathways, as measured using nuclear magnetic resonance analysis in combination with 13 C-labeled glucose. Conclusions: These data demonstrate how endothelial glycocalyx function and functional adaptation to shear is coupled to KLF2-mediated regulation of endothelial glycolysis.

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Abstract 116: Arterial Endothelial Cell Has Stronger Angiogenic Potential Compared To Venous Endothelial Cell Through Up-regulation Of Endogenous FGF2 And FGF5 Expression In 3D Microfluidic System

Circulation Research ◽

10.1161/res.115.suppl_1.116 ◽

2014 ◽

Vol 115 (suppl_1) ◽

Author(s):

Ha-Rim Seo ◽

Hyo Eun Jeong ◽

Hyung Joon Joo ◽

Seung-Cheol Choi ◽

Jong-Ho Kim ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Molecular Mechanisms ◽

Umbilical Vein ◽

Assay System ◽

Vascular Density ◽

Angiogenic Potential ◽

Angiogenesis Assay

Background: Human body contains many kinds of different type of endothelial cells (EC). However, cellular difference of their angiogenic potential has been hardly understood. We compared in vitro angiogenic potential between arterial EC and venous EC and investigated its underlying molecular mechanisms. Method: Used human aortic endothelial cells (HAEC) which was indicated from arterial EC and human umbilical vein endothelial cells (HUVEC) indicated from venous EC. To explore angiogenic potential in detail, we adopted a novel 3D microfluidic angiogenesis assay system, which closely mimic in vivo angiogenesis. Results: In 3D microfluidic angiogenesis assay system, HAEC demonstrated stronger angiogenic potential compared to HUVEC. HAEC maintained its profound angiogenic property under different biophysical conditions. In mRNA microarray sorted on up- regulated or down-regulated genes, HAEC demonstrated significantly higher expression of gastrulation brain homeobox 2 (GBX2), fibroblast grow factor 2 (FGF2), FGF5 and collagen 8a1. Angiogenesis-related protein assay revealed that HAEC has higher secretion of endogenous FGF2 than HUVEC. HAEC has only up-regulated FGF2 and FGF5 in this part of FGF family. Furthermore, FGF5 expression under vascular endothelial growth factor-A (VEGF-A) stimulation was higher in HAEC compared to HUVEC although VEGF-A augmented FGF5 expression in both HAEC and HUVEC. Those data suggested that FGF5 expression in both HAEC and HUVEC is partially dependent to VEGF-A stimulate. HUVEC and HAEC reduced vascular density after FGF2 and FGF5 siRNA treat. Conclusion: HAEC has stronger angiogenic potential than HUVEC through up-regulation of endogenous FGF2 and FGF5 expression

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Laminar Shear Stress Inhibits Endothelial Cell Metabolism via KLF2-Mediated Repression of PFKFB3

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvbaha.114.304277 ◽

2015 ◽

Vol 35 (1) ◽

pp. 137-145 ◽

Cited By ~ 117

Author(s):

Anuradha Doddaballapur ◽

Katharina M. Michalik ◽

Yosif Manavski ◽

Tina Lucas ◽

Riekelt H. Houtkooper ◽

...

Keyword(s):

Shear Stress ◽

Endothelial Cell ◽

Cell Metabolism ◽

Laminar Shear Stress ◽

Laminar Shear ◽

Endothelial Cell Metabolism

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SENCR stabilizes vascular endothelial cell adherens junctions through interaction with CKAP4

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1810729116 ◽

2018 ◽

Vol 116 (2) ◽

pp. 546-555 ◽

Cited By ~ 28

Author(s):

Qing Lyu ◽

Suowen Xu ◽

Yuyan Lyu ◽

Mihyun Choi ◽

Christine K. Christie ◽

...

Keyword(s):

Shear Stress ◽

Endothelial Cell ◽

Noncoding Rna ◽

Rna Binding ◽

Adherens Junctions ◽

Vascular Endothelial Cell ◽

Membrane Integrity ◽

Laminar Shear Stress ◽

Loss Of Function ◽

Laminar Shear

SENCR is a human-specific, vascular cell-enriched long-noncoding RNA (lncRNA) that regulates vascular smooth muscle cell and endothelial cell (EC) phenotypes. The underlying mechanisms of action of SENCR in these and other cell types is unknown. Here, levels of SENCR RNA are shown to be elevated in several differentiated human EC lineages subjected to laminar shear stress. Increases in SENCR RNA are also observed in the laminar shear stress region of the adult aorta of humanized SENCR-expressing mice, but not in disturbed shear stress regions. SENCR loss-of-function studies disclose perturbations in EC membrane integrity resulting in increased EC permeability. Biotinylated RNA pull-down and mass spectrometry establish an abundant SENCR-binding protein, cytoskeletal-associated protein 4 (CKAP4); this ribonucleoprotein complex was further confirmed in an RNA immunoprecipitation experiment using an antibody to CKAP4. Structure–function studies demonstrate a noncanonical RNA-binding domain in CKAP4 that binds SENCR. Upon SENCR knockdown, increasing levels of CKAP4 protein are detected in the EC surface fraction. Furthermore, an interaction between CKAP4 and CDH5 is enhanced in SENCR-depleted EC. This heightened association appears to destabilize the CDH5/CTNND1 complex and augment CDH5 internalization, resulting in impaired adherens junctions. These findings support SENCR as a flow-responsive lncRNA that promotes EC adherens junction integrity through physical association with CKAP4, thereby stabilizing cell membrane-bound CDH5.

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Endothelial Barrier Function is co-regulated at Vessel Bifurcations by Fluid Forces and Sphingosine-1-Phosphate

10.1101/2020.08.18.256586 ◽

2020 ◽

Author(s):

Ehsan Akbari ◽

Griffin B. Spychalski ◽

Miles M. Menyhert ◽

Kaushik K. Rangharajan ◽

Shaurya Prakash ◽

...

Keyword(s):

Shear Stress ◽

Fluid Flow ◽

Barrier Function ◽

Endothelial Barrier ◽

Laminar Shear Stress ◽

Endothelial Barrier Function ◽

Fluid Forces ◽

Sphingosine 1 Phosphate ◽

Laminar Shear ◽

Vessel Bifurcations

AbstractSphingosine-1-phosphate (S1P) is a blood-borne bioactive lipid mediator of endothelial barrier function. Prior studies have implicated mechanical stimulation due to intravascular laminar shear stress in co-regulating S1P signaling in endothelial cells (ECs). Yet, vascular networks in vivo consist of vessel bifurcations, and this geometry produces hemodynamic forces that are distinct from laminar shear stress. However, the role of these forces at vessel bifurcations in regulating S1P-dependent endothelial barrier function is not known. In this study, we implemented a microfluidic platform that recapitulates the flow dynamics of vessel bifurcations with in situ quantification of the permeability of microvessel analogues. Co-application of S1P with impinging bifurcated fluid flow, which was characterized by approximately zero shear stress and 38 dyn cm-2 stagnation pressure at the vessel bifurcation point, promotes vessel stabilization. Similarly, co-treatment of carrier-free S1P with 3 dyn cm-2 laminar shear stress is also protective of endothelial barrier function. Moreover, it is shown that vessel stabilization due to laminar shear stress, but not bifurcated fluid flow, is dependent on S1P receptor 1 or 2 signaling. Collectively, these findings demonstrate the endothelium-protective function of fluid forces at vessel bifurcations and their involvement in coordinating S1P-dependent regulation of vessel permeability.

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