scholarly journals Laminar shear stress upregulates endothelial Ca2+-activated K+ channels KCa2.3 and KCa3.1 via a Ca2+/calmodulin-dependent protein kinase kinase/Akt/p300 cascade

2013 ◽  
Vol 305 (4) ◽  
pp. H484-H493 ◽  
Author(s):  
Jun Takai ◽  
Alexandra Santu ◽  
Haifeng Zheng ◽  
Sang Don Koh ◽  
Masanori Ohta ◽  
...  
Keyword(s):  
Shear Stress ◽  
Transcriptional Activation ◽  
Static Condition ◽  
Fold Increase ◽  
Laminar Shear Stress ◽  
Human Coronary Artery ◽  
Intracellular Ca ◽  
Kinase Cascade ◽  
Dependent Protein ◽  
Laminar Shear

In endothelial cells (ECs), Ca2+-activated K+ channels KCa2.3 and KCa3.1 play a crucial role in the regulation of arterial tone via producing NO and endothelium-derived hyperpolarizing factors. Since a rise in intracellular Ca2+ levels and activation of p300 histone acetyltransferase are early EC responses to laminar shear stress (LS) for the transcriptional activation of genes, we examined the role of Ca2+/calmodulin-dependent kinase kinase (CaMKK), the most upstream element of a Ca2+/calmodulin-kinase cascade, and p300 in LS-dependent regulation of KCa2.3 and KCa3.1 in ECs. Exposure to LS (15 dyn/cm2) for 24 h markedly increased KCa2.3 and KCa3.1 mRNA expression in cultured human coronary artery ECs (3.2 ± 0.4 and 45 ± 10 fold increase, respectively; P < 0.05 vs. static condition; n = 8–30), whereas oscillatory shear (OS; ± 5 dyn/cm2 × 1 Hz) moderately increased KCa3.1 but did not affect KCa2.3. Expression of KCa2.1 and KCa2.2 was suppressed under both LS and OS conditions, whereas KCa1.1 was slightly elevated in LS and unchanged in OS. Inhibition of CaMKK attenuated LS-induced increases in the expression and channel activity of KCa2.3 and KCa3.1, and in phosphorylation of Akt (Ser473) and p300 (Ser1834). Inhibition of Akt abolished the upregulation of these channels by diminishing p300 phosphorylation. Consistently, disruption of the interaction of p300 with transcription factors eliminated the induction of these channels. Thus a CaMKK/Akt/p300 cascade plays an important role in LS-dependent induction of KCa2.3 and KCa3.1 expression, thereby regulating EC function and adaptation to hemodynamic changes.

scholarly journals Role of Myoendothelial Gap Junctions in the Regulation of Human Coronary Artery Smooth Muscle Cell Differentiation by Laminar Shear Stress

2016 ◽  
Vol 39 (2) ◽  
pp. 423-437 ◽  
Author(s):  
Zongqi Zhang ◽  
Yizhu Chen ◽  
Tiantian Zhang ◽  
Lingyu Guo ◽  
Wenlong Yang ◽  
...  
Keyword(s):  
Shear Stress ◽  
Smooth Muscle ◽  
Coronary Artery ◽  
Gap Junctions ◽  
Phenotypic Switching ◽  
Laminar Shear Stress ◽  
Human Coronary Artery ◽  
Laminar Shear ◽  
Synthetic Phenotype

Background/Aims: Smooth muscle cells may dedifferentiate into the synthetic phenotype and promote atherosclerosis. Here, we explored the role of myoendothelial gap junctions in phenotypic switching of human coronary artery smooth muscle cells (HCASMCs) co-cultured with human coronary artery endothelial cells (HCAECs) exposed to shear stress. Methods: HCASMCs and HCAECs were seeded on opposite sides of Transwell inserts, and HCAECs were exposed to laminar shear stress of 12 dyn/cm2 or 5 dyn/cm2. The myoendothelial gap junctions were evaluated by using a multi-photon microscope. Results: In co-culture with HCAECs, HCASMCs exhibited a contractile phenotype, and maintained the expression of differentiation markers MHC and H1-calponin. HCASMCs and HCAECs formed functional intercellular junctions, as evidenced by colocalization of connexin(Cx)40 and Cx43 on cellular projections inside the Transwell membrane and biocytin transfer from HCAECs to HCASMCs. Cx40 siRNA and 18-α-GA attenuated protein expression of MHC and H1-calponin in HCASMCs. Shear stress of 5 dyn/cm2 increased Cx43 and decreased Cx40 expression in HCAECs, and partly inhibited biocytin transfer from HCAECs to HCASMCs, which could be completely blocked by Cx43 siRNA or restored by Cx40 DNA transfected into HCAECs. The exposure of HCAECs to shear stress of 5 dyn/cm2 promoted HCASMC phenotypic switching, manifested by morphological changes, decrease in MHC and H1-calponin expression, and increase in platelet-derived growth factor (PDGF)-BB release, which was partly rescued by Cx43 siRNA or Cx40 DNA or PDGF receptor signaling inhibitor. Conclusions: The exposure of HCAECs to shear stress of 5 dyn/cm2 caused the dysfunction of Cx40/Cx43 heterotypic myoendothelial gap junctions, which may be replaced by homotypic Cx43/Cx43 channels, and induced HCASMC transition to the synthetic phenotype associated with the activation of PDGF receptor signaling, which may contribute to shear stress-associated arteriosclerosis.

Abstract 255: Laminar Shear Stress Enhances the Activity and Localization of RNA Polymerase II on the eNOS Gene

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Martina Weber ◽  
Jeffrey P Moore ◽  
Charles D Searles
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Fold Increase ◽  
Mrna Processing ◽  
Enos Gene ◽  
Laminar Shear Stress ◽  
Enos Mrna ◽  
Rnap Ii ◽  
Induced Changes ◽  
Laminar Shear

We have previously found that laminar shear enhances eNOS mRNA stability and translation by altering endothelial NO synthase (eNOS) mRNA 3′ polyadenylation. Transcription is tightly coupled to pre-mRNA processing, and is coordinated by RNA polymerase (RNAP) II. We assessed whether laminar shear stress alters the activity and localization of RNAP II on the eNOS gene. We found that endothelial cells exposed to laminar shear stress had a 2-fold (n=3, p < 0.05) increase in total RNAP II protein levels compared to control; this effect was dose-dependent (0–15 dynes/cm2). Since three different RNAP II phosphoisoforms are associated with different stages of mRNA synthesis, we examined whether shear stress affected protein expression of these particular phosphoisoforms. We found that cells exposed to laminar shear had a 3-fold increase (p<0.05) in expression of RNAP II phosphorylated at serine 2, which is associated with transcription elongation and 3′ polyadenylation. In contrast, shear stress did not alter expression of the other phosphoisoforms. We performed chromatin immunoprecipitation (ChIP) analysis to examine shear-induced changes in RNAP II localization on the eNOS gene. Using antibody against total RNAP II, eNOS sequences along the entire gene were amplified. Using the phosphoserine 2 specific RNAP II antibody, we found active RNAP II predominantly bound to the 3′UTR (exon 26) and downstream sequence of eNOS in sheared cells. These findings were confirmed by quantitative ChIP (3 fold increase, n = 8, p = 0.0378). This suggests that shear-induced changes in RNAP II phosphorylation enhance its ability to polyadenylate eNOS mRNA. Serine 2 phosphorylation is dependent on cyclin-dependent kinase 9 (CDK 9), and shear-induced recruitment of CDK 9 to the eNOS gene was examined. We found that endothelial cells exposed to laminar shear had enhanced localization of CDK 9 to the eNOS promoter and exons 8 and 22, and diminished localizationto exon 26 and in the downstream sequence. This indicates that shear recruits CDK 9 while RNAP II is bound to the promoter and early exons to activate RNAP by phosphorylating serine 2. In conclusion, laminar shear stress enhances eNOS mRNA processing and increases gene expression through recruitment of CDK 9 and activation of RNAP II.

Differential regulation of urokinase-type plasminogen activator expression by fluid shear stress in human coronary artery endothelial cells

2004 ◽  
Vol 287 (5) ◽  
pp. H2027-H2034 ◽  
Author(s):  
Takaaki Sokabe ◽  
Kimiko Yamamoto ◽  
Norihiko Ohura ◽  
Hideki Nakatsuka ◽  
Kairong Qin ◽  
...  
Keyword(s):  
Shear Stress ◽  
Half Life ◽  
Turbulent Shear ◽  
Laminar Shear Stress ◽  
Human Coronary Artery ◽  
Turbulent Shear Stress ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Upa Mrna ◽  
Laminar Shear

Atherosclerotic plaques preferentially localize at arterial regions exposed to turbulent low-shear flow. Urokinase-type plasminogen activator (uPA) plays a role in vascular remodeling by facilitating smooth muscle cell migration and proliferation in addition to the proteolysis of extracellular matrix, and the expression of uPA is elevated in atherosclerotic lesions. In this study, we analyzed the effects of laminar and turbulent shear stress on uPA expression in cultured human coronary artery endothelial cells. The application of laminar shear stress (1.5 or 15 dyn/cm2) significantly decreased the amount of uPA mRNA as well as the secretion of uPA protein. In contrast, turbulent shear stress (average intensity, 1.5 dyn/cm2) markedly increased uPA gene expression and protein secretion. Laminar shear stress downregulated uPA gene expression transcriptionally and posttranscriptionally; laminar shear stress activated transcription factor GATA6, which binds to a GATA consensus element located between −692 and −687 bp in the uPA promoter, thereby inhibiting uPA gene transcription. Laminar shear stress also accelerated the degradation of uPA mRNA; the half-life of uPA mRNA decreased to about half of the static control's half-life. Although turbulent shear stress had no effect on the transcription of uPA, it significantly increased uPA mRNA stability; the half-life of uPA mRNA increased by about two times the static control's half-life. Our results suggest that endothelial uPA expression is flow sensitive and differentially regulated by laminar and turbulent shear stress in vitro. We speculate that this effect may contribute to the local nature of atherosclerosis.

Epigenetic histones modification and cardiovascular lineage programming in mouse embryonic stem cells exposed to laminar shear stress

2006 ◽  
Vol 45 (3) ◽  
pp. e56
Author(s):  
Barbara Illi ◽  
Alessandro Scopece ◽  
Simona Nanni ◽  
Antonella Farsetti ◽  
Liliana Morgante ◽  
...  
Keyword(s):  
Stem Cells ◽  
Shear Stress ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Laminar Shear Stress ◽  
Laminar Shear

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

2015 ◽  
Vol 35 (1) ◽  
pp. 137-145 ◽  
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

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

1991 ◽  
Vol 260 (6) ◽  
pp. H1992-H1996 ◽  
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.

scholarly journals SENCR stabilizes vascular endothelial cell adherens junctions through interaction with CKAP4

2018 ◽  
Vol 116 (2) ◽  
pp. 546-555 ◽  
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.

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

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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