Abstract 927: The Role Of Peroxiredoxins As Mechanosensitive Antioxidants In Endothelial Cells

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Amy Mowbray ◽  
Sang Won Kang ◽  
Sue Goo Rhee ◽  
Hanjoong Jo
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Subcellular Localization ◽  
Molecular Mechanisms ◽  
Immunofluorescent Staining ◽  
Dependent Manner ◽  
Laminar Shear Stress ◽  
Arterial Tree ◽  
Aortic Endothelial Cells ◽  
Stress Dependent

Atherosclerosis is an inflammatory disease occurring primarily in curved or branching regions of the arterial tree where disturbed flow patterns, such as oscillation, exist. We have previously shown that oscillatory shear stress (OS) increases reactive oxygen species (ROS) levels in endothelial cells, while laminar shear stress (LS) reduces ROS compared to static controls. OS stimulation of ROS has been shown to occur in an NADPH oxidase-dependent manner. However, the mechanism by which LS removes ROS remains unclear. Peroxiredoxins (PRX) are a family of antioxidant proteins that have been linked to the prevention of oxidative stress and inflammation, but their role in atherosclerosis is unknown. Here, we hypothesize that shear stress regulates ROS levels in endothelial cells by controlling antioxidant peroxiredoxins. To test this hypothesis, bovine aortic endothelial cells (BAEC) were subjected to static, laminar, and oscillatory fluid flow conditions via cone-and-plate viscometer. Western blot analysis and immunofluorescent staining were used to evaluate the expression and subcellular localization of six known mammalian peroxiredoxins (PRX I-VI). Immunoblots indicated that BAEC express all six isoforms of peroxiredoxin proteins and that LS upregulated PRX I levels significantly compared to static controls and OS. Immunofluorescence also showed a distinct subcellular localization of each PRX: PRX I, II, IV, V and VI in the cytoplasm, PRX I, IV and V in the Golgi, PRX III in the mitochondria, and PRX I in the nucleus. These results indicate that peroxiredoxins are mechanosensitive antioxidants, removing ROS in a subcellular-specific manner. Based on these data, we suggest that peroxiredoxin antioxidants are likely involved in the molecular mechanisms that control shear stress-dependent atherosclerotic plaque development.

Expression of cathepsin K is regulated by shear stress in cultured endothelial cells and is increased in endothelium in human atherosclerosis

2007 ◽  
Vol 292 (3) ◽  
pp. H1479-H1486 ◽  
Author(s):  
Manu O. Platt ◽  
Randall F. Ankeny ◽  
Guo-Ping Shi ◽  
Daiana Weiss ◽  
J. D. Vega ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Molecular Mechanisms ◽  
Cathepsin K ◽  
Immunohistochemical Analysis ◽  
Cysteine Proteases ◽  
Laminar Shear Stress ◽  
Gelatinase Activity ◽  
Aortic Endothelial Cells ◽  
Expression And Activity

Cathepsins, the lysosomal cysteine proteases, are involved in vascular remodeling and atherosclerosis. Genetic knockout of cathepsins S and K in mice has shown to reduce atherosclerosis, although the molecular mechanisms remain unclear. Because atherosclerosis preferentially occurs in arteries exposed to disturbed flow conditions, we hypothesized that shear stress would regulate cathepsin K expression and activity in endothelial cells. Mouse aortic endothelial cells (MAEC) exposed to proatherogenic oscillatory shear (OS, ± 5 dyn/cm2 for 1 day) showed significantly higher cathepsin K expression and activity than that of atheroprotective, unidirectional laminar shear stress (LS, 15 dyn/cm2 for 1 day). Western blot and active-site labeling studies showed an active, mature form of cathepsin K in the conditioned medium of MAEC exposed to OS but not in that of LS. Functionally, MAEC exposed to OS significantly increased elastase and gelatinase activity above that of LS. The OS-dependent elastase and gelatinase activities were significantly reduced by knocking down cathepsin K with small-interfering (si) RNA, but not by a nonsilencing siRNA control, suggesting that cathepsin K is a shear-sensitive protease. In addition, immunohistochemical analysis of atherosclerotic human coronary arteries showed a positive correlation between the cathepsin K expression levels in endothelium and elastic lamina integrity. These findings suggest that cathepsin K is a mechanosensitive, extracellular matrix protease that, in turn, may be involved in arterial wall remodeling and atherosclerosis.

Shear Stress Modulates the Expression of Thrombospondin-1 and CD36 in Endothelial Cells in vitro and during Shear Stress-Induced Angiogenesis in vivo

2006 ◽  
Vol 19 (1) ◽  
pp. 205873920601900 ◽  
Author(s):  
M. Bongrazio ◽  
L. DA Silva-Azevedo ◽  
E.C. Bergmann ◽  
O. Baum ◽  
B. Hinz ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Western Blot ◽  
Dependent Manner ◽  
Vascular Networks ◽  
Thrombospondin 1 ◽  
Matrix Bound ◽  
Stress Dependent

Binding of thrombospondin-1 (TSP-1) to the CD36 receptor inhibits angiogenesis and induces apoptosis in endothelial cells (EC). Conversely, matrix-bound TSP-1 supports vessel formation. In this study we analyzed the shear stress-dependent expression of TSP-1 and CD36 in endothelial cells in vitro and in vivo to reveal its putative role in the blood flow-induced remodelling of vascular networks. Shear stress was applied to EC using a cone-and-plate apparatus and gene expression was analyzed by RT-PCR, Northern and Western blot. Angiogenesis in skeletal muscles of prazosin-fed (50 mg/1 drinking water; 4 d) mice was assessed by measuring capillary-to-fiber (C/F) ratios. Protein expression in whole muscle homogenates (WMH) or BS-1 lectin-enriched EC fractions (ECF) was analyzed by Western blot. Shear stress down-regulated TSP-1 and CD36 expression in vitro in a force- and time-dependent manner sustained for at least 72 h and reversible by restoration of no-flow conditions. In vivo, shear stress-driven increase of C/F in prazosin-fed mice was associated with reduced expression of TSP-1 and CD36 in ECF, while TSP-1 expression in WMH was increased. Down-regulation of endothelial TSP-1/CD36 by shear stress suggests a mechanism for inhibition of apoptosis in perfused vessels and pruning in the absence of flow. The increase of extra-endothelial (e.g. matrix-bound) TSP-1 could support a splitting type of vessel growth.

Integrin mechanotransduction stimulates caveolin-1 phosphorylation and recruitment of Csk to mediate actin reorganization

2005 ◽  
Vol 288 (2) ◽  
pp. H936-H945 ◽  
Author(s):  
C. Radel ◽  
V. Rizzo
Keyword(s):  
Shear Stress ◽  
Dependent Manner ◽  
Laminar Shear Stress ◽  
Integrin Activation ◽  
Actin Reorganization ◽  
Aortic Endothelial Cells ◽  
Caveolin 1 ◽  
Domain Peptide ◽  
Mlc Phosphorylation

To identify the role of caveolin-1 in integrin mechanotransduction, we exposed bovine aortic endothelial cells to 10 dyn/cm2 of laminar shear stress. Caveolin-1 was acutely and transiently phosphorylated with shear, occurring downstream of β1-integrin activation as the β1-integrin blocking antibody JB1A was inhibitory. In manipulating Src family kinase (SFK) activity with knockdown of Csk or type 1 protein phosphatase (PP1) treatment, we observed coordinate increase and decrease in shear-induced caveolin-1 phosphorylation, respectively. Hence, shear-stimulated caveolin-1 phosphorylation is regulated by SFKs. Shear-induced recruitment and phosphorylation of caveolin-1 occurred at β1-integrin sites in a β1-integrin- and SFK-dependent manner. Csk, described to interact with pY14-caveolin-1 and integrins, bound to an increased pool of phosphorylated caveolin-1 after shear corresponding with elevated Csk at β1-integrin sites. Like caveolin-1, treatment with JB1A and PP1 attenuated shear-induced Csk association with β1-integrins. Csk function was assayed with transfection of a caveolin-1 phosphorylation domain peptide. The peptide attenuated shear-induced association of Csk at β1-integrin sites, as well as colocalization of Csk with paxillin and phosphorylated caveolin-1. Because integrin and Csk activity regulate cytoskeletal reorganization, we evaluated the role of this mechanism in shear-induced myosin light chain (MLC) phosphorylation. Knockdown of Csk expression was sufficient to reduce MLC diphosphorylation due to shear. Disruption of Csk-integrin association by peptide treatment was also inhibitory of the MLC diphosphorylation response. Together these data indicate that integrin activation with shear stress results in SFK-regulated caveolin-1 phosphorylation that, in turn, mediates Csk association at integrin sites, where it plays a role in downstream, shear-stimulated MLC diphosphorylation.

scholarly journals Flow-dependent Smad2 phosphorylation and TGIF nuclear localization in human aortic endothelial cells

2011 ◽  
Vol 301 (1) ◽  
pp. H98-H107 ◽  
Author(s):  
Robert D. Shepherd ◽  
Stephanie M. Kos ◽  
Kristina D. Rinker
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Shear Stress ◽  
Fluid Flow ◽  
Nuclear Localization ◽  
Aortic Endothelial Cells ◽  
Linker Region ◽  
Stress Dependent ◽  
Human Aortic Endothelial Cells ◽  
Aortic Endothelial

Endothelial cells respond to fluid flow stimulation through transient and sustained signal pathway activation. Smad2 is a signaling molecule and transcription factor in the Smad signaling pathway, traditionally associated with TGF-β. Although phosphorylation of Smad2 in the receptor-dependent COOH-terminal region is the most appreciated way Smad2 is activated to affect gene expression, phosphorylation may also occur in the MH1-MH2 linker region (L-psmad2). Here, we show that in human aortic endothelial cells (HAEC), Smad2 was both preferentially phosphorylated in the linker region and localized to the nucleus in a flow-dependent manner. The Smad corepressor transforming growth interacting factor (TGIF) was also found to have flow-dependent nuclear localization. Tissue studies confirmed this L-psmad2 generation trend in rat aorta, indicating likely importance in arterial tissue. HAEC-based inhibitor studies demonstrated that L-psmad2 levels were not related to MAPK phosphorylation, but instead followed the pattern of pAkt473, both with and without the phosphatidylinositol 3-kinase inhibitor PI-103. Akt and Smad species were also shown to directly interact under flow relative to static controls. To further evaluate impacts of PI-103 treatment, expression profiles for two TGF-β and shear stress-dependent genes were determined and showed that mRNAs were lower from untreated 10 dyn/cm2than 2 dyn/cm2average shear stress cultures. However, upon exposure to PI-103, this trend was reversed, with a stronger response observed at 10 dyn/cm2. Taken together, the results of this work suggest that fluid flow exposure may influence endothelial gene expression by a novel mechanism involving Akt, L-psmad2, and TGIF.

Sustained JNK activation induces endothelial apoptosis: studies with colchicine and shear stress

1999 ◽  
Vol 277 (4) ◽  
pp. H1593-H1599 ◽  
Author(s):  
Ying-Li Hu ◽  
Song Li ◽  
John Y.-J. Shyy ◽  
Shu Chien
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Colchicine Treatment ◽  
Laminar Shear Stress ◽  
Aortic Endothelial Cells ◽  
Bovine Aortic Endothelial Cells ◽  
Jnk Activation ◽  
Laminar Shear ◽  
Sustained Activation ◽  
Aortic Endothelial

The disruption of microtubules by treating bovine aortic endothelial cells with 10−7–10−5M colchicine caused apoptosis, as evidenced by DNA laddering and TdT-mediated dUTP nick end labeling fluorescence staining. Colchicine treatment also induced a sustained activation of c-Jun NH2-terminal kinase (JNK) that lasted for ≥12 h. The blockade of JNK activity by using the negative interfering mutant JNK(K-R) markedly decreased the apoptosis induced by colchicine. Exposure of bovine aortic endothelial cells to laminar shear stress (12 dyn/cm2) caused a transient (<2 h) activation of JNK, and there was no induction of apoptosis. The sustained activation of JNK may play a significant role in the apoptosis induced by colchicine.

scholarly journals Shear-induced tyrosine phosphorylation in endothelial cells requires Rac1-dependent production of ROS

1999 ◽  
Vol 276 (4) ◽  
pp. C838-C847 ◽  
Author(s):  
Li-Hong Yeh ◽  
Young J. Park ◽  
Riple J. Hansalia ◽  
Imraan S. Ahmed ◽  
Shailesh S. Deshpande ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Map Kinase ◽  
Tyrosine Phosphorylation ◽  
Protein Detection ◽  
Pyrrolidine Dithiocarbamate ◽  
Dependent Manner ◽  
Aortic Endothelial Cells ◽  
Bovine Aortic Endothelial Cells ◽  
Aortic Endothelial

The shear-induced intracellular signal transduction pathway in vascular endothelial cells involves tyrosine phosphorylation and activation of mitogen-activated protein (MAP) kinase, which may be responsible for the sustained release of nitric oxide. MAP kinase is known to be activated by reactive oxygen species (ROS), such as H2O2, in several cell types. ROS production in ligand-stimulated nonphagocytic cells appears to require the participation of a Ras-related small GTP-binding protein, Rac1. We hypothesized that Rac1 might serve as a mediator for the effect of shear stress on MAP kinase activation. Exposure of bovine aortic endothelial cells to laminar shear stress of 20 dyn/cm2for 5–30 min stimulated total cellular and cytosolic tyrosine phosphorylation as well as tyrosine phosphorylation of MAP kinase. Treating endothelial cells with the antioxidants N-acetylcysteine and pyrrolidine dithiocarbamate inhibited in a dose-dependent manner the shear-stimulated increase in total cytosolic and, specifically, MAP kinase tyrosine phosphorylation. Hence, the onset of shear stress caused an enhanced generation of intracellular ROS, as evidenced by an oxidized protein detection kit, which were required for the shear-induced total cellular and MAP kinase tyrosine phosphorylation. Total cellular and MAP kinase tyrosine phosphorylation was completely blocked in sheared bovine aortic endothelial cells expressing a dominant negative Rac1 gene product (N17rac1). We concluded that the GTPase Rac1 mediates the shear-induced tyrosine phosphorylation of MAP kinase via regulation of the flow-dependent redox changes in endothelial cells in physiological and pathological circumstances.

Endothelin receptor B-mediated induction of c-jun and AP-1 in response to shear stress in human endothelial cellsThis article is one of a selection of papers published in the special issue (part 2 of 2) on Forefronts in Endothelin.

2008 ◽  
Vol 86 (8) ◽  
pp. 499-504 ◽  
Author(s):  
H. Morawietz ◽  
A.H. Wagner ◽  
M. Hecker ◽  
W. Goettsch
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Endothelin Receptor ◽  
Laminar Shear Stress ◽  
Human Endothelial Cells ◽  
Endothelin 1 ◽  
Subtype B ◽  
Short Term Exposure ◽  
Stress Dependent ◽  
Laminar Shear

In vivo, endothelial cells are constantly exposed to shear stress by flowing blood. Short-term exposure of endothelial cells to shear stress has been shown to induce endothelin-1 release. It is currently unknown, however, whether this shear stress-dependent endothelin-1 release affects the expression and activity of transcription factors. In this study, primary cultures of human endothelial cells from the umbilical vein were exposed to laminar shear stress in a cone-and-plate viscometer. Laminar shear stress for 30 min induced a 2-fold increase in mRNA expression of c-jun , but not c-fos, in human endothelial cells. Blockade of endothelin receptor subtype B (ETB) with BQ788 prevented this shear stress-dependent induction of c-jun expression. The induction of c-jun by shear stress involved protein kinase C and endothelial NO synthase. In addition, exposure of endothelial cells to arterial laminar shear stress for 1 h increased the binding of transcription factor AP-1 to its consensus sequence by 1.7-fold in electrophoretic mobility shift assays. This induction was also mediated by an ETB-dependent pathway. Supershift analysis supports an AP-1 complex containing c-jun, but not c-fos, in human endothelial cells. In conclusion, our data suggest endothelin-1-mediated induction of c-jun expression and activation of AP-1 (possibly as a c-jun homodimer) by laminar shear stress in human endothelial cells.

scholarly journals Acute limb heating improves macro- and microvascular dilator function in the leg of aged humans

2017 ◽  
Vol 312 (1) ◽  
pp. H89-H97 ◽  
Author(s):  
Steven A. Romero ◽  
Daniel Gagnon ◽  
Amy N. Adams ◽  
Matthew N. Cramer ◽  
Ken Kouda ◽  
...  
Keyword(s):  
Shear Stress ◽  
Young Adults ◽  
Lower Limb ◽  
Local Heating ◽  
Reactive Hyperemia ◽  
Arterial Occlusion ◽  
Lower Limbs ◽  
Dependent Manner ◽  
Arterial Tree ◽  
Stress Dependent

Local heating of an extremity increases blood flow and vascular shear stress throughout the arterial tree. Local heating acutely improves macrovascular dilator function in the upper limbs of young healthy adults through a shear stress-dependent mechanism but has no such effect in the lower limbs of this age group. The effect of acute limb heating on dilator function within the atherosclerotic prone vasculature of the lower limbs of aged adults is unknown. Therefore, the purpose of this study was to test the hypothesis that acute lower limb heating improves macro- and microvascular dilator function within the leg vasculature of aged adults. Nine young and nine aged adults immersed their lower limbs at a depth of ~33 cm into a heated (~42°C) circulated water bath for 45 min. Before and 30 min after heating, macro (flow-mediated dilation)- and microvascular (reactive hyperemia) dilator functions were assessed in the lower limb, following 5 min of arterial occlusion, via Doppler ultrasound. Compared with preheat, macrovascular dilator function was unchanged following heating in young adults ( P = 0.6) but was improved in aged adults ( P = 0.04). Similarly, microvascular dilator function, as assessed by peak reactive hyperemia, was unchanged following heating in young adults ( P = 0.1) but was improved in aged adults ( P < 0.01). Taken together, these data suggest that acute lower limb heating improves both macro- and microvascular dilator function in an age dependent manner. NEW & NOTEWORTHY We demonstrate that lower limb heating acutely improves macro- and microvascular dilator function within the atherosclerotic prone vasculature of the leg in aged adults. These findings provide evidence for a potential therapeutic use of chronic lower limb heating to improve vascular health in primary aging and various disease conditions.

Mechanism and functional impact of CD40 ligand-induced von Willebrand factor release from endothelial cells

2015 ◽  
Vol 113 (05) ◽  
pp. 1095-1108 ◽  
Author(s):  
Kerstin Möller ◽  
Oliver Adolph ◽  
Jennifer Grünow ◽  
Julia Elrod ◽  
Miruna Popa ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Von Willebrand Factor ◽  
Cd40 Ligand ◽  
Dependent Manner ◽  
Vascular Remodelling ◽  
String Formation ◽  
Stress Dependent ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryCo-stimulation via CD154 binding to CD40, pivotal for both innate and adaptive immunity, may also link haemostasis to vascular remodelling. Here we demonstrate that human platelet-bound or recombinant soluble CD154 (sCD154) elicit the release from and tethering of ultra-large (UL) von Willebrand factor (vWF) multimers to the surface of human cultured endothelial cells (ECs) exposed to shear stress. This CD40-mediated ULVWF multimer release from the Weibel-Palade bodies was triggered by consecutive activation of TRAF6, the tyrosine kinase c-Src and phospholipase Cγ1 followed by inositol-1,4,5 tris-phosphate-mediated calcium mobilisation. Subsequent exposure to human washed platelets caused ULVWF multimer-platelet string formation on the EC surface in a shear stress-dependent manner. Platelets tethered to these ULVWF multimers exhibited P-selectin on their surface and captured labelled monocytes from the superfusate. When exposed to shear stress and sCD154, native ECs from wild-type but not CD40 or vWF-deficient mice revealed a comparable release of ULVWF multimers to which murine washed platelets rapidly adhered, turning P-selectin-positive and subsequently capturing monocytes from the perfusate. This novel CD154-provoked ULVWF multimerplatelet string formation at normal to fast flow may contribute to vascular remodelling processes requiring the perivascular or intravascular accumulation of pro-inflammatory macrophages such as arteriogenesis or atherosclerosis.

Phosphatidylinositol 3-kinase γ mediates shear stress-dependent activation of JNK in endothelial cells

1998 ◽  
Vol 275 (5) ◽  
pp. H1898-H1904 ◽  
Author(s):  
Young-Mi Go ◽  
Heonyong Park ◽  
Matthew C. Maland ◽  
Victor M. Darley-Usmar ◽  
Borislav Stoyanov ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Phosphatidylinositol 3 Kinase ◽  
Signaling Mechanism ◽  
Jnk Pathway ◽  
Aortic Endothelial Cells ◽  
Dependent Form ◽  
Stress Dependent ◽  
Phosphatidylinositol 3

Shear stress differentially activates extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) by mechanisms involving Gαi2 and Gβ/γ proteins, respectively, in bovine aortic endothelial cells (BAEC). The early events in this signaling mechanism by which G proteins regulate ERK and JNK in response to shear stress have not been defined. Here we show that BAEC endogenously express a G protein-dependent form of phosphatidylinositol 3-kinase, PI3Kγ, and its activity is stimulated by shear stress. PI3Kγ activity was measured in vitro using BAEC that were transiently transfected with an epitope-tagged PI3Kγ (vsv-PI3Kγ). Exposure of BAEC to shear stress rapidly and transiently stimulated the activity of vsv-PI3Kγ (maximum by 15 s, with a return to basal after 1-min exposure to 5 dyn/cm2 shear stress). Activity of vsv-PI3Kγ was stimulated by shear stress intensities as low as 0.5 dyn/cm2. Treatment of BAEC with an inhibitor of PI3K, wortmannin, inhibited shear-dependent activation of JNK but had no effect on that of ERK. Furthermore, expression of a kinase-inactive mutant (PI3KγK799R) in BAEC inhibited the shear-dependent activation of JNK but not ERK. Taken together, these results suggest that PI3Kγ selectively regulates the shear-sensitive JNK pathway. This differential and novel signaling pathway may be responsible for coordinating various mechanosensitive events in endothelial cells.

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