Gene expression profiling of human aortic endothelial cells exposed to disturbed flow and steady laminar flow

2002 ◽  
Vol 9 (1) ◽  
pp. 27-41 ◽  
Author(s):  
Alan R. Brooks ◽  
Peter I. Lelkes ◽  
Gabor M. Rubanyi
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Laminar Flow ◽  
Mrna Levels ◽  
Monocyte Adhesion ◽  
Disturbed Flow ◽  
Aortic Endothelial Cells ◽  
Human Aortic Endothelial Cells ◽  
Steady Laminar

Subtraction cloning and cDNA arrays were used to compare steady-state mRNA levels in cultured human aortic endothelial cells (HAEC) exposed for up to 24 h to either high-shear (13 dyn/cm2) steady laminar flow (LF), an established representation of “atheroprotective” flow conditions, or low-shear (<1 dyn/cm2), pulsatile, nonsteady, non-unidirectional flow (disturbed flow, DF) that simulates conditions in the atherosclerosis-prone areas of the arterial circulation. More than 100 genes not previously known to be flow regulated were identified. Analysis of selected genes by quantitative RT-PCR confirmed the results obtained from the microarrays. These data demonstrate that DF is not simply the absence of LF but in fact represents a distinct biomechanical stimulus that has a profound impact upon the gene expression profile of HAEC in culture. In line with previous studies, many of the changes in mRNA levels induced by LF are atheroprotective. In contrast, DF upregulated the mRNA levels of a plethora of proatherosclerotic genes including proinflammatory, proapoptotic, and procoagulant molecules. For some of the genes whose expression was altered by DF and LF, corresponding changes in EC function (proliferation and monocyte adhesion) could be demonstrated. Specifically, the sustained upregulation of VCAM-1 and increased monocyte adhesion to EC exposed to DF was similar to that found in EC in vivo at atherosclerosis-prone regions, confirming the relevance of our model system for in vivo conditions. Distinct differences in the cellular response induced by TNFα and DF suggest that the effects of DF are not mediated entirely by the same signaling pathways that activate NF-κB. These studies demonstrate extensive and pathophysiologically relevant changes in sustained gene expression patterns in aortic EC exposed to DF compared with LF which are predicted to induce a proatherogenic EC phenotype.

scholarly journals Thrombospondin 2 Regulates Cell Proliferation Induced by Rac1 Redox-Dependent Signaling

2003 ◽  
Vol 23 (15) ◽  
pp. 5401-5408 ◽  
Author(s):  
Neuza Lopes ◽  
David Gregg ◽  
Sanjay Vasudevan ◽  
Hamdy Hassanain ◽  
Pascal Goldschmidt-Clermont ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cells ◽  
Matricellular Protein ◽  
Mrna Levels ◽  
Experimental Conditions ◽  
Aortic Endothelial Cells ◽  
Blocking Antibodies ◽  
Human Aortic Endothelial Cells ◽  
Proliferative Ability

ABSTRACT Thrombospondin 2 (TSP2) is a matricellular protein controlling the apoptosis-proliferation balance in endothelial cells. Little is known about its transcriptional regulation compared with that of TSP1. We found that overexpression of a constitutively active mutant of Rac (RacV12) specifically increases TSP2 mRNA levels without affecting TSP1 in human aortic endothelial cells (HAEC). Moreover, TSP2 induction by RacV12 is dependent upon reactive oxygen species (ROS) production, as gp91ds-tat peptide, an inhibitor of NADPH oxidase, and the flavoprotein inhibitor diphenylene iodinium (DPI) block TSP2 synthesis. Furthermore, we found that increasing RacV12 expression results in a biphasic proliferative curve, with proliferation initially increasing as RacV12 expression increases and then returning to levels less than that of control cells at higher expression. This growth inhibition is mediated by TSP2, as either DPI treatment, which blocks TSP2 synthesis, or pan-TSP blocking antibodies restore the proliferative ability of HAEC with high expression. Mechanistically, we show that the effect of TSP2 on cell proliferation is independent of the antiangiogenic TSP2 Hep1 sequence, which is capable of altering actin cytoskeletal reorganization but not proliferation in our experimental conditions. Finally, we show in vivo that Rac-induced TSP2 expression is observed in the aorta of transgenic mice selectively expressing RacV12 in smooth muscle cells. These results identify Rac-induced ROS as a new pathway involved in the regulation of TSP2 expression.

Laminar Flow on Endothelial Cells Suppresses eNOS O-GlcNAcylation to Promote eNOS Activity

2021 ◽  
Author(s):  
Sarah Basehore ◽  
Samantha Bohlman ◽  
Callie Weber ◽  
Swathi Swaminathan ◽  
Yuji Zhang ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Shear Stress ◽  
Laminar Flow ◽  
No Production ◽  
Disturbed Flow ◽  
Enos Activity ◽  
Enos Phosphorylation ◽  
Steady Laminar ◽  
In 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.

Abstract 376: Berry Anthocyanins and Their Metabolite Ameliorate High Glucose-Induced Adhesion of Monocytes to Human Aortic Endothelial Cells by Modulating the Cross-Talk Between eNOS and NFκB Signaling

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Anandh Babu Pon Velayutham ◽  
Eui Min Kim ◽  
Leena Panneerseelan-Bharath ◽  
Vasanthi Muthuswamy ◽  
Thunder Jalili ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
High Glucose ◽  
Vascular Inflammation ◽  
Ros Generation ◽  
Monocyte Adhesion ◽  
Enos Expression ◽  
Aortic Endothelial Cells ◽  
Berry Anthocyanins ◽  
Berry Fruits ◽  
Human Aortic Endothelial Cells

Introduction: In diabetes, hyperglycemia-induced vascular inflammation, resulting in the adhesion of monocytes to vascular endothelial cells and their subsequent migration into the subendothelial space, plays a major role in the development of atherosclerosis. ROS induce the inflammatory events involved in diabetic vascular disease by suppressing endothelial nitric oxide synthase (eNOS) through activation of Inhibitor κB kinase (IKKβ), an activator of nuclear factor-κB (NFκB). Aim: To determine the effects of cyanidin-3-glucoside (a major anthocyanin abundant in berry fruits; cyn-3-glu), protocatechuic acid (a major metabolite of anthocyanins; PCA), and bilberry anthocyanins (a mixture of 15 anthocyanins present in bilberry; BBA) on endothelial inflammation induced by hyperglycemia. Methods: Human aortic endothelial cells (HAEC) were cultured in normal (5 mM) or high-glucose (25 mM) ± 1 nM - 10 μM cyn-3-glu, PCA or BBA for 72 h. Cell adhesion (assessed via fluorescent probe labeled THP-1, a human monocytic cell line), intracellular ROS (assessed via DCFH-DH), mRNA expression of eNOS, NFκB-p65, and IKKβ (assessed via qPCR), and cell viability (assessed via CellTiter-Blue assay) were quantified in response to the described treatments. Results: Relative to results obtained from HAEC exposed to 5 mM glucose, ROS generation (42±4%), monocyte adhesion (35±2%), NFκB-p65 expression (80±13%), and IKKβ expression (50±11%) were elevated (p<0.05), whereas eNOS expression was suppressed (50±2%; p<0.05), in cells challenged with high-glucose (n=3-4). Cell viability was maintained regardless of the treatment. The effects of high-glucose on ROS generation, monocyte adhesion, and NFκB-p65, IKKβ and eNOS expression were ameliorated by concurrent treatment with cyn-3-glu or PCA (100 nM). Further, BBA treatment improved the effects of high glucose on ROS and monocyte adhesion. Conclusion: Berry anthocyanins and their metabolite, at concentrations achievable in human plasma by consumption of berry fruits, attenuate glucotoxic vascular inflammation in HAEC by ameliorating NFκB mediated suppression of eNOS signaling. Anthocyanin consumption might be a novel ancillary treatment to prevent vascular complications associated with diabetes.

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.

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