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MicroRNA-663 upregulated by oscillatory shear stress plays a role in inflammatory response of endothelial cells.

ABSTRACT: The mechanisms by which oscillatory shear stress (OS) induces, while high laminar shear stress (LS) prevents, atherosclerosis are still unclear. Here, we examined the hypothesis that OS induces inflammatory response, a critical atherogenic event, in endothelial cells by a microRNA (miRNA)-dependent mechanism. By miRNA microarray analysis using total RNA from human umbilical vein endothelial cells (HUVECs) that were exposed to OS or LS for 24 h, we identified 21 miRNAs that were differentially expressed. Of the 21 miRNAs, 13 were further examined by quantitative PCR, which validated the result for 10 miRNAs. Treatment of HUVECs with the miR-663 antagonist (miR-663-locked nucleic acids) blocked OS-induced monocyte adhesion, but not apoptosis. In contrast, overexpression of miR-663 increased monocyte adhesion in LS-exposed cells. Subsequent mRNA expression microarray study using HUVECs treated with miR-663-locked nucleic acids and OS revealed 32 up- and 3 downregulated genes, 6 of which are known to be involved in inflammatory response. In summary, we identified 10 OS-sensitive miRNAs, including miR-663, which plays a key role in OS-induced inflammatory responses by mediating the expression of inflammatory gene network in HUVECs. These OS-sensitive miRNAs may mediate atherosclerosis induced by disturbed flow.

SUBMITTER: Ni CW

PROVIDER: S-EPMC3094086 | biostudies-other | 2011 May

REPOSITORIES: biostudies-other

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MicroRNA-663 upregulated by oscillatory shear stress plays a role in inflammatory response of endothelial cells.

Ni Chih-Wen CW Qiu Haiwei H Jo Hanjoong H

American journal of physiology. Heart and circulatory physiology 20110304 5

The mechanisms by which oscillatory shear stress (OS) induces, while high laminar shear stress (LS) prevents, atherosclerosis are still unclear. Here, we examined the hypothesis that OS induces inflammatory response, a critical atherogenic event, in endothelial cells by a microRNA (miRNA)-dependent mechanism. By miRNA microarray analysis using total RNA from human umbilical vein endothelial cells (HUVECs) that were exposed to OS or LS for 24 h, we identified 21 miRNAs that were differentially ex ...[more]

PMID: 21378144

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Project description:BackgroundCerebral arteriovenous malformations (cAVM) are a significant cause of intracranial hemorrhagic stroke and brain damage. The arteriovenous junctions in AVM nidus are known to have hemodynamic disturbances such as altered shear stress, which could lead to endothelial dysfunction. The molecular mechanisms coupling shear stress and endothelial dysfunction in cAVMs are poorly understood. We speculated that disturbed blood flow in artery-vein junctions activates Notch receptors and promotes endothelial mesenchymal plasticity during cAVM formation.MethodsWe investigated the expression profile of endothelial mesenchymal transition (EndMT) and cell adhesion markers, as well as activated Notch receptors, in 18 human cAVM samples and 15 control brain tissues, by quantitative real-time PCR (qRT-PCR) and immunohistochemical evaluation. Employing a combination of a microfluidic system, qRT-PCR, immunofluorescence, as well as invasion and inhibitor assays, the effects of various shear stress conditions on Notch-induced EndMT and invasive potential of human cerebral microvascular endothelial cells (hCMEC/d3) were analyzed.ResultsWe found evidence for EndMT and enhanced expression of activated Notch intracellular domain (NICD3 and NICD4) in human AVM nidus samples. The expression of transmembrane adhesion receptor integrin α9/β1 is significantly reduced in cAVM nidal vessels. Cell-cell adhesion proteins such as VE-cadherin and N-cadherin were differentially expressed in AVM nidus compared with control brain tissues. Using well-characterized hCMECs, we show that altered fluid shear stress steers Notch3 nuclear translocation and promotes SNAI1/2 expression and nuclear localization. Oscillatory flow downregulates integrin α9/β1 and VE-cadherin expression, while N-cadherin expression and endothelial cell invasiveness are augmented. Gamma-secretase inhibitor RO4929097, and to a lesser level DAPT, prevent the mesenchymal transition and invasiveness of cerebral microvascular endothelial cells exposed to oscillatory fluid flow.ConclusionsOur study provides, for the first time, evidence for the role of oscillatory shear stress in mediating the EndMT process and dysregulated expression of cell adhesion molecules, especially multifunctional integrin α9/β1 in human cAVM nidus. Concomitantly, our findings indicate the potential use of small-molecular inhibitors such as RO4929097 in the less-invasive therapeutic management of cAVMs.

Project description:Aim: To survey the alteration of endothelial transcriptome under different types of shear stress and find novel shear stress-sensitive protein-coding genes as well as non-coding RNAs as potential therapeutic targets for the treatment of atherosclerosis Method: After exposed to LS, OS and ST for 24 hours (n=4, respectively), ECs were harvested by scraping, and RNA was isolated and purified. Double-stranded cDNA was generated from 100 to 150 ng of total qualified RNA using selective priming and prepared for the final library. Quantitative PCR (qPCR) was performed to quantify the library using a KAPA kit for Illumina sequencing platforms. Twelve samples were then clustered on the cBot and sequenced on a 50-cycle single end on a HiSeq 2000 to generate 50 bp paired-end reads, using TruSeq SBS v3 reagents according to manufacturer’s protocols. RNA-seq fastq files were aligned to the human genome primary assembly (GRCh38) using the Subread package. Read counts on each gene were determined by featureCounts function in the Subread package using GeneCode human GTF file version 23 (http://www.gencodegenes.org/). Differential expression analysis between endothelial cells under different types of shear stress were performed using the limma package with the voom transformation. P values were adjusted by default Benjamini-Hochberg procedure. Results: RNA-seq data provided a broad view of endothelial transcriptome under shear stress. We mapped about 70 million reads and discovered over 16,000 genes that are well-expressed in human coronary artery endothelial cells. RNA-seq results were consistent with previously reported DNA microarray data and we also confirmed the expression of genes that are previously reported and novel genes with qRT–PCR results. Comparing the endothelial transcriptomes under different types of shear stress, we found that there are approximately 50% genes that are differentially expressed in ECs under ST vs. LS and OS vs. LS while 10% under ST vs. OS. Conclusion: RNA-seq data provide a framework for investigating endothelial transcriptome under shear stress. We found that endothelial cells under LS condition profoundly differs from those under either ST or OS conditions. The profiling also reveals a large amount of novel shear sensitive genes and lncRNAs that are not reported before. It provides the community with potential targets for anti-atherosclerosis drug development considering the determinant role of shear stress in the distribution of atherosclerotic lesions in the arterial tree.

Project description:Atherosclerosis is a multifactorial disease that preferentially develops in specific regions in the arterial tree. This characteristic is mainly attributed to the unique pattern of hemodynamic shear stress in vivo. High laminar shear stress (LS) found in straight lumen exerts athero-protective effects. Low or oscillatory shear stress (OS) present in regions of lesser curvature and arterial bifurcations predisposes arterial intima to atherosclerosis. Shear stress-regulated endothelial function plays an important role in the process of atherosclerosis. Most in vitro research studies focusing on the molecular mechanisms of endothelial function are performed in endothelial cells (ECs) under cultured static (ST) condition. Some findings, however, are not recapitulated in subsequent translational studies, mostly likely due to the missing biomechanical milieu. Here, we profiled the whole transcriptome of primary human coronary arterial endothelial cells (HCAECs) under different shear stress conditions with RNA sequencing. Among 16,313 well-expressed genes, we detected 8,177 that were differentially expressed in OS vs. LS conditions and 9,369 in ST vs. LS conditions. Notably, only 1,618 were differentially expressed in OS vs. ST conditions. Hierarchical clustering of ECs demonstrated a strong similarity between ECs under OS and ST conditions at the transcriptome level. Subsequent pairwise heat mapping and principal component analysis gave further weight to the similarity. At the individual gene level, expressional analysis of representative well-known genes as well as novel genes showed a comparable amount at mRNA and protein levels in ECs under ST and OS conditions. In conclusion, the present work compared the whole transcriptome of HCAECs under different shear stress conditions at the transcriptome level as well as at the individual gene level. We found that cultured ECs are significantly different from those under LS conditions. Thus using cells under ST conditions is unlikely to elucidate endothelial physiology. Given the revealed high similarities of the endothelial transcriptome under OS and ST conditions, it may be helpful to understand the underlying mechanisms of OS-induced endothelial dysfunction from static cultured endothelial studies.

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MicroRNA-663 upregulated by oscillatory shear stress plays a role in inflammatory response of endothelial cells.

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MicroRNA-663 upregulated by oscillatory shear stress plays a role in inflammatory response of endothelial cells.

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