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Detection of frequency-dependent endothelial response to oscillatory shear stress using a microfluidic transcellular monitor.

ABSTRACT: The endothelial microenvironment is critical in maintaining the health and function of the intimal layer in vasculature. In the context of cardiovascular disease (CVD), the vascular endothelium is the layer of initiation for the progression of atherosclerosis. While laminar blood flows are known to maintain endothelial homeostasis, disturbed flow conditions including those the endothelium experiences in the carotid artery are responsible for determining the fate of CVD progression. We present a microfluidic device designed to monitor the endothelium on two fronts: the real-time monitoring of the endothelial permeability using integrated electrodes and the end-point characterization of the endothelium through immunostaining. Our key findings demonstrate endothelial monolayer permeability and adhesion protein expression change in response to oscillatory shear stress frequency. These changes were found to be significant at certain frequencies, suggesting that a frequency threshold is needed to elicit an endothelial response. Our device made possible the real-time monitoring of changes in the endothelial monolayer and its end-point inspection through a design previously absent from the literature. This system may serve as a reliable research platform to investigate the mechanisms of various inflammatory complications of endothelial disorders and screen their possible therapeutics in a mechanistic and high-throughput manner.

SUBMITTER: Sei YJ

PROVIDER: S-EPMC5577378 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Detection of frequency-dependent endothelial response to oscillatory shear stress using a microfluidic transcellular monitor.

Sei Yoshitaka J YJ Ahn Song Ih SI Virtue Theodore T Kim Taeyoung T Kim YongTae Y

Scientific reports 20170830 1

The endothelial microenvironment is critical in maintaining the health and function of the intimal layer in vasculature. In the context of cardiovascular disease (CVD), the vascular endothelium is the layer of initiation for the progression of atherosclerosis. While laminar blood flows are known to maintain endothelial homeostasis, disturbed flow conditions including those the endothelium experiences in the carotid artery are responsible for determining the fate of CVD progression. We present a ...[more]

PMID: 28855638

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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:The objective of this study was to advance the understanding of how in vivo arterial shear forces affect vascular endothelial gene expression. Complicated blood flow patterns at arterial branches create small regions that experience fluctuations in shear stress at frequencies higher than the heart rate. To assess whether such temporal variations in shear stress can affect endothelial gene expression, a series of in vitro microarray experiments was performed. The effects of three sinusoidal waveforms (1, 2, and 3 Hz) and one physiological waveform were compared to the expression profiles under steady flow. At each frequency, three levels of mean shear stress (0, 7.5, and 15 dyn/cm2) were used. Porcine aortic endothelial cells were exposed for 24 hours to each combination, replicated four times. Following shear exposure, phase contrast images of the cells were acquired, and RNA was extracted for microarray analysis against about 10,000 porcine oligonucleotides. Cell alignment with the flow was positively correlated with mean shear (p < 0.001) and independent of frequency. A two-way ANOVA identified 232 genes that were differentially regulated by frequency. The frequency sensitive genes were clustered to identify groups of genes exhibiting similar frequency responses. The largest response was seen at 2 Hz. At this frequency, several inflammatory molecules were upregulated, including VCAM, CTGF, TGF-beta2, c-jun, and IL-8, indicating a potential endothelial atherosusceptibility at this frequency. Mean shear significantly affected the expression of ~3,000 genes. Purely oscillatory flow (zero mean shear) enhanced the expression of several growth factors and adhesion molecules (E-selectin, VCAM, MCP-1, IL-8, c-jun), relative to non-reversing flow (15 dyn/cm2 mean shear). The 2 Hz upregulation of certain atherogenic molecules such as VCAM, c-jun, and IL-8 was enhanced as the mean shear was reduced. Thus, the inflammatory response evoked at certain frequencies appears to be exacerbated by low, oscillatory shear. Keywords: Shear stress response

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.

Dataset Information

Detection of frequency-dependent endothelial response to oscillatory shear stress using a microfluidic transcellular monitor.

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Detection of frequency-dependent endothelial response to oscillatory shear stress using a microfluidic transcellular monitor.

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