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:Giantsos-Adams2013 - Growth of glycocalyx under static conditions This model is described in the article: Heparan Sulfate Regrowth Profiles Under Laminar Shear Flow Following Enzymatic Degradation. Giantsos-Adams KM, Koo AJ, Song S, Sakai J, Sankaran J, Shin JH, Garcia-Cardena G, Dewey CF. Cell Mol Bioeng 2013 Jun; 6(2): 160-174 Abstract: The local hemodynamic shear stress waveforms present in an artery dictate the endothelial cell phenotype. The observed decrease of the apical glycocalyx layer on the endothelium in atheroprone regions of the circulation suggests that the glycocalyx may have a central role in determining atherosclerotic plaque formation. However, the kinetics for the cells' ability to adapt its glycocalyx to the environment have not been quantitatively resolved. Here we report that the heparan sulfate component of the glycocalyx of HUVECs increases by 1.4-fold following the onset of high shear stress, compared to static cultured cells, with a time constant of 19 h. Cell morphology experiments show that 12 h are required for the cells to elongate, but only after 36 h have the cells reached maximal alignment to the flow vector. Our findings demonstrate that following enzymatic degradation, heparan sulfate is restored to the cell surface within 12 h under flow whereas the time required is 20 h under static conditions. We also propose a model describing the contribution of endocytosis and exocytosis to apical heparan sulfate expression. The change in HS regrowth kinetics from static to high-shear EC phenotype implies a differential in the rate of endocytic and exocytic membrane turnover. This model is hosted on BioModels Database and identified by: MODEL1609100001. To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

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Homo sapiens

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