Abstract
The dysregulation of vascular endothelial cells by thrombin has been implicated in the development of a number of pathologic disorders such as inflammatory conditions, cancer, diabetes, coronary heart disease. However, transcriptional regulation of vascular endothelial cells by thrombin is not completely understood. In the present study, Illumina RNA-seq was used to profile the transcriptome in human pulmonary microvascular endothelial cells (HMVEC-L) treated with thrombin for 6 h to gain insight into thrombin's direct effects on the endothelial function. Out of 100 million total reads from a paired end sequencing assay, 91–94% of the reads were aligned to over 16,000 genes in the reference human genome. Thrombin upregulated 150 known genes and 480 known isoforms, and downregulated 2,190 known genes and 3,574 known isoforms by at least 2 fold. Of note, thrombin upregulated 1,775 previously unknown isoforms and downregulated 12,202 previously unknown isoforms by at least 2 fold. Many genes displayed isoform specific differential expression levels and different usage of transcriptional start sites after the thrombin treatment. The cross comparisons between our RNA-seq data and those of DNA microarray analysis of either 6 h thrombin treated HUVEC or 5 h TNFα treated HMVEC have provided a significant overlapping list of differentially expressed genes, supporting the robust utility of our dataset. Further in-depth follow-up analysis of the transcriptional regulation reported in this study may shed light on molecular pathogenic mechanisms underlying thrombin mediated endothelial dysfunction in various diseases and provide new leads of potential therapeutic targets.
Highlights
Endothelial cells lining the inner surface of microvessels form a semipermeable barrier that actively participates in blood–tissue exchange of plasma fluid, proteins and cells
Our study provides the first comprehensive insight into the transcriptome of human pulmonary microvascular endothelial cells treated with thrombin using RNA-seq, a powerful nextgeneration DNA sequencing platform, since a literature search for the PubMed database with the combination of key words of thrombin and RNA-seq retrieves zero records as of Sept. 16, 2011
We obtained a mean value of 102,568,132 reads per sample, which is more than enough to deliver sufficient sequence coverage for transcriptome profiling according to a previous report by Sultan et al [18]
Summary
Endothelial cells lining the inner surface of microvessels form a semipermeable barrier that actively participates in blood–tissue exchange of plasma fluid, proteins and cells. Besides its central role in the coagulation cascade acting as both a coagulant factor and an anticoagulant factor, thrombin can trigger important cellular effects via a protease-activated receptor pathway or receptor independent pathways [2]. Thrombin can stimulate endothelial cells and regulate the expression, release and activation of a number of biological mediators. Thrombin plays an important role in angiogenesis and is involved in tumor progression and metastasis [4]. The list of physiological and pathological roles of thrombin is still expanding. The understanding of these events at a molecular level will provide new targets of therapeutic interventions in disease states
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