The role of microRNAs for the regulation of stress-induced tissue factor isoform expression of human endothelial cells and mononuclear leukocytes

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Tissue Factor (TF), the initiator of blood coagulation, is a contributor to the pathogenesis of cardiovascular disease and thrombotic events. Alternative splicing leads to the genesis of two TF isoforms. Full length TF is a membrane-bound protein that determinates the procoagulability of blood and vessel wall. The alternatively spliced TF isoform is a soluble protein with pro-angiogenic characteristics. Recently, we described the mechanism of alternative splicing of TF-pre-mRNA in cytokine-stimulated human endothelial cells in detail. Cdc2-like kinases and DNA-topoisomerase I regulate the cytokine-induced TF isoform expression in endothelial cells by modulating the activity of serin/arginin-rich (SR) proteins. This led to alterations of the TF activity and, thus, the procoagulability of endothelial cells under defined rheological conditions. MicroRNAs (miRNA) are short non-coding RNAs and important post-transcriptional regulators of the gene expression. The miRNAs, that control the stress-induced expression of the TF isoform in endothelial cells, are unknown. In silico analyses were performed by utilizing different data file banks in order to identify possible miRNAs, which might be able to regulate the expression of proteins involved in the alternative splicing process of TF-pre-mRNA. Several different candidate miRNAs were identified that possibly contribute to the regulation of the TF isoform expression. Furthermore, we showed in a first proof-of-principal experiment that miR-181b regulated the endothelial expression of the two TF isoforms in a differential manner. In this study proposal, we want to examine how the pre-identified candidate miRNAs regulate the TF isoform expression with a special focus on the alternative splicing of TF. The interactions between the candidate miRNAs, such as miR-181b, and the respective target-mRNAs of relevant splice factors, such as Cdc2-like Kinases, DNA-topoisomerase I or SR-proteins, will be characterized via interaction studies with the mRNA-target sequences as well as co-immunoprecipitation analyses with effector proteins of the argonaute (Ago) family. Moreover, the effect of the miRNA-dependent regulation of the TF isoform expression on the endothelial procoagulability will be studied ex vivo in a laminar flow chamber under defined rheological conditions. The expression pattern of the candidate miRNAs will be assessed in mononuclear cells of the peripheral blood (PBMC) of patients with inflammatory and ischemic cardiomyopathy with respect to their possible usefulness for estimating the clinical prognosis. In summary, the proposed study will enable us to obtain new insights into central regulatory mechanisms of the cardiovascular hemostasis and will possibly lead to the identification of new targets for the development of anti-thrombotic strategies.