Elucidation of the signalling mechanisms involved in TF-mediated apoptosis in endothelial cells

Ethaeb, Ali Mahdi

Abstract

Tissue factor (TF) is the main initiator of blood coagulation. In addition to its procoagulant property, TF has the ability to regulate various functions within cells including proliferation, angiogenesis and apoptosis. These outcomes appear to depend on the amount of TF with which the cell comes into contact with. In this study, human dermal blood endothelial cells (HDBEC) were transfected to express wild-type TF which is released following the activation of PAR2 in a normal physiological response. In addition, a model for the accumulation of TF in vascular disease and cancer was used by expressing a mutant form of TF (TFAla253-tGFP) which although expressed is not released by the cells and therefore it accumulates intracellularly. Initially, the phosphorylation of Src1 and Rac1 were monitored in order to determine any difference in phosphorylation patterns following PAR2 activation of cells. Phosphorylation of Src1, but not Rac1 was prolonged on expression of TF and was further enhanced on intracellular accumulation of TF. Therefore, the role of Src1 as a mediator of TF-induced apoptosis was examined next. Either inhibition of Src using pp60c-srcpeptide, or suppression of Src1 expression using siRNA prevented the TF-induced p38 MAPK activation and subsequent cellular apoptosis. Following confirmation of the role of Src1 in this process, an attempt was then made to delineate upstream intermediaries involved in this pathway. By using an inhibitory antibody (AIIB2), β1-integrin was shown to participate in TF-induced Src1 activation. In contrast, prevention of Src1-FAK complex formation using FAK inhibitor-14 did not interfere with the TF-mediated Src1 activation, despite a clear reduction in Src1 phosphorylation. Furthermore, TF-induced apoptosis did not appear to require Src1-FAK binding. In conclusion, this study has established further steps in the pathway by which TF can induce cellular apoptosis, and suggests a mechanism by which the increased amount of TF during inflammation can have detrimental outcome on the vascular system.

Citation

Ethaeb, A. M. Elucidation of the signalling mechanisms involved in TF-mediated apoptosis in endothelial cells. (Thesis). University of Hull. https://hull-repository.worktribe.com/output/4220989

• Thesis Type: Thesis
• Deposit Date: Nov 12, 2018
• Publicly Available Date: Feb 23, 2023
• Keywords: Biological sciences
• Public URL: https://hull-repository.worktribe.com/output/4220989
• Additional Information: School of Biological, Biomedical and Environmental Sciences, The University of Hull
• Award Date: Sep 1, 2018