An investigation into the mechanisms of tissue factor-mediated apoptosis in endothelial cells

ElKeeb, Azza M.

Abstract

Cells are known to express and release tissue factor (TF) following activation. In addition, endothelial cells are capable of acquiring TF carried by circulating microvesicles. Accumulation of TF in the endothelium contributes to chronic pathological conditions including cardiovascular disease. The aim of this study was to investigate the mechanisms that regulate the release of TF within microvesicles. In addition, the effects of accumulation of TF on the mediators of cell proliferation and apoptosis were examined and the underlying mechanisms explored. Throughout this study, endothelial cells were transfected to express TF or, alternatively, incubated with TF-containing microvesicles to permit accumulation of TF within cells. Activation of PAR2 receptor in TF-bearing cells resulted in prolonged activation of p38 which was enhanced by preventing the phosphorylation of Ser253 within the cytoplasmic domain of TF through Ala-substitution. Moreover, the inhibition of p38α resulted in decreased Ser258 phosphorylation and increased TF release as microvesicles. Expression of wild-type TF or Asp253-substituted TF induced cell proliferation via a mechanism that involves the up-regulation of Cyclin D1. In contrast, increased cellular apoptosis was observed in cells expressing Ala253-substituted TF, but only following cell activation. The level of p53 protein, p53-phosphorylation at Ser33, p53 nuclear localisation and transcriptional activity, but not p53 mRNA, were increased in cells expressing the wild-type and Ala253-substituted TF. These observations were reversed by inhibition of p38α using either SB202190 or siRNA-mediated suppression. The expression of bax and p21 mRNA and Bax protein was also increased in cells expressing Ala253-substituted TF, but not in cells expressing wild-type TF. Inhibition of transcriptional activity of p53 with pifthrin-α or inhibition of p38α suppressed the expression of bax. These data suggest that the activation of endothelial cells expressing TF prolongs p38α activation which in turn phosphorylates TF at Ser258 and terminates TF release within microvesicles. This leads to the accumulation of TF within cells and can induce cell apoptosis. The mechanism of apoptosis is mediated via the activation of p38α which in turn phosphorylates p53 at Ser33 and stabilises p53 within the nucleus. This study has shown a mechanism associating increases in circulating TF-containing microvesicles with endothelial depletion and the progression of vascular disease.

Citation

ElKeeb, A. M. (2014). An investigation into the mechanisms of tissue factor-mediated apoptosis in endothelial cells. (Thesis). University of Hull. Retrieved from https://hull-repository.worktribe.com/output/4217145

• Thesis Type: Thesis
• Deposit Date: Oct 1, 2015
• Publicly Available Date: Feb 23, 2023
• Keywords: Biological sciences
• Public URL: https://hull-repository.worktribe.com/output/4217145
• Additional Information: Department of Biological, Biomedical and Environmental Sciences, The University of Hull
• Award Date: Jun 1, 2014