| Summary: | In response to injury, the collagen-producing cells fibroblasts and myofibroblasts play an important role in promoting extracellular matrix deposition and release of inflammatory mediators in order to repair the damaged tissue. However, persistence and over-activity of these cells is associated with excessive collagen deposition and hence development of brosis, a pathological scarring process that leads to destruction of organ architecture and impairment of organ function. Removal of fibroblasts and myofibroblasts during fibrogenesis by apoptosis is therefore required for the normal resolution of tissue repair responses. Proteinase-activated protein 4 (PAR4) belongs to a subfamily of multifunctional G protein-coupled receptors which are activated by the proteolytic unmasking of a tethered peptide ligand that resides within their N-terminus. In addition to its well-studied role in platelet function, PAR4, like the other PAR family members, is thought to be involved in pro-inflammatory responses and fibroproliferative processes. However, the role of PAR4 in these processes remains largely unknown. The hypothesis of this thesis is that during injury, PAR4 expression is upregulated and this somehow contributes to apoptosis. To test this, the expression of PAR4 was examined in two types of fibroblasts, cultured from human lung and renal explants. Results indicate that PAR4 is not expressed in these cultured fibroblasts. Surprisingly, contradictory to a previous observation, exposure of lung fibroblasts to lipopolysaccharide did not consistently induce PAR4 expression. As an alternative to the use of PAR4-expressing fibroblasts, a stable PAR4-expressing human cell line (HEK-PAR4) was generated. Using these cells in an in vitro signalling assay, PAR4 was found to confer responsiveness to PAR4 activating peptide but not to a serine protease cathepsin G, which was previously shown to activate PAR4 in platelets. Interestingly, the observation that cathepsin G-treated cells elicit a reduced signalling response to the stimulatory action of PAR4 activating peptide suggests that cathepsin G might cleave PAR4 at the ECL2 domain and cause PAR4 to be unresponsive to the PAR4 AP. Next, serine proteases cathepsin G and trypsin were demonstrated as apoptosis inducers for human lung and renal fibroblasts. They induced pronounced morphologic changes in the fibroblasts (which do not express PAR4). In HEK-PAR4 cells, cathepsin G induced similar apoptotic level in comparison to empty vector control. Together, these findings suggest that cathepsin G can trigger apoptosis independently of PAR4. On the other hand, PAR4 activating peptide triggered higher percentage of apoptosis in HEK-PAR4 cells compared to empty vector control, suggesting that apoptosis is mediated by PAR4 signalling in this case. Epithelium has a role in modulating a variety of inflammatory processes through the production and release of inflammatory cytokines. Apoptosis of damaged epithelium during inflammation has been reported to promote fibroblast growth and collagen deposition. In the last part of this thesis, media derived from confluent epithelial cell cultures were demonstrated to induce proliferation of lung and renal fibroblasts in a concentration dependent manner. This observation preliminarily supports the idea that epithelial cell injury may promote the proliferation of fibroblasts via the secretion of multiple cytokines. In conclusion, this thesis demonstrates that apoptosis can be induced, first, by cathepsin G and trypsin in human primary bronchial fibroblasts and human primary renal fibroblasts independently of PAR4, and second, by activation of PAR4 in a non-fibroblastic PAR4-expressing cell line.
|
|---|
