AbstractThe bronchial epithelium serves as a protective cellular barrier and the first site of contact for damaging inflammatory and physical stimuli from the environment. Previous in vivo studies in guinea pigs have demonstrated that normal bronchial epithelial repair is both rapid, and dependent on the formation of a provisional fibrin matrix that is exclusively plasma-derived. This study tests the hypothesis that local release of coagulation factors from bronchial epithelial cells and subsequent fibrin formation supports bronchial epithelial repair independently of plasma proteins.
Cells of the 16HBE 140- cell line and primary normal human bronchial epithelial (NHBE) cells were cultured in serum-free medium to confluence and mechanically wounded. The expression of coagulation factors in response to wounding was examined at the protein and mRNA level using immunohistochemistry, immunoblot, western blot and RT-PCR. The role of coagulation factors in epithelial repair was investigated using neutralising antibodies and specific inhibitors and monitored by both standard light and kinetic video microscopy. The role of PARs in fibrin formation and epithelial repair was investigated using specific PAR-l and PAR-2 peptide agonists.
Active TF was constitutively expressed in 16HBE 140- cells and initiated the coagulation cascade that was essential for epithelial repair. Fibrinogen and FXIII were rapidly released, within 20 minutes in response to wounding and were essential for wound repair. Fibrin formation and turnover was demonstrated by the release of D-dimers from epithelial cell layers in proportion to the degree of wounding. Expression of tissue factor (TF) and factor VII (FVII) mRNA increased post-wounding over an extended time course up to 12 hours. The bronchial epithelium is therefore a source of preformed coagulation factors released rapidly in response to mechanical wounding and the autonomous formation of a cross-linked fibrin matrix is essential for wound repair.
In asthma, the bronchial epithelium has increased susceptibility to injury and normal repair mechanisms are compromised, contributing to bronchial hyper-reactivity. The data presented in this thesis suggests novel therapeutic strategies to enhance repair of the damaged bronchial epithelium in asthma, such as the delivery of recombinant FXIII by inhalation or the use of specific agonists of PAR-2, which is associated with cytoprotection in the lung.
|Date of Award||Sept 2007|
|Supervisor||Jan Shute (Supervisor)|