| الملخص: |
Endogenous anti-inflammatory mediators form a complex network triggered in the host to dampen cell activation and promote resolution of inflammation. The glucocorticoidregulated 37-kDa protein Annexin A1 is one such endogenous checkpoint effector, acting on human neutrophils via a specific GPCR termed FPR like-1 (FPRL1). FPRL1 belongs to the family of formyl-peptide receptors, of which FPR (the receptor for formylated peptides) is the prototype. However, very little information is available on the mechanisms governing the export of this protein from activated cells, hence the machinery required to activate the counter-regulatory circuit centred on Annexin A1. Furthermore, little data is available describing the status of Annexin A1 and the FPR receptor family in human disease conditions. These questions have been addressed in my thesis. The analysis of PMNs and monocytes from both Wegener granulomatosis and Giant cell arthritis (GCA) patients provided strong evidence for a deregulation in the Annexin A1 pathway as I observed an increased expression of both FPRL-1 and FPR, along with an elevated cell surface Annexin A1 expression. Moreover, Western blotting against Annexin A1 membrane expression also evidenced elevated protein cleavage suggesting that this is potentially the mechanism responsible for the reported hyper-activated status of the PMNs in these pathologies, a feature I could confirm also in the flow chamber assay, where marked adhesion to endothelial cell monlayers was measured. Significantly, pharmacological manipulation of the Annexin A1 axis could correct cell behaviour, further supporting the notion of a deregulated Annexin A1 system in these conditions. Interestingly, very little difference was observed at the mRNA levels for both the FPRL-1 and Annexin A1 genes in samples analysed from patients suffering from Wegener granulomatosis whilst there was a significant increase in expression of both these genes in the GCA samples when compared to aged matched healthy volunteers. An observation, which suggests that the underlying mechanisms governing the regulation in these two conditions, and their potential impact on the Annexin A1 pathway, might be different. Annexin A1 lacks a signal peptide but nonetheless is abundantly released from activated PMNs; our understanding of the route that is employed for its release is still modest. The analysis of PMN derived microparticles confirmed the presence of the Annexin A1 protein in these microstructures; I was then able to demonstrate that this protein was responsible for microparticle-induced inhibition of PMN recruitment to an 4 activated endothelium in vitro and into the airpouch in vivo. Furthermore, when monitoring levels of Annexin A1 positive microparticles in plasma samples from a number of human inflammatory disease, I could observe that both these microparticle subsets were altered when compared to those found in healthy age matched controls, with higher extent of PMN-derived microparticles (CD62L positive) and Annexin A1 positive microparticles. I had the opportunity to monitor these microparticles longitudinally in RA patients treated with prednisolone over a 2-week period: both CD62L and Annexin A1 positive microparticles were restored back to the values (as percentage and median fluorescence intensity) of healthy volunteers. This result occurred in parallel to an amelioration of the clinical symptoms. The final part of my project involved assessing the anti-inflammatory properties of 5 novel Annexin A1 N-terminal derived peptides developed in collaboration with Unigene (Fairfield, NJ), that are modification of peptide Ac2-26 (which conserves the natural amino acid sequence). In vitro analyses of these peptides identified two peptides as the ones with the highest anti-inflammatory capabilities. In radioligand binding assays, I observed these peptides possessed similar binding affinities to the FPRL-1 as the natural peptide. More in detail, peptide 57 did not bind to FPR in a dose dependent faishon as opposed to peptide 84, even though when assessing p-ERK activity it was noted that both peptides equally activated ERK. When tested in vivo it was observed that both peptides were able to inhibit PMN recruitment into an inflamed mouse airpouch, with peptide 84 showing the highest potency. The findings of this thesis provided evidence for a deregulated Annexin A1 system in the human inflammatory pathologies under observation, hence vasculitis and RA. Treatment of patients with an acute glucocorticoid regimen modulated the Annexin A1 pathway suggesting that the re-establishment of this effector of anti-inflammation, likely to occur also at the functional level, could contribute – at least partly – to the positive clinical effect of glucocorticoids in RA patients. Captivatingly, this acute glucocorticoid treatment was also observed to restore the plasma Annexin A1 positive microparticle levels to those observed in healthy age-matched volunteers, suggesting that these microstructures can potentially be used both as biomarkers of disease and also a measure of treatment effectiveness. Finally I have provided evidence for the antiinflammatory properties of two novel Annexin A1 N-terminal derived peptides that may serve as guidance for the development of novel treatments for inflammatory disorders, depicted on the biology of this intriguing protein that is Annexin A1. |
