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Glavini, Aikaterini
Languages: English
Types: Doctoral thesis
Subjects: RE
Corneal dystrophies are inherited bilateral diseases that compromise the transparency of the cornea. They are difficult to treat and re-occur in corneal transplants. A number of anterior stromal and Bowman's layer dystrophies have been linked to keratoepithelin. This is an extracellular matrix protein, synthesised and secreted by the epithelium and deposited in the accumulations found in the epithelial and stromal areas of the affected individuals. Keratoepithelin binds to different types of integrins on the cell surface and has been shown to regulate migration, proliferation adhesion and tumourgenicity. The gene encoding keratoepithelin, 3ig-h3, is induced by growth factor TGF-p and has been involved with growth, differentiation, development and wound healing. The aim of this study was to explore the effects the wild type and mutant forms of this gene and protein have on primary corneal cells. pig-h3 cDNA was synthesised from fibroblast mRNA and in vitro mutagenesis was carried out to produce mutant constructs encountered in dystrophies Granular Groenouw Type I and Thiel Behnke, R555W and R555Q respectively. Replication deficient adenoviruses were constructed to achieve overexpression of the wild type and mutant {3ig-h3 genes in cultured cells. Native recombinant keratoepithelin protein was expressed and purified corresponding to the wild type and mutant cDNA sequences. Primary corneal epithelial cells and keratocytes were cultured in the presence of the recombinant constructs and the effects on adhesion, proliferation, migration and apoptosis were monitored. Wild type recombinant keratoepithelin promoted significantly the adhesion of both epithelial cells and keratocytes (p<0.001). The mutant proteins had the same effect as the wild type on epithelial cell adhesion, but keratocytes showed a decreased adhesion (p>0.05). The corneal epithelial cells overexpressing wild type 3ig-h3 adhered significantly better (p<0001) than the control and the keratocytes showed higher adhesion (p<0.001) during overexpression of the mutant R555W 3ig-h3. Epithelial proliferation was increased in response to the recombinant proteins (50.27 1.93% in the wild type compared to 35.59 6.26% in the control) and overexpression of pig-h3 genes (p<0.001), whereas the keratocyte proliferation was unaffected. R555W mutant protein was associated with slower migration rate of epithelial cells compared to wild type protein, whereas the keratocytes migration did not seem to be affected in any case. Difficulties were encountered when using adenoviral constructs to assess the effects of overexpression of pig-h3 transgenes on cell migration and proliferation. Annexin-V and propidium iodide staining suggested there may be an increase in the apoptosis of cells in the presence of the mutant recombinant proteins and adenoviruses. However, the apoptosis assay could only provide an indication of the effect of the recombinant constructs since the data could not be accurately quantified and analysed, due to a discrepancy on the setup of the FACS analyser. These findings indicate the feasibility of using recombinant keratoepithelin proteins and pig-h3 overexpressing adenoviruses to assay their effects on primary corneal cells. The diversity of the behavioural responses of corneal cells to mutant and wild type keratoepithelin and (3ig-h3 gene can provide useful insight in the pathology of the anterior corneal dystrophies.
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