Elucidating the regulatory mechanisms of pterygium

Fang, Chunlai (2013) Elucidating the regulatory mechanisms of pterygium. Doctoral thesis, University of East Anglia.

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Purpose: Pterygium is characterised as invasive, proliferative fibrovascular altered conjunctival tissue. The extensive vascular network is likely to significantly contribute to the progression of the disease. In the present study we investigated the effects of reduced serum (to mimic a suppressed blood supply) and treatment with transforming growth factor β on cell signalling and function of pterygial derived fibroblasts.
Methods: Pure fibroblast cultures were established from cell outgrowths of pterygial tissue. Immunochemistry techniques were used to identify cell phenotype, tissue features and cell signalling. Growth and migration of pterygial-derived fibroblast were evaluated using a patch growth assay, MTS assay and a scratch wound assay. Intracellular calcium levels were determined using Fura-2 detection in response to ligand stimulation using a 96-well plate format. RT-PCR and Western blot were utilized to detect cell transdifferentiation. Human angiogenesis protein array was used for investigating angiogenic activities in pterygial fibroblasts. Gene microarray was employed to provide a global profile of gene expression in unstimulated and treated (1ng/ml TGFβ2 and 10% serum) conditions.
Results: A progressive increase in serum level resulted in promotion of pterygial cell growth. A significant increase in intracellular calcium level was observed in response to histamine, ATP, acetylcholine and epidermal growth factor in serum-maintained cells. However, no significant changes were observed when cells were maintained in serum-free medium. 1M thapsigargin induced a significantly greater increase in intracellular calcium level in the serum maintained group relative to serum starved cells. Pre-incubation of cells with 1M thapsigargin ablated ligand-induced calcium responses. Disruption of calcium signalling through thapsigargin treatment significantly perturbed cell growth and migration. Smad2/Smad3 translocated to the nucleus in response to TGFβ in pterygial fibroblasts. TGFβ2 stimulated transdifferentiation of pterygial fibroblasts to myofibroblasts. Proteome Profiler™ Array data revealed that pterygial fibroblasts release angiogenic factors including IL-8 and VEGF following treatment with 10% serum and TGFβ2. Gene microarray demonstrated that a total of 103 genes were up-regulated and 53 genes downregulated by more than 2 fold in pterygial fibroblast treated with 10% serum. A total of 198 genes were up-regulated and 197 genes were down-regulated by more than 2 fold in pterygial fibroblast exposed to TGFβ2.
Conclusions: Calcium signalling was suppressed in pterygial--derived fibroblasts in response to serum-deprivation. The store plays a key role in cell growth and migration of pterygial derived fibroblasts. TGFβ induce the Smad signalling pathway and transdifferentiation in pterygial derived fibroblasts. Pterygial fibroblasts release a number of angiogenic factors and up-regulate transdifferentiation genes in response to serum or TGFβ2. Serum level/blood supply and TGFβ2 are likely to play a key role in the pathogenesis of pterygium.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Users 2593 not found.
Date Deposited: 16 Jan 2014 15:26
Last Modified: 16 Jan 2014 15:26
URI: https://ueaeprints.uea.ac.uk/id/eprint/47282

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