Abstract
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 mimick a suppressed blood supply) on cell signalling events in cultured pterygial derived fibroblasts.
Methods: :
Pterygium specimens were cultured in vitro and cellular outgrowths were phenotypically characterised as fibroblasts using vimentin and cytokeratin staining. Growth was evaluated using the MTS assay. Intracellular calcium levels were determined using Fura-2 detection methods in a 96-well plate format using a BMG labtech FluoStar Plate reader. To assess the effect of serum deprivation on calcium signalling we used three known calcium mobilising agonists; histamine (1, 10 and 100µM), EGF (0.1, 1 and 10ng/ml) and ATP (1, 10 and 100µM). These were applied to cells that had been maintained in 10% FCS supplemented EMEM or serum-starved for the 24 hours prior to analysis.
Results: :
Pure cultures of pterygial-derived fibroblasts were established, which were vimentin positive and cytokeratin negative. Peak growth detected using the MTS assay was obtained with 10% FCS, but significant reduction in growth was not observed until serum concentrations fell to ≤ 2% FCS. Transfer to serum-free medium reduced detection levels to 54.6% of the peak response. A significant increase in intracellular calcium was observed with addition of 1, 10 and 100µM histamine to serum maintained cells; however no significant change was observed in serum-starved cells. EGF did not induce a calcium response in serum-starved cells, but in serum maintained cells a significant elevation of intracellular calcium was detected with 1 and 10ng/ml EGF. ATP was found to significantly induce intracellular calcium in both serum-maintained and serum-starved cells; however, the response in serum maintained cells remained significantly higher than their serum starved counterparts.
Conclusions: :
Pterygial-derived fibroblasts are sensitive to serum-deprivation, such that a reduction of growth is observed in conjunction with reduced calcium signalling activity of known stimuli. The current data therefore support the strategic reduction of the vascular network in pterygium as a therapeutic approach.
Keywords: pterygium • calcium • blood supply