Pterygia are ocular surface lesions that originate from the limbal region of the eye and grow across the cornea. They consist of a fibrovascular stroma covered by an epithelial layer, and typically occur on the nasal rather than temporal side. Progression of the lesion can result in visual loss upon migration over the central cornea; less advanced pterygia can impair vision through astigmatism and cause discomfort. ? ? Although the etiology of pterygia is not fully understood, it is clear that the development of pterygial lesions is associated with exposure to sunlight. The incidence of pterygia is significantly elevated in populations near the equator or at high altitudes, and it is also common among those who work outdoors (for example, farmers, postal workers, Chesapeake Bay watermen).? ? In order to identify specific genes that may play a role in pterygium pathogenesis, a cDNA library of nine, pooled, surgically removed pterygia was produced and analyzed. DNA sequence was obtained from 2298 randomly picked clones and analyzed using GRIST to group and identify sequence tags. There were 1856 unique clusters, representing single genes. The most abundant transcripts are for eukaryotic translation elongation factor1 alpha 1, clusterin, keratin 13, Ig light chain, calgranulin B, spermidine/spermine N1-acetyltransferase and keratin 4, each representing 0.4-1 percent of the total transcripts. Markers for both conjunctiva (such as keratin 13 and AQP3) and corneal epithelium (such as keratin 12 and AQP 5) are present with conjunctival markers predominating. Several highly expressed genes, including spermidine/spermine N1-acetyltranferase (SSAT) are associated with cell migration. The high levels of SSAT expression led us to examine further the role of this enzyme in pterygium. Immunohistochemistry confirmed that SSAT is indeed abundantly expressed in pterygia. ? ? Previous studies have demonstrated the use of novel spermine analogs to affect SSAT enzyme activity in various cell types through feedback regulation. Two such analogs, N1,N11-diethylnorspermine (BENSpm), and (S)-N1-(2-methyl-1-butyl)-N11-ethyl-4,8-diazaundecane (IPENSpm), were used to study pterygium cell migration. Migration upon treatment with SSAT analogs was quantitatively determined with various concentrations of either BENSpm or IPENSpm (0-10 microM). Migration assays were run for 18h in a CO2 humidified incubator, treated with fluorescent dye and measured using a Victor3 multi-label reader. Our findings suggest that modulation of the enzyme SSAT can significantly affect the migration of cultured pterygium cells based on the treatment with SSAT analogs. Treatment with BENSpm appears to increase pterygial cell migration, while IPENSpm treatment has an opposite effect, consequently reducing migration levels.? ? Polyamine analogs, such as BENSpm, have been shown to induce SSAT mRNA transcription in human melanoma cells, resulting in increased protein synthesis and activity. We show that treatment with BENSpm increases pterygial cell migration, consistent with a role for SSAT in enhancing cell migration. However, our data also show that IPENSpm decreases cell migration, suggesting that IPENSpm may inhibit SSAT?s ability to interact functionally with the integrin alpha9 cytoplasmic domain. Previous work with a human tumor cell line has shown that IPENSpm and BENSpm behave similarly by reducing polyamine amounts through an increase in SSAT activity. Although synthetic polyamine analogs alter the enzymatic activity of SSAT, the possibility cannot be ruled out that conformational changes in SSAT may affect its interaction with integrin membrane proteins to cause changes in migration. Further study will be needed to determine whether this kind of interplay could explain the differences we have observed in the effects of the two analogs.? ? By reducing the level of pterygium migration over the central cornea, specifically-selected and screened analogs could prove to have important therapeutic benefits. We are currently using siRNA knockdown to reduce SSAT gene expression in pterygium cells so that the enyzme?s role can be further elucidated.
Showing the most recent 10 out of 12 publications
