The trabecular meshwork of the eye is a principal site of outflow resistance to the aqueous humor. The trabecular meshwork is subjected to stretching by the ciliary muscle. In an effort to understand the effects of mechanical stretch on the trabecular meshwork, we have grown human trabecular meshwork cells on silicone sheets and then stretched them by 10%. The cells have an altered morphology and are elongated when their support is stretched. Besides a rapid change in tyrosine phosphorylation on several proteins, there is a prominent change in the organization of actin within the cytoplasm. One of the proteins closely lined to actin polymerization, alpha B-crystallin, rapidly disappears from the cell. Over the next six hours, the cellular shape and actin organization returns to the appearance of unstretched cell. The level of alpha B-crystallin starts to return to control levels at this time. This crystallin may be an important component in stabilizing actin filaments. Another protein called myocilin has been linked genetically to some forms of glaucoma. We have studied the expression of this protein in trabecular meshwork slices as well as human trabecular meshwork cells grown in tissue culture. This protein has a molecular mass of around 57kDa. In organ cultured human meshwork, the mRNA level of this protein increases. We have also seen an increase in the mRNA levels in cultured cells treated with dexamethasone, heat, and oxidative stress. Because of the difficulty in obtaining primary human trabecular cells in culture, we have attempted to get a mouse cell line established. The mouse trabecular meshwork is similar to human and contains a collecting canal reminiscent of Schlemm's canal. We have been able to get a cloned line of mouse trabecular meshwork cells by using the """"""""immorto"""""""" transgenic mouse. The animal carries a temperature sensitive large T-antigen from the SV-40 virus linked to a promoter activated by gamma interferon. By growing cells under permissive conditions, we are able to maintain an actively dividing population of cells. When switched to higher temperatures without interferon, the cells stop dividing and start to express certain proteins that are present in trabecular meshwork. One of these proteins is myocilin. The mouse myocilin protein is slightly smaller than the human one. Fortunately the antibodies that we produced using a peptide fragment from the human also cross-react with the mouse. These cells are currently being used to investigate other properties of the trabecular meshwork.
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