HTM Cells: Steroid Effects on Function and mRNA Expression

Normally, aqueous humor drains through outflow channels within a “self-cleaning” filter composed of cells and connective tissue before returning to the venous circulation. This filter is called the trabecular meshwork. In glaucoma, the drainage of aqueous is impeded and pressure builds up in the eye. Administration of steroid medications (glucocorticoids) to treat inflammation in the eye can cause elevation of intraocular pressure (IOP) and lead to a “steroid” glaucoma, but the mechanism by which aqueous outflow is blocked is unknown, for this and for many other types of glaucoma. Our laboratories have been investigating the basis for steroid glaucoma and other glaucomas through the use of lines of cells grown from human trabecular meshwork (HTM cells). One focus of our research has been to explore the effect of steroid treatment on the ability of normal HTM cells to engulf debris and other material which can clog the outflow channels. This cell function is called phagocytosis (cellular “eating”); our experiments with cultured HTM cells indicate that steroid treatment reduces this ability by 40-60%. Our proposed studies include investigation of possible steroid-induced alterations which reduce phagocytosis, both in the machinery inside the cells and in the proteins and other elements which reside on the cell surfaces. For comparison, we also propose to study phagocytosis in other special lines of HTM cells grown from tissue obtained from patients with steroid glaucoma and with primary open angle glaucoma (the most frequent type of glaucoma). Another important function of HTM cells may be to regulate the passage of aqueous humor. We have established a method for measurement of fluid flow through a single layer of HTM cells grown on a filter-support system, and have shown that important glaucoma medications, such as epinephrine, can cause increased flow through the cell layer, a change which may be related to cell shape alterations which produce spaces between the cells and allow for increased flow. With AHAF support, we now propose to extend these studies to include the effects of steroid treatment on flow. Our preliminary observations indicate an intriguing 500% decrease in flow through cell-filter cultures treated with glucocorticoids. We plan to extend these studies to monitor changes over several weeks of glucocorticoid treatment, and to examine the treated cells by electron microscopy to look for changes in cell shape, size and modifications in the spaces between cells. Special immunological labels will be used to investigate possible changes in the cell junctions and cytoskeletal structures. Biochemical studies in our laboratories have also shown that there is a progressive induction of certain cellular and secreted proteins by HTM cells during a 10-day glucocorticoid treatment with a time-dependence similar to clinical steroid effects on IOP. We have applied the latest molecular biology methods to isolate particular induced messenger RNA’s and to produce the proteins in a cell-free system. Through the support of this AHAF grant, we have isolated one special genetic clone, and we have demonstrated that this clone, which is a unique gene sequence, is induced progressively over the ten-day period. This is the first demonstration of a delayed, progressive steroid effect on gene regulation in HTM cells which parallels the clinical course of IOP increase. Our proposed studies seek to understand whether the progressive induction is a primary effect of the steroid treatment, or whether protein synthesis is required. We also plan to investigate whether the time effect is related to stability of the messenger RNA. Finally, we will analyze the gene sequence of this message to identify elements which could contribute to its stability or its progressive induction by glucocorticoid treatment In summary, we will use a multifaceted approach which is designed to explore glucocorticoid-induced alterations at cellular, biochemical and molecular biological levels. These studies should provide significant insight into the steroid-induced changes in aqueous outflow which may suggest new modes of prevention or therapy for steroid and other glaucomas.

2nd Year

In the normal eye, aqueous humor drains through outflow channels within a “self-cleaning” filter composed of cells and connective tissue before it returns to the venous circulation. In a glaucomatous eye, aqueous drainage is impeded and pressure within the eye (intraocular pressure or IOP) builds up. IOP elevation can be caused by administration of steroid medications (glucocorticoids) used for treatment of inflammation in the eye. However, the mechanism by which aqueous outflow is blocked is unknown. Our laboratories have been investigating the basis for steroid and other glaucomas through the use of lines of cells grown from human trabecular meshwork (HTM cells). The hypothesis we are testing is that if we can identify specific responses to steroid treatment in these cells, which have a time course like that of “steroid” glaucoma, then we may discover the basis for this disease and perhaps that of other glaucomas as well. The steroid given in these experiments is one of those commonly given by ophthalmologists as eye drops to treat irritation and inflammation of the eye. Our research program has three major aims. One goal is to apply recently developed molecular biological and other biochemical techniques to identify a pattern of proteins (and the “messenger” RNA molecules which code for them) which may be responsible for steroid glaucoma. Major alterations in the production of messages for certain types of proteins normally made by HTM cells, as well as for enzymes which digest these proteins were found. Our findings demonstrate a delayed, progressive steroid effect on certain gene products in HTM cells which parallels the clinical course of steroid effects on IOP in patients. A second goal is to test the effect of steroid treatment on the ability of HTM cells to “clean” the aqueous outflow channels of debris. We examined this phagocytosis (cellular “eating”) in HTM cells and found that steroid treatment reduces this important cell function by about 50%. Recently, we have also determined that adding a certain cell attachment glycoprotein (laminin) to the cultures could reverse the steroid effect. Investigations to determine whether only cell surface changes, as indicated by the laminin results, or other factors found within cells, such as cytoskeletal proteins, are involved in the phagocytic effects are now underway. The third goal is to see whether steroid addition affects the regulation of the passage of aqueous humor, which may be another very important function of HTM cells. We have developed a model system to measure the fluid flow through a single layer of HTM cells grown on filter suppor ts. New long-term studies indicate that there is a profound reduction (3-5-fold) in this flow in response to glucocorticoid treatment. A major response occurs after 10 days and is maintained for as long as 43 days. During the next year, we plan to use special labeling techniques to determine whether cell volume changes are involved in this response, and to apply electron microscopy and immunological techniques to look for changes in cell junctions and cytoskeleton. This broad-based approach to the study of steroid induced changes in aqueous outflow involves several levels of examination. Investigations at cellular, biochemical and molecular biological levels should enable us to determine the basis of the steroid response and may suggest new treatment or prevention modalities for steroid and other glaucomas.