Transfer of an Actin Regulator Gene to Outflow Cells
About the Research Project
Program
Award Type
Standard
Award Amount
$50,000
Active Dates
April 01, 1997 - March 31, 1999
Grant ID
G1997409
Summary
Glaucoma, a disease that affects a high percent of the adult population, is commonly characterized by an elevated intraocular pressure that if uncontrolled, may result in damage to the optic nerve and irreversible blindness. Glaucoma is the second most important cause of nonremedial blindness in the United States. During normal physiological conditions, the fluid inside the eye is produced by the cells of the so called ciliary body and leaves the eye through a sponge-like tissue (called trabecular meshwork) situated toward the front of the eye. The precise flow of this fluid, just a little over one ml per hour, helps maintain the necessary pressure inside the eye. Theoretically, when one of the two mechanisms that control the proper flow, either an overproduction of the fluid by the ciliary body or a blockage of its drain in the trabecular meshwork, the pressure inside the eye rises. Clinically, it is the blockage of the drain mechanism the one mainly responsible for the raise in intraocular pressure. The trabecular meshwork tissue acts a filter through which the eye fluid percolates on its way out of the eye. As such, it is not densely packed if not formed by a mixture of cells and extracellular material rearranged in a loose manner. The cells mastermind the whole architecture of the tissue. Under certain stimulus, they are able to secrete more or less extracellular material. They have phagocytic activity, or in other words, are able to ingest and process passing clogging material and thus contribute to removal of cell debris. They are also subject to change their shape and by doing that, influence the shape and size of the pathway through which the fluid flows out. We believe that changes in cell shape are very important for the definition of the pathway and adequate draining process. Our hypothesis is that by temporarily provoking changes in cell shape we can counteract some of the blockage to the fluid and thus contribute to better outflow rates and reduction of intraocular pressure. One of the ways to induce changes in cell shape is by trying to alter the configuration of the cell’s own skeleton (cytoskeleton). There are two main players in the formation of the cytoskeletal network, the actin and the microtubules. Both constitute the scaffold that supports the cell body. Furthermore, they are dynamic. Microtubules and actin fibers are in constant restructuring mode with some of these changes being the consequence of external stimulus. Numerous reports in the literature support our hypothesis. Different drugs known to have an effect on the rates of flow of the eye fluid, affect also cell shape and cytoskeletal organization. One of these drugs, cytochalasin, has been intensively studied. Cytochalasin, on one hand interferes with the formation of actin and, on the other has been proven to facilitate the fluid draining in experiments where living monkeys were treated locally with the drug. However, pharmacological agents are far from being specific to one pathway. Our goal, is to specifically determine which of the drug induced effects has a direct influence on the increase rate observed. In this proposal, we aim to manipulate the configuration of the actin cytoskeleton by using the new, highly specific, gene transfer/gene therapy technology. As alternative way to use conventional drugs, gene transfer techniques offer the possibility of temporarily provide a given tissue with proteins that will target a very specific cell function. Each gene delivered inside the cell acts as a mold for the production of a particular protein. Of the available carriers to deliver genes to cellular tissues only viruses have evolved to naturally transfer their genetic information to host cells. Among the recombinant viral systems available, replication-deficient adenovirus has the ability to take its genes into the tissue of the trabecular meshwork. A major breakthrough during the last few years has been the discovery of a family of proteins, the Rho family, whose function is specifically to regulate the organization of the actin cytoskeleton in eukaryotic cells. Studies conducted in prestigious laboratories have shown that when Rho is experimentally introduced into a cell, the cell undergoes dramatic shape changes, including formation of actin fibers and evident contractility. It is because of these discoveries that we want to apply the study of Rho to the cells of the trabecular meshwork. Our specific aim includes to genetically engineer the Rho gene into the carrier (the adenoviral vector) and to deliver this gene to the cells of the trabecular meshwork. We plan to use experimental systems with both isolated cells growing in plastic dishes and whole organs, where the tissue is not disrupted and is maintained within its original architecture. We will then measure the rate of the eye fluid through the draining tissue before and after transferring the gene Rho (that in turn will make the protein Rho inside the cell). We will try to establish a correlation between the two phenomena and search for conditions that could provide an increase of the flow rate of the eye fluid. We think that even if these experiments do not result in the expected effect, they will provide an invaluable step toward the understanding of the molecular and cellular mechanisms underlying the occurrence of glaucoma.
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