The Cellular Basis of Aqueous Outflow Resistance

Glaucoma is a disease of the eye in which damage to the optic nerve causes progressive loss of sight, leading to blindness. The nerve damage is caused by a period of elevated pressure in the eye. Eye pressure (called intraocular pressure) results from the constant production of fluid, termed aqueous humor. The aqueous humor supplies the correct environment and nutritional requirements for the cells that form the lining of the eye. In addition, a certain minimum pressure is as important in the normal eye as it is in the blood system, acting to keep the eye “inflated.” To maintain normal pressure levels the aqueous humor must pass through a resistance to flow. Supplying the needed resistance is one of the functions of the outflow pathway, particularly the trabecular mesh work and the canal of Schlemm. In glaucoma, production of aqueous humor is about normal, but the resistance to outflow has increased, resulting in a destructive elevation of pressure.   Through many years of searching for remedies to glaucoma, chemical agents that lower intraocular pressure have been found. Most do not work well, or for very long, and are difficult to administer. But since they do work to some degree, they offer important clues as to what systems are involved in lowering resistance to flow, and may also offer clues as to what causes resistance to be too high in the first place (i.e. the cause of glaucoma). Our laboratory is interested in understanding how drugs that lower intraocular pressure exert their effects, and we are interested in understanding these effects at the level of the individual cell. Our goal is to find drugs that are more effective, specific, and longer lasting than those currently available for the treatment of glaucoma. The outflow pathway for aqueous humor consists of a meshwork, or sieve-like structure, and a closed channel, the canal of Schlemm. The meshwork and canal are lined by flat, elongated cells called endothelial cells, the same cell type as the cells that line blood vessels. The cells most likely to be the main contributor to resistance are those lining the canal of Schlemm. The aqueous humor must cross an actual continuous layer of endothelial cells to get into the canal, whereas it merely flows past the cells of the mesh work. Consequently, our long term goals are to determine how the layer of endothelial cells in the outflow pathway can allow normal flow across itself and provide normal resistance to flow, to determine how these processes are altered in glaucoma, and to refine our search for new drugs that might specifically decrease resistance and lower eye pressure.   Unfortunately, it is not easy to look at cell shape changes in response to drug treatment in the whole eye. The techniques, although capable of high resolution, are time consuming and difficult. An alternative is to look at changes in cells of similar or identical type to those found in the meshwork or canal of Schlemm, only grown in cell culture. One can grow various kinds of endothelial cells in culture and study the effects of drugs much more conveniently. Then the goal becomes to establish a relationship between effects seen in culture and effects measured in the whole eye. In the past, work in the laboratory of David Epstein, our senior investigator, has shown that ethacrynic acid, a drug that also works as a diuretic (increasing the production of urine), acts to decrease outflow resistance in intact monkey and human eyes. Other studies in Dr. Epstein’s laboratory have shown specific alterations in endothelial cells in culture after treatment with ethacrynic acid. These changes include a rapid breakdown or disassembly of what is known as the cytoskeleton, or the “bones” of a cell. [The cell skeleton is, in reality, usually in flux, particularly in the case of dividing or growing cells. Cells that have reached maturity, however, only rarely divide and are inhibited from growth by close contact with neighboring cells. They do not have as dynamic a cell skeleton.] Our immediate goal is to test whether the breakdown of the cytoskeleton in response to ethacrynic acid, as seen in endothelial cells in culture, produces the decrease in outflow resistance in the whole eye. If so, it could be that an inability of the meshwork or canal cells to periodically disrupt their own cytoskeletal components is part of the basis of some kinds of glaucoma.

To try to determine the nature of the association between cell shape, the cytoskeleton and outflow resistance, we need to establish at least the general pathway by which ethacrynic acid makes these changes happen. When drugs have effects on cells, especially global changes like causing them to change shape, they can act by directly binding to and disrupting the physical, structural components of the cell responsible for maintaining shape, or they could perturb the signalling processes that continually tell the cellular components what to do. Calcium is normally kept in very low abundance in the cell fluid (the “cytoplasm”), and increases in calcium are a powerful signals that can alter many cellular processes. We think ethacrynic acid may cause an increase in calcium in the cytoplasm, which is part of the pathway by which it induces its beneficial effects. If true, this would tell us a great deal in itself, and it would also open up many new approaches to lowering eye pressure. For example, it could be that the calcium rise is sufficient in itself to generate the changes we see, in which case other calcium activating drugs might also be effective glaucoma drugs. If calcium ion is not affected, on the other hand, it is still interesting and potentially important, in that other signalling pathways, and another set of drugs, could then be tested.

Glaucoma is a disease of the eye in which damage to the optic nerve causes progressive loss of sight. The nerve damage is caused by a period of elevated pressure in the eye due to increased obstruction or resistance to the exit of aqueous humor, the fluid that bathes and supports the eye. In glaucoma, production of aqueous humor is about normal but the resistance to outflow, which takes place through a sieve-like structure called the trabecular meshwork, has increased, resulting in a destructive elevation of IOP. The meshwork is lined by flat, elongated cells called trabecular meshwork cells. Our long term goals are to determine how the endothelial cells of the outflow pathway can provide normal resistance to flow, to determine how resistance is altered in glaucoma, and to refine our search for new drugs that might specifically decrease resistance and lower eye pressure. In the past, work in the laboratory of our senior investigator, David Epstein, has shown that ethacrynic acid, a drug that is commonly used as a diuretic, acts also to decrease outflow resistance in intact monkey and human eyes. It is not known, however, how ethacrynic acid acts on the cellular level to lower outflow resistance. We formed the hypothesis that ethacrynic acid acts by increasing the levels of calcium in the cells of the outflow pathway. [The level of calcium in cells is very often used as a signal to tum· on new processes, or to begin growing, or for muscle cells to contract] We planned to test this hypothesis by monitoring cellular calcium levels during exposure to ethacrynic acid and comparing responses to those of known calcium-active drugs. We also planned to test ethacrynic acid in experiments that measure 1M cells ability to contract, and to test calcium-active drugs in experiments measuring outflow resistance in whole eyes. In this way we hoped to establish whether calcium activation played a role in the ability of ethacrynic acid to lower outflow resistance. In the first year of the our study we have determined that 1M cells are indeed contractile, and that ECA does induce 1M cell contraction, with a similar time course as drugs that cause an increase in calcium. However, we have also shown that ECA does not cause a rise in calcium in other endothelial and porcine (pig) 1M cells. We have also demonstrated that agents that produce a rise in cell calcium do not mimic the effects of ethacrynic acid. Thus we have eliminated one of the most common pathways for drug action. This has allowed us to focus on other signalling pathways as potential mediators of the effects of ethacrynic acid on outflow. We have since tested stimulators of three other common signalling pathways to see if they could produce effects similar to those of ethacrynic acid. We have found that stimulators of the cyclin AMP, cyclin GMP and protein kinase C pathways also· do not produce effects similar to those seen after treatment with ethacrynic acid. We have seen positive results in the realm of still another of the most common sign pathways, however. Blockade of certain kinases have been shown to block the effects of ethacrynic acid. This points us to a set of pathways that may be very important in causing the beneficial effects of ethacrynic acid, and opens up a new and promising area for study.

2nd Year

Aqueous humor is produced inside the eye where it bathes and nourishes the eye tissues. The aqueous leaves the eye primarily through the trabecular meshwork and the wall of Schlemm’s canal. These structures filters the aqueous humor and also supplies a needed resistance to the outflow, allowing pressure in the eye to be higher than in the veins. In many types of glaucoma, the pressure inside the eye is too high, and this produces damage to the optic nerve, eventually causing blindness. In primary open angle glaucoma, there is no obvious obstruction to aqueous flow. The inner wall of Schlemm’ s canal, like the walls of ones blood vessels is composed of interlocking cells. We believe that the answer to the increased resistance found in primary open angle glaucoma will be found in the interaction between these cells. Specifically, the cells must maintain a balance between stiffness and flexibility, so that they can provide some resistance to aqueous outflow without generating too much. Our idea is the perhaps the cells in the wall of Schlemm’s canal of glaucoma patients have lost flexibility critical to maintaining the low resistance to flow necessary for health. In the past, work in the laboratory of our senior investigator, David Epstein, has shown that ethacrynic acid, a drug that is commonly used as a diuretic, acts also to decrease outflow resistance in intact monkey and human eyes. It is not known, however, how ethacrynic acid acts on the cellular level to lower outflow resistance. In cells grown in culture, ethacrynic acid causes cells to separate from each other, which may be a correlate to the opening of spaces in the outflow pathway (lowering outflow resistance). We formed the hypothesis that ethacrynic acid acts by increasing the levels of calcium in the cells of the outflow pathway. [The level of calcium in cells is very often used as a signal to turn on new processes, or to begin growing, or for muscle cells to contract.] If this were true, we could then look for other drugs that might work better or more safely. We grew cells from the trabecular meshwork, the filtering cells in the eye to test our hypothesis.

We tested the levels of calcium after exposure to ethacrynic acid, and found that these levels did not increase. We showed that we could see calcium increases after exposure to certain drugs that are known to produce such increases, so we knew our system was working and that we knew how to use it We found that the ethacrynic acid interfered with the dye used to see calcium, and switched to a second dye to be absolutely sure of our result. We then went on to test our second specific aim, which was to continue to look for clues as to the signalling systems most affected by ethacrynic acid to produce its results on cell shape. Calcium is one of the major signals used by the cell to begin a number of important behaviors. Other signalling systems in cells include cyclin AMP, cyclin GMP and protein kinase C. Instead of testing the levels of each of these after ethacrynic acid exposure, we tested drugs that are known to act primarily by stimulating each of these systems, and asked whether, in our cultured cell system, any of these drugs mimicked the activity of ethacrynic acid on cell shape. These results were presented at our annual research meeting in 1994, and basically none of the stimulators of these specific pathways did really mimic the effects of ethacrynic acid.   Although this result was again negative, it helped us focus on some of the remaining systems that could be affected by ECA, and this has lead to some real progress. In the last year we have looked carefully at another important signalling system that is present at the sites of cell attachment. This system is dependent on the addition of phosphate groups onto the structural proteins that comprise the junctions themselves. We had to learn some new techniques to test whether ethacrynic acid caused changes in phosphotyrosine early enough after drug addition to think it might be important. We found that certain proteins are definitely dephosphorylated after ethacrynic acid addition, and that this takes place very rapidly after drug addition. We can now ask a number od related questions to determine. whether this is indeed the transduction step we have been looking for. These results were presented at our annual research meeting this spring.