Effects of Oxidative Stress On Trabecular Meshwork

It has been proposed by a number of investigators that the disease of glaucoma is related to oxidative damage which occurs with age in the trabecular meshwork (TM). The TM resides in a potentially damaging oxidative environment as a result of continuous exposure to oxidants present in the aqueous humor. For example the aqueous contains significant concentrations of hydrogen peroxide and ascorbic acid, both of which can lead to generation of highly reactive oxygen species. The aqueous humor also contains oxygen and photosensitizers which in combination with light arc capable of producing a highly reactive, potentially damaging compound called superoxide anion. It is well-established that active species of oxygen such as those which may challenge the TM can be damaging to a number of cell constituents including proteins, lipids and DNA. However little is known about the possible harmful effects of these reactive compounds on the TM and the possible relationship to glaucoma. One reason that oxidation is believed to play a possible role in the pathogenesis of glaucoma is the fact that healthy TM possesses a number of mechanisms to defend against oxidative damage. For example TM has been shown to have an active glutathione redox cycle, a system of cellular enzymes which is capable of detoxifying potentially harmful compounds such as hydrogen peroxide and superoxide anion. Similarly, TM has high activities of other antioxidant enzymes such as catalase and superoxide dismutase. Although the TM is known to possess significant antioxidant activity the functions and relative roles of the antioxidant systems in the tissue are not clearly understood. A significant portion of the aqueous outflow pathway is lined by cells of the TM. It has been suggested that the metabolic activities of these cells may be important in regulating aqueous outflow dynamics and that the cells may be associated with underlying causes of glaucoma. Trabecular cell cultures are a useful means of evaluating antioxidant systems present in TM and of investigating oxidative effects which may be related to the development of glaucoma. By culturing cells from calf and cow TM it may be possible to investigate age-related oxidative effects on the tissue. An additional reason for using cultured bovine trabecular cells in the planned studies is that a significant amount of information has been collected during the past several years on the biochemical composition of isolated bovine trabecular tissue. Very little information is available on oxidant detoxification mechanisms present in cultured trabecular cells. Studies are needed concerning the levels of activity of the glutathione redox cycle and other antioxidant systems in cultured TM cells. It will be important to compare values in young and old TM cells with corresponding results for cultured lens epithelium which is known to possess exceptional antioxidant activity. In addition it will be important to evaluate the ability of the TM cells to tolerate oxidative stress. Oxidative effects on DNA in TM cells and the possible relationship to the development of glaucoma have also not been studied. DNA of cultured lens epithelial cells has been reported to be damaged by H2O2 concentrations only slightly higher than normal physiological levels found in the aqueous humor. The first goal of this research project is to culture TM cells from calf and cow eyes and establish parameters which will be employed in oxidation experiments. The parameters will include passage number, cell density and cell number. Baseline values will be determined for various antioxidant mechanisms present in the cells including levels and activities of various components of the glutathione redox cycle. The ability of TM cells to tolerate oxidative stress will be evaluated by exposing the cells to maintained and pulsed levels of hydrogen peroxide, various pressures of oxygen and various concentrations of ascorbic acid. The ability of the cells to maintain normal biochemistry under oxidative stress will be determined. Cell viability will be monitored by various techniques including scanning electron microscopy. Factors evaluated will include cell growth, activity of the enzyme Na+, K⁺-ATPase, cation transport, synthesis of proteins and DNA, DNA damage and effects on soluble, membrane and cytoskeletal proteins as evaluated by electrophoretic techniques. Our laboratory has considerable research experience concerning oxidative processes taking place in the ocular lens. We believe that similar mechanisms may be operative in other types of cells and it is opportune to extend our knowledge in this area to TM which has a crucial role in the outflow mechanism of the aqueous humor and hence to the pathogenesis of glaucoma. Our experience is this general area offers a unique opportunity to extend these studies to TM cells. The broad, long-term objective of this project is to investigate a possible link between oxidative damage to the TM and the progression of glaucoma. A discovery of this type would stimulate development of antioxidant drugs which might lead to a cure for the disease.

2nd Year

It has been proposed by a number of investigators that the disease of glaucoma is related to oxidative damage which occurs with age in the trabecular meshwork (TM). The TM resides in a potentially damaging oxidative environment as a result of continuous exposure to oxidants present in the aqueous humor. For example the aqueous contains significant concentrations of hydrogen peroxide and ascorbic acid, both of which can lead to generation of highly reactive oxygen species. The aqueous humor also contains oxygen and photosensitizers which in combination with light are capable of producing a highly reactive, potentially damaging compound called superoxide anion. It is well-established that active species of oxygen such as those which may challenge the TM can be damaging to a number of cell constituents including proteins, lipids and DNA. However little is known about the possible harmful effects of these reactive compounds on the TM and the possible relationship to glaucoma. One reason that oxidation is believed to play a possible role in the pathogenesis of glaucoma is the fact that healthy TM possesses a number of mechanisms to defend against oxidative damage. For example TM has been shown to have an active glutathione redox cycle, a system of cellular enzymes which is capable of detoxifying potentially harmful compounds such as hydrogen peroxide and superoxide anion. Glutathione (GSH) is a small antioxidant compound made up of three different amino acids. GSH is present in high concentration in most cells including TM. TM also has high activities of antioxidant enzymes such as catalase and superoxide dismutase. Although the TM is known to possess significant antioxidant activity the functions and relative roles of the antioxidant systems in the tissue are not clearly understood. A significant portion of the aqueous outflow pathway is lined by cells of the TM. It has been suggested that the metabolic activities of these cells may be important in regulating aqueous outflow dynamics and that the cells may be associated with underlying causes of glaucoma. Trabecular cell cultures are a useful means of evaluating antioxidant systems present in TM and of investigating oxidative effects which may be related to the development of glaucoma. An additional reason for using cultured bovine trabecular cells in the planned studies is that a significant amount of information has been collected during the past several years on the biochemical composition of isolated bovine trabecular tissue. Very little information is available on oxidant detoxification mechanisms present in cultured trabecular cells. Studies are needed concerning the levels of activity of the glutathione redox cycle and other antioxidant systems in cultured TM cells. It will be important to evaluate the ability of the TM cells to tolerate oxidative stress. Oxidative effects on DNA in TM cells and the possible relationship of DNA damage to the development of glaucoma have also not been studied. Our progress to date has included the investigation of various antioxidant properties of cultured calf TM cells. The cells were found to contain a high level of GSH, no oxidized glutathione and significant activities of the antioxidant enzymes glutathione peroxidase, glucose-6-phosphate dehydrogenase, glutathione reductase, catalase and superoxide dismutase (SOD). The cells also exhibited a significant activity of the hexose monophosphate shunt (HMPS), an essential component of the glutathione redox cycle. In general, the antioxidant activity of the TM cells, on a per mg protein basis, was comparable to that present in cultured rabbit lens epithelial cells. During the last grant period we carried out other experiments which demonstrated the importance of the glutathione redox cycle in cultured TM cells. We found that the cells could tolerate exposure to a maintained level of 0.02mM H2O2 (a concentration similar to that present in normal aqueous humor) without damage but if the activity of the enzyme glutathione reductase was inhibited and the cells were then treated with the same level of H2O2, they were rapidly killed. Similar studies using brief pulses of higher levels of H2O2 demonstrated an inhibition in the rate of growth of the cells and damage to DNA The results indicate that TM cells possess efficient mechanisms for detoxifying physiological levels of H2O2 and for repairing oxidative damage to DNA. However, if the antioxidant systems are inhibited or the level of oxidant becomes too high, significant damage to the cells can result. We also tested a promising new antioxidant compound called TEMPOL which is able to detoxify a toxic cellular oxidant, superoxide anion. We found that TEMPOL was very effective in preventing damage to DNA in cultured TM cells caused by exposure to a high level of H2O2. The results indicate that compounds called free radicals and metals such as iron are involved in the process by which DNA becomes damaged. It is of interest that higher than normal plasma levels of iron have recently been implicated in other clinical disorders such as heart disease. The results of recent studies suggest that it might not be wise for someone to take iron supplements on a regular basis. In more recent work we investigated TM proteins which may link together by disulfide bonds (two sulfer atoms joining together) when exposed to oxidative stress. Interest in protein sulfur groups of TM has been stimulated by the potential for new glaucoma drug development. It is believed that the oxidation and reduction of sulfur groups in the TM may be associated with the control of flow of aqueous humor through the TM (faulty flow of aqueous humor may lead to glaucoma). We have found that the sulfur groups of certain proteins called cytoskeletal proteins in the TM can be oxidized and reduced and we have identified the specific proteins which are involved by using a technique involving the use of antibodies. Two types of cytoskeletal proteins called alpha and beta tubulin appear to form disulfide-crosslinked proteins when either cultured TM cells or perfused bovine eyes are challenged by oxidative stress. The oxidation and reduction of the proteins correlate with changes in the flow of aqueous humor. Understanding the significance of this finding and the role that cytoskeletal proteins such as tubulin may play in the outflow process will require further investigation which will be carried out in the second year of funding from the AHAF. The broad, long-term objective of this project is to investigate a possible link between oxidative damage to the TM and the progression of glaucoma. A discovery of this type would stimulate development of antioxidant drugs which might lead to a cure for the disease.