Even with all the historical research, it was difficult to understand how the ALDH story and the increased activity of degradative enzymes were related to each other. Work in our laboratory (Cedars-Sinai Medical Center, Los Angeles) examined another pathway by which the cornea can process the free radicals: a series of interactions with nitric oxide.
(Fig. 3)
Nitric oxide is formed when arginine (an amino acid) interacts with nitric oxide synthase (an enzyme) to form citrulline and nitric oxide. Nitric oxide can diffuse a short distance through tissue and reacts with superoxides to form peroxynitrites. These peroxynitrites are also harmful to tissues and can be identified by using antibodies to nitrotyrosine. Our experiments show that KC corneas have an increased amount of nitroytrosine staining compared to normal corneas or other diseased corneas (Fig 3). This increased staining in KC corneas suggests that they have additional destructive substances in the tissue.
If it is true that the KC corneas possess higher levels of oxidative damage, this could lead to irreversible damage of some of the KC cells. When cells are damaged in this way they undergo “programmed cell death,” called apoptosis. Apoptosis as a process has been known for some time; however, the connection of apoptosis to KC is new. Dr. Steve Wilson’s laboratory provided evidence that some of the KC cells do indeed undergo apoptosis. Normally, the human cornea has very little apoptosis occurring. Dr. Wilson demonstrated that KC corneas have apoptosis occurring in the anterior stroma and epithelium, especially in the areas of breaks in Bowman’s layer. (Fig 1)
Our laboratory’s molecular studies support this finding. We have demonstrated that cells in KC corneas produce a unique enzyme that is not found in normal corneas or corneas with other diseases. This enzyme is called leukocyte common antigen related protein (LAR). This transmembrane protein (part of it is within the cell and the other part is outside the cell membrane) belongs to a family of proteins called phosphotyrosine phosphatases. These enzymes are responsible for the removal of phosphatases from the amino acid, tyrosine. They play a role in cell to cell interactions, cell-matrix interactions, cell differentiation and proliferation.
The addition or removal of phosphatases from tyrosine is a method by which cells activate or inhibit different biochemical pathways. The enzymes responsible for the addition of the phosphates are called kinases, and the enzymes that remove phosphates are called phosphatases. Normally the cell has a balance between these enzymes, both of which play an important role in cell regulation. Our molecular studies show that there is an increased expression of LAR in the KC cells and corneas. This implies that the KC corneas are sending signals for biochemical processes that are not found in normal corneas or other diseases. We believe that the appearance of LAR in KC corneas also supports the notion of apoptosis in KC corneas because other investigators have shown in other systems that LAR expression plays a role in apoptosis.
But what happens to the cells that are only partially damaged by oxidative stress or destructive substances? We believe that these cells undergo a process of wound healing. Studies by numerous investigators support this. They show that various components of wound healing (such as transcription factors, cytokines, and their receptors) are reported to be increased in the KC corneas. For example, IL-1 receptor, prostaglandin E2 and the transcription factor, SP1, are increased in KC. TGF-beta, IL1 and heat shock protein27 are also increased in KC corneas and other diseased corneas. In addition, reports of localized areas of fibrosis and scarring are reported in KC corneas.
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Reviewed 2012
Dr. Kenney is Professor and Director of Ophthalmology Research, Ophthalmology School of Medicine at the University of California-Irvine in Irvine, CA.
