Eyeworld

EW CORNEA 64 August 2018 Keratoconus patient Healthy patient Source: Jesper Hjortdal, MD Cultured cells from both keratoconus and non-keratoconus patients were exposed to a crosslinking procedure to determine any changes in metabolites of the cells afterward. Source: Dimitrios Karamichos, PhD by Liz Hillman EyeWorld Senior Staff Writer Research compared metabolites in healthy cells vs. keratoconic cells pre- and post-crosslinking T he fact that corneal cross- linking (CXL) can increase the ability of collagen fibrils to form stronger bonds with each other and thus stop progressive kerato- conus (KC) is well documented in published literature, but just what is going on metabolically in the keratoconic cells pre- and post-cross- linking has not been studied. Research out of the University of Oklahoma Health Science Center, Oklahoma City, and Aarhus Uni- versity Hospital, Aarhus, Denmark, evaluated metabolites from human corneal fibroblasts, isolated from healthy donors and keratoconic corneas that had been removed in transplants, both before and after crosslinking. 1 What Sharif et al. found ana- lyzing 276 metabolites from both groups was that the most affected by the crosslinking process were glutathione disulfide, ascorbic acid, proline, and lysine. There was a decrease in pro-inflammatory biomarkers and downregulation of some amino acids, lactate levels, and water-soluble metabolites. What does all this mean? In short, Dimitrios Karamichos, PhD, associate professor of ophthalmol- ogy and cell biology, Dean McGee Eye Institute, Oklahoma City, and corresponding author of the study, said it shows that crosslinking is working. compared to those analyzed prior to crosslinking. The researchers found that the oxidative stress-related path- ways were significantly affected by crosslinking. The accumulation of reactive oxygen species, as seen in keratoconic corneas pre-crosslink- ing, can have a negative effect in the cornea, Dr. Karamichos said, which in turn, the study authors noted, can affect mitochondrial function. Dr. Karamichos said cross- linking seems to be doing a good job of recovering the glutathione ratio (low levels can be an indicator of oxidative stress) in keratoconic Effects of crosslinking on keratoconus metabolism reported for the first time in new study "There are a lot of positives [showing that] crosslinking is good for the cells," Dr. Karamichos said. To reach these conclusions, the investigators obtained donor corneas from both healthy and keratoconic patients. Stromal tissue was isolated and the primary human corneal fibroblasts were cultured in an established 3-D model, which Dr. Karamichos explained allowed the cells to create their own extra- cellular matrix over several weeks. This model with the cultured cells and extracellular matrix was then exposed to crosslinking conditions and the cellular metabolites were cells and played a role in reducing lactate, which has been reported as elevated in keratoconus as well. "Based on our results in this study, CXL seems to have a positive impact on KC microenvironment. We showed that CXL can impact different metabolic processes, such as antioxidant changes and lactate with inflammation, changes in the hydroxylated products of CXL, and amino acid levels," Sharif et al. wrote. Dr. Karamichos said while there have been a lot of studies on the biomechanical effects of crosslink- ing, there has not been a lot of work done at the cellular level outside his lab. He thinks continued research to assess the effects of crosslinking at this level could lead to person- alized crosslinking treatments that could be more efficacious and/or could open the procedure to more patients. "I think in the long run we can provide a more targeted treatment, more specific to the patient, to the severity of the disease," Dr. Karam- ichos said. "If we understand the cell mechanisms, we can (a) open it up to the early stages of the disease and (b) personalize the amount of crosslinking that the cells can take. A younger keratoconic cornea may not require the same UV-A power, length of treatment, etc., so we think we could personalize it based on what the cells like or don't like." Sharif et al. also noted in their paper that the metabolic profile differences identified through this research could help with diagnosis of keratoconus and disease progres- sion prediction. Dr. Karamichos said research going forward will look at cultures of epithelial cells and simulate epi-on vs. epi-off conditions in vitro, varying the depth of the crosslinking. EW Reference 1. Sharif R, et al. Effects of collagen cross-linking on the keratoconus metabolic network. Eye (Lond). 2018;32:1271–1281. Editors' note: Dr. Karamichos has no financial interests related to his com- ments. Contact information Karamichos: Dimitrios-Karamichos@ouhsc.edu

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