Abstract
Oxidative stress may play an important role in the pathogenesis of keratoconus (KC). Mitochondrial DNA (mtDNA) is involved in mitochondrial function, and the mtDNA content, integrity, and transcript level may affect the generation of reactive oxygen species (ROS) and be involved in the pathogenesis of KC. We designed a case-control study to research the relationship between KC and mtDNA integrity, content and transcription. One-hundred ninety-eight KC corneas and 106 normal corneas from Chinese patients were studied. Quantitative real-time PCR was used to measure the relative mtDNA content, transcript levels of mtDNA and related genes. Long-extension PCR was used to detect mtDNA damage. ROS, mitochondrial membrane potential and ATP were measured by respective assay kit, and Mito-Tracker Green was used to label the mitochondria. The relative mtDNA content of KC corneas was significantly lower than that of normal corneas (P = 9.19×10−24), possibly due to decreased expression of the mitochondrial transcription factor A (TFAM) gene (P = 3.26×10−3). In contrast, the transcript levels of mtDNA genes were significantly increased in KC corneas compared with normal corneas (NADH dehydrogenase subunit 1 [ND1]: P = 1.79×10−3; cytochrome c oxidase subunit 1 [COX1]: P = 1.54×10−3; NADH dehydrogenase subunit 1, [ND6]: P = 4.62×10−3). The latter may be the result of increased expression levels of mtDNA transcription-related genes mitochondrial RNA polymerase (POLRMT) (P = 2.55×10−4) and transcription factor B2 mitochondrial (TFB2M) (P = 7.88×10−5). KC corneas also had increased mtDNA damage (P = 3.63×10−10), higher ROS levels, and lower mitochondrial membrane potential and ATP levels compared with normal corneas. Decreased integrity, content and increased transcript level of mtDNA are associated with KC. These changes may affect the generation of ROS and play a role in the pathogenesis of KC.
Highlights
Keratoconus (KC) is a degenerative corneal disease, which is characterized by corneal ectasia, thinning, and cone-shaped protrusion, resulting in reduced vision, irregular astigmatism, and corneal scarring [1, 2]
The keratoconus corneal fibroblast (KCF) cells had significantly higher reactive oxygen species (ROS) levels compared to the normal corneal fibroblast cells (Fig 1A and 1D)
The mitochondrial membrane potential and ATP levels of the KCF cells were significantly lower than that of the normal corneal fibroblast cells (Fig 1C–1E). These results showed that KCF cells had greater ROS production, lower mitochondrial fluorescence intensity, and lower mitochondrial membrane potential and ATP levels compared to normal corneal fibroblast cells
Summary
Keratoconus (KC) is a degenerative corneal disease, which is characterized by corneal ectasia, thinning, and cone-shaped protrusion, resulting in reduced vision, irregular astigmatism, and corneal scarring [1, 2]. Oxidative phosphorylation in mitochondria is the major source of endogenous reactive oxygen species (ROS) [9] Mitochondria have their own genome, mitochondrial DNA (mtDNA), which encodes 13 subunits of respiratory complexes I, III, IV, and V [10, 11]. In an American population, Atilano et al reported that KC corneas had a lower mtDNA-to-nDNA (nuclear DNA) ratio and more mtDNA damage than do normal corneas [13] These results suggest that mtDNA variations may be involved in the pathogenesis of KC, but as of yet no one had attempted to study the relationship between mtDNA and KC systematically in order to uncover the underlying mechanisms. To further validate these results in larger cornea samples and study the underlying mechanisms, we carried out this study
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