Aims: Fuchs endothelial corneal dystrophy (FECD), a leading cause of age-related corneal edema requiring transplantation, is characterized by rosette formation of corneal endothelium with ensuing apoptosis. We sought to determine whether excess of mitochondrial reactive oxygen species leads to chronic accumulation of oxidative DNA damage and mitochondrial dysfunction, instigating cell death. Results: We modeled the pathognomonic rosette formation of postmitotic corneal cells by increasing endogenous cellular oxidative stress with menadione (MN) and performed a temporal analysis of its effect in normal (HCEnC, HCECi) and FECD (FECDi) cells and ex vivo specimens. FECDi and FECD ex vivo specimens exhibited extensive mtDNA and nDNA damage as detected by quantitative PCR. Exposure to MN triggered an increase in mitochondrial superoxide levels and led to mtDNA and nDNA damage, while DNA amplification was restored with NAC pretreatment. Furthermore, MN exposure led to a decrease in DCm and adenosine triphosphate levels in normal cells, while FECDi exhibited mitochondrial dysfunction at baseline. Mitochondrial fragmentation and cytochrome c release were detected in FECD tissue and after MN treatment of HCEnCs. Furthermore, cleavage of caspase-9 and caspase-3 followed MN-induced cytochrome c release in HCEnCs. Innovation: This study provides the first line of evidence that accumulation of oxidative DNA damage leads to rosette formation, loss of functionally intact mitochondria via fragmentation, and subsequent cell death during postmitotic cell degeneration of ocular tissue. Conclusion: MN induced rosette formation, along with mtDNA and nDNA damage, mitochondrial dysfunction, and fragmentation, leading to activation of the intrinsic apoptosis via caspase cleavage and cytochrome c release.

In previous studies, we have shown that heme oxygenase (HO)-2 null [HO-2(Ϫ/Ϫ)] mice exhibit a faulty response to injury; chronic inflammation and massive neovascularization replaced resolution of inflammation and tissue repair. Endothelial cells play an active and essential role in the control of inflammation and the process of angiogenesis. We examined whether HO-2 deletion affects endothelial cell function. Under basal conditions, HO-2(Ϫ/Ϫ) aortic endothelial cells (mAEC) showed a 3-fold higher expression of vascular endothelial growth factor receptor 1 and a marked angiogenic response compared with wild-type (WT) cells. Compared with WT cells, HO-2(Ϫ/Ϫ) mAEC showed a 2-fold reduction in HO activity and marked increases in levels of gp91 phox /NADPH oxidase isoform, superoxide, nuclear factor B activation, and expression of inflammatory cytokines, including interleukin (IL)-1␣ and IL-6. HO-2 deletion transforms endothelial cells from a "normal" to an "activated" phenotype characterized by increases in inflammatory, oxidative, and angiogenic factors. This switch may be the result of reduced HO activity and the associated reduction in the cytoprotective HO products, carbon monoxide and biliverdin/bilirubin, because addition of biliverdin to HO-2(Ϫ/Ϫ) cells attenuated angiogenesis and reduced superoxide production. This transformation underscores the importance of HO-2 in the regulation of endothelial cell homeostasis.The integrity of the vascular endothelium is critical for the maintenance of vascular homeostasis. This layer of cells actively participates in the regulation of vascular tone, blood fluidity, growth of vascular smooth muscle cells, and local inflammation by synthesizing and releasing paracrine factors in response to humoral, mechanical, and neural stimuli. Under normal conditions, the endothelium maintains a vasodilatory, antithrombotic, and anti-inflammatory state. One of the systems that contribute to the maintenance of this state is the heme-heme oxygenase (HO) system. HO is the rate-limiting enzyme in heme catabolism. It cleaves heme into iron, sequestered by ferritin, carbon monoxide (CO), and biliverdin, which is reduced to bilirubin by biliverdin reductase (Abraham and Kappas, 2008). Two isoforms, HO-1 and HO-2, are the major source of HO activity in most tissues. Both are alike in terms of mechanisms of heme oxidation, cofactor and substrate specificity, and susceptibility to inhibition by porphyrins (Maines, 1988;Abraham and Kappas, 2008). They differ in their postulated function; HO-2 functions as the constitutive HO activity contributing to cell homeostasis, whereas HO-1 expression is relatively low in most normal tissues. After injury, however, HO-1 expression is greatly enhanced to play a significant role in cytoprotection (Abraham and Kappas, 2008). The catalytic activity of HO is considered the underlying principle of HO cytoprotective actions. Heme functions as a double-edged sword in that in moderate quantities and when bound to protein, heme forms an essential element for ...

Human corneal endothelial cells (HCEnCs) are terminally differentiated cells that have limited regenerative potential. The large numbers of mitochondria in HCEnCs are critical for pump and barrier function required for corneal hydration and transparency. Fuchs Endothelial Corneal Dystrophy (FECD) is a highly prevalent late-onset oxidative stress disorder characterized by progressive loss of HCEnCs. We previously reported increased mitochondrial fragmentation and reduced ATP and mtDNA copy number in FECD. Herein, carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-induced mitochondrial depolarization decreased mitochondrial mass and Mfn2 levels, which were rescued with mitophagy blocker, bafilomycin, in FECD. Moreover, electron transport chain complex (I, V) decrease in FECD indicated deficient mitochondrial bioenergetics. Transmission electron microscopy of FECD tissues displayed an increased number of autophagic vacuoles containing degenerated and swollen mitochondria with cristolysis. An elevation of LC3-II and LAMP1 and downregulation of Mfn2 in mitochondrial fractions suggested that loss of fusion capacity targets fragmented mitochondria to the pre-autophagic pool and upregulates mitophagy. CCCP-induced mitochondrial fragmentation leads to Mfn2 and LC3 co-localization without activation of proteosome, suggesting a novel Mfn2 degradation pathway via mitophagy. These data indicate constitutive activation of mitophagy results in reduction of mitochondrial mass and abrogates cellular bioenergetics during degeneration of post-mitotic cells of ocular tissue.