Aged Lens Epithelial Cells Suppress Proliferation and Epithelial-Mesenchymal Transition-Relevance for Posterior Capsule Opacification.

Posterior capsule opacification (PCO) is a frequent complication after cataract surgery, and advanced PCO requires YAG laser (Nd: YAG) capsulotomy, which often gives rise to more complications. Lens epithelial cell (LEC) proliferation and transformation (i.e., epithelial-mesenchymal transition (EMT)) are two critical elements in PCO initiation and progression pathogenesis. While PCO marginally impacts aged cataract surgery patients, PCO incidences are exceptionally high in infants and children undergoing cataract surgery. The gene expression of lens epithelial cell aging and its role in the discrepancy of PCO prevalence between young and older people have not been fully studied. Here, we conducted a comprehensive differentially expressed gene (DEG) analysis of a cell aging model by comparing the early and late passage FHL124 lens epithelial cells (LECs). In vitro, TGFß2, cell treatment, and in vivo mouse cataract surgical models were used to validate our findings. We found that aged LECs decelerated rates of cell proliferation accompanied by dysregulation of cellular immune response and cell stress response. Surprisingly, we found that LECs systematically downregulated epithelial-mesenchymal transition (EMT)-promoting genes. The protein expression of several EMT hallmark genes, e.g., fibronectin, αSMA, and cadherin 11, were gradually decreased during LECs aging. We then confirmed these findings in vitro and found that aged LECs markedly alleviated TGFß2-mediated EMT. Importantly, we explicitly confirmed the in vitro findings from the in vivo mouse cataract surgery studies. We propose that both the high proliferation rate and EMT-enriched young LECs phenotypic characteristics contribute to unusually high PCO incidence in infants and children.

Cellular FLICE-like inhibitory protein (cFLIP) critically maintains apoptotic resistance in human lens epithelial cells.

The present study aims to understand the mechanism of the lens epithelial cell's strong anti-apoptotic capacity and survival in the mature human lens that, on the one hand, maintains lens transparency over several decades, while on the other hand, increases the risk of posterior capsule opacification (PCO). Here we compared FHL124 cells and HeLa cells, spontaneously immortalized epithelial cell lines derived from the human lens and cervical cancer cells, respectively, of their resistance to TNFα-mediated cell death. TNFα plus cycloheximide (CHX) triggered almost all of HeLa cell death. FHL124 cells, however, were unaffected and able to block caspase-8 activation as well as prevent caspase-3 and PARP-1 cleavage. Interestingly, despite spontaneous NFκB and AP-1 activation and upregulation of multiple cell survival/anti-apoptotic genes in both cell types, only FHL124 cells were able to survive the TNFα challenge. After screening and comparing the cell survival genes, cFLIP was found to be highly expressed in FHL124 cells and substantially upregulated by TNFα stimulation. FHL124 cells with a mild cFLIP knockdown manifested a profound apoptotic response to TNFα stimulus similar to HeLa cells. Most importantly, we confirmed these findings in an ex vivo lens capsular bag culture system. In conclusion, our results show that cFLIP is a critical gene that is regulating lens epithelial cell survival.

Aging lens epithelium is susceptible to ferroptosis.

Age-related cataracts (ARC) are the primary cause of blindness worldwide, and oxidative stress is considered the central pathogenesis of age-related cataractogenesis. Interestingly, ample evidence suggests that there is no remarkable apoptosis present in aged and cataractous human lenses despite the profound disruption of redox homeostasis, raising an essential question regarding the existence of other cell death mechanisms. Here we sought to explore the lens epithelial cell's (LEC) susceptibility to ferroptosis after documentation has concluded that aged and cataractous human lenses manifest with increased reactive oxygen species (ROS) formation, elevated lipid peroxidation, and accumulative intracellular redox-active iron, constituting the three hallmarks of ferroptosis during aging and cataractogenesis. Here we show that very low concentrations of system Xc- inhibitor Erastin (0.5 µM) and glutathione peroxidase 4 (GPX4) inhibitor RSL3 (0.1 µM) can drastically induce human LEC (FHL124) ferroptosis in vitro and mouse lens epithelium ferroptosis ex vivo. Depletion of intracellular glutathione (GSH) in human LECs and mouse lens epithelium significantly sensitizes ferroptosis, particularly under RSL3 challenge. Intriguingly, both human LECs and the mouse lens epithelium demonstrate an age-related sensitization of ferroptosis. Transcriptome analysis indicates that clusters of genes are up-or down-regulated in aged LECs, impacting cellular redox and iron homeostases, such as downregulation of both cystine/glutamate antiporter subunits SLC7A11 and SLC3A2 and iron exporter ferroportin (SLC40A1). Here, for the first time, we are suggesting that LECs are highly susceptible to ferroptosis. Moreover, aged and cataractous human lenses may possess more pro-ferroptotic criteria than any other organ in the human body.