The first two authors contributed equally to this work.Silence of p120-catenin has shown promise in inducing proliferation in human corneal endothelial cells (HCECs), but there is concern regarding off-target effects in potential clinical applications. We aimed to develop ex vivo expansion of HCECs using natural compounds, and we hypothesized that lysophosphatidic acid (LPA) can unlock the mitotic block in contact-inhibited HCECs via enhancing nuclear translocation of yes-associated protein (YAP). Firstly, we verified that exogenous YAP could induce cell proliferation in contact-inhibited HCEC monolayers and postconfluent B4G12 cells. In B4G12 cells, enhanced cyclin D1 expression, reduced p27KIP1/p21CIP1 levels, and the G1/S transition were detected upon transfection with YAP. Secondly, we confirmed that LPA induced nuclear expression of YAP and promoted cell proliferation. Moreover, PI3K and ROCK, but not ERK or p38, were required for LPA-induced YAP nuclear translocation. Finally, cells treated with LPA or transfected with YAP remained hexagonal in shape, in addition to unchanged expression of ZO-1, Na/K-ATPase, and smooth muscle actin (SMA), suggestive of a preserved phenotype, without endothelial–mesenchymal transition. Collectively, our findings indicate an innovative strategy for ex vivo cultivation of HCECs for transplantation and cell therapy.

Carbonic anhydrase 8 (CA8), a member of the carbonic anhydrase family, is one of the three isozymes that do not catalyze the reversible hydration of carbon dioxide due to the lack of one important histidine. In the present study, we observed increased expression of CA8 in more aggressive types of human osteosarcoma (OS) cells and found that CA8 expression is correlated with disease stages, such that more intense expression occurs in the disease late stage. We also demonstrated that overexpression of CA8 in human OS (HOS) cells significantly increased cell proliferation both in vitro and in vivo. Downregulated CA8 sensitized cells to apoptotic stress induced by staurosporine and cisplatin, suggesting a specific role of CA8 to protect cells from stresses. In addition, downregulation of CA8 in HOS cells reduced cell invasion and colony formation ability in soft agar and further decreased matrix metalloproteinase 9 and focal adhesion kinase expression, indicating that CA8 might facilitate cancer cell invasion via the activation of FAK-MMP9 signaling. Interestingly, HOS cells with CA8 knockdown showed a significant decrease in glycolytic activity and cell death under glucose withdrawal, further indicating that CA8 may be involved in regulating aerobic glycolysis and enhancing cell viability. Knockdown of CA8 significantly decreased phosphorylated Akt expression suggesting that the oncogenic role of CA8 may be mediated by the regulation of Akt activation through p-Akt induction. Importantly, the inhibition of glycolysis by 2-deoxyglucose sensitized CA8 HOS-CA8-myc cells to cisplatin treatment under low glucose condition, highlighting a new therapeutic option for OS cancer.

To avoid xenogeneic infection, we report a novel protocol for producing animal-derived component-free oral mucosal epithelial cells (OMECs) sheet for transplantation, in which collagenase was used to replace dispase II/trypsin-EDTA for digesting oral mucosal tissue, and human platelet-derived PLTMax to replace fetal bovine serum. The resulting epithelial aggregates were expanded on de-epithelialized amniotic membranes without 3T3 feeder cells, and serum-free EpiLife was used to reduce contamination by submucosal mesenchymal cells. The OMEC sheets thus generated showed similar positive keratin 3/76-positive and keratin 8-negative staining patterns compared with those generated by the original protocol. Colony formation efficiency assay, BrdU label retention assay, and p63 and p75NTR immunostaining results indicated that higher proliferative potentials and more progenitor cells were preserved by the modified protocol. TaqMan array analysis revealed that the transcription of integrin-linked kinase (ILK) was up-regulated along with an increase in β-catenin signaling and its downstream cell cycle modulators, cyclin D1 and p27KIP1. Furthermore, ILK silencing led to the inhibition of nuclear β-catenin accumulation, suppressed p63 expression, and reduced the expression of cyclin D1 and p27KIP1; these observations suggest that ILK/β-catenin pathway may be involved in cell proliferation regulation during the ex vivo expansion of OMECs for transplantation purposes.

Compromised pumping function of the corneal endothelium, due to loss of endothelial cells, results in corneal edema and subsequent visual problems. Clinically and experimentally, oxidative stress may cause corneal endothelial decompensation after phacoemulsification. Additionally, in vitro and animal studies have demonstrated the protective effects of intraoperative infusion of ascorbic acid (AA). Here, we established a paraquat-induced cell damage model, in which paraquat induced reactive oxygen species (ROS) production and apoptosis in the B4G12 and ARPE-19 cell lines. We demonstrate that oxidative stress triggered autophagic flux blockage in corneal endothelial cells and that addition of AA ameliorated such oxidative damage. We also demonstrate the downregulation of Akt phosphorylation in response to oxidative stress. Pretreatment with ascorbic acid reduced the downregulation of Akt phosphorylation, while inhibition of the PI3K/Akt pathway attenuated the protective effects of AA. Further, we establish an in vivo rabbit model of corneal endothelial damage, in which an intracameral infusion of paraquat caused corneal opacity. Administration of AA via topical application increased its concentration in the corneal stroma and reduced oxidative stress in the corneal endothelium, thereby promoting corneal clarity. Our findings indicate a perioperative strategy of topical AA administration to prevent oxidative stress-induced damage, particularly for those with vulnerable corneal endothelia.

No abstract