Yi-Jen Hsueh

Lysophosphatidic acid induces YAP-promoted proliferation of human corneal endothelial cells via PI3K and ROCK pathways

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.

STAT3 Regulates the Proliferation and Differentiation of Rabbit Limbal Epithelial Cells via a ΔNp63-Dependent Mechanism

PURPOSE To explore the roles of STAT3 in the regulation of ΔNp63-dependent proliferation and differentiation of rabbit limbal keratinocytes. METHODS siRNAs were designed to specifically suppress the expression of STAT3 and ΔNp63, and their effects on limbal epithelial cell proliferation and differentiation were examined. Ectopically expressed ΔNp63 was used to compensate for the decreased endogenous ΔNp63. Immunoblot was used to examine the expressions of STAT3, ΔNp63, K3, integrin β1, and involucrin. RESULTS Limbal tissue expresses higher level of phosphorylated and nuclear translocated STAT3 compared with that of the cornea. Knockdown of STAT3 expression reduces the expression of ΔNp63, inhibits the expansion of limbal epithelial outgrowth, suppresses the expression of integrin β1, and promotes the expression of involucrin. CONCLUSIONS STAT3 enhances the proliferation of limbal keratinocytes through a ΔNp63-dependent mechanism. Suppression of this pathway inhibits cell proliferation with a concomitant increase of cell differentiation.

Preservation of human limbal epithelial progenitor cells on carbodiimide cross-linked amniotic membrane via integrin-linked kinase-mediated Wnt activation.

UNLABELLED The Wnt pathway is a major signaling pathway that regulates corneal epithelial stem cells. However, little is known about how the ultrastructure of the limbal epithelial basement membrane (EBM) affects Wnt activity. Due to its enhanced matrix stability, the cross-linked amniotic membrane (AM) has gained increasing interest in the field of regenerative medicine. For the first time, we used EDC/NHS cross-linked denuded AM (CLDAM) as a simulated EBM substrate to investigate this mechanism. Human limbal epithelial (HLE) cells were cultured on dishes (HLE/dish), denuded AM (HLE/DAM) or CLDAM (HLE/CLDAM). Compared with HLE/dish or HLE/DAM cultures, HLE/CLDAM cultures showed greater BrdU retention and colony formation efficiency and expressed higher levels of p63, ABCG2, integrin β1, and integrin-linked kinase (ILK). Nuclear β-catenin and TCF-4 levels were higher in HLE/CLDAM cultures compared with HLE cells cultured on collagen IV, laminin, Matrigel, or DAM. Silencing of ILK in HLE/CLDAM cultures resulted in decreased levels of nuclear β-catenin, TCF-4 and deltaNp63α, whereas cytokeratin 12 expression increased. Over-expression of ILK in HLE/dish cultures had the opposite effects. Accordingly, we proposed that the CLDAM matrix, with its higher rigidity and rougher ultrastructure, better preserved HLE progenitor cells in vitro, possibly by activating integrin β1/ILK, which indirectly activated Wnt/β-catenin and subsequently deltaNp63α. Crosstalk between the integrin β1/ILK and Wnt/β-catenin pathways appears to play a crucial role in limbal progenitor cell survival on EBM. STATEMENT OF SIGNIFICANCE We demonstrated the superior capability of carbodiimide cross-linked denuded amniotic membrane (CLDAM) than natural DAM to preserve limbo-corneal epithelial progenitor cells in vitro, then we used CLDAM as a simulated epithelial basement membrane (EBM) to study how EBM maintains limbal epithelial stem cells (LESCs). We found that integrin-linked kinase (ILK) is an important mediator that transfers survival signals detected by integrin β1 to the Wnt/β-catenin pathway, which in turn up-regulates deltaNp63α, a master gene that regulates LESC function. The rougher surface of the limbal EBM suggests that the surface complexity of the LESC niche may be important in regulating LESC function, which is triggered by the recognition of topographic cues by integrin β1, followed by activation of the ILK/Wnt/β-catenin/p63 cascade.

Development of gelatin/ascorbic acid cryogels for potential use in corneal stromal tissue engineering

To offer an ideal hospitable environment for corneal keratocyte growth, the carrier materials can be functionalized with incorporation of signaling molecules to regulate cell biological events. This study reports, for the first time, the development of gelatin/ascorbic acid (AA) cryogels for keratocyte carriers in vitro and in vivo. The cryogel samples were fabricated by blending of gelatin with varying amounts of AA (0-300 mg) and carbodiimide cross-linking via cryogelation technique. Hydrophilic AA content in the carriers was found to significantly affect cross-linking degree and pore dimension of cryogels, thereby dictating their mechanical and biological stability and AA release profile. The cryogel carriers with low-to-moderate AA loadings were well tolerated by rabbit keratocyte cultures and anterior segment eye tissues, demonstrating good ocular biocompatibility. Although higher incorporated AA level contributed to enhanced metabolic activity and biosynthetic capacity of keratocytes grown on cryogel matrices, the presence of excessive amounts of AA molecules could lead to toxic effect and limit cell proliferation and matrix production. The cytoprotective activity against oxidative stress was shown to be strongly dependent on AA release, which further determined cell culture performance and tissue reconstruction efficiency. With the optimum AA content in carrier materials, intrastromally implanted cell/cryogel constructs exhibited better capability to enhance tissue matrix regeneration and transparency maintenance as well as to mitigate corneal damage in an alkali burn-induced animal model. It is concluded that understanding of antioxidant molecule-mediated structure-property-function interrelationships in gelatin/AA cryogels is critical to designing carrier materials for potential use in corneal stromal tissue engineering. STATEMENT OF SIGNIFICANCE Multifunctional cryogel material can offer an ideal hospitable environment for cell-mediated tissue reconstruction. To our knowledge, this is the first report describing the use of gelatin/ascorbic acid (AA) cryogels as keratocyte carriers for corneal stromal tissue engineering. The AA loading during cryogel fabrication is found to have a significant effect on cross-linking degree and pore dimension, mechanical and biological stability, ocular biocompatibility, cell culture performance, and cytoprotective activity, giving comprehensive insight into fine-tuning the structure-property-function interrelationships of keratocyte carrier material. Using an alkali burn-induced animal model, we present evidence that with the optimum AA loading into cryogel materials, intrastromally implanted cell/carrier constructs exhibited better capability to enhance tissue matrix regeneration and transparency maintenance as well as to mitigate corneal damage.

Preservation of epithelial progenitor cells from collagenase-digested oral mucosa during ex vivo cultivation

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.

Surgical outcome of deep anterior lamellar keratoplasty with air-assisted manual dissection for corneas with previous inflammation or fibrosis

PURPOSE: To report our experience in air-assisted manual dissection deep anterior lamellar keratoplasty (DALK) for the treatment of corneal scar with previous inflammation and fibrosis. MATERIALS AND METHODS: We retrospectively reviewed the medical history of 21 patients (male:female = 13:8 mean age 41.9 years old) with corneal pathology from previous infection and inflammation. Trephination diameter ranged from 7.0 to 8.0 mm, and the graft was oversized by 0.25–0.50 mm. Debulking technique was performed to expose Descemets membrane after filling stroma with air. Starting from postoperative 3 months, selective suture removal was performed to reduce corneal astigmatism. RESULTS: The mean follow-up period was 59.9 ± 19.8 (20–96) months. Intraoperative microperforation occurred in 2 eyes (9.5%); however, there was no shift to penetrating keratoplasty. Air-bubble tamponade was performed in 7 eyes (33.3%) for postoperative gapping of the graft. There were 2 failed grafts (9.5%) due to corneal ulcer while all the other grafts remained clear throughout follow-up. The mean preoperative best-corrected visual acuity (BCVA) was 1.84 ± 0.66 logMAR, which improved to 0.74 ± 0.63 (P < 0.01). The average sphere power was − 0.88 ± 3.88 diopter (D), average cylinder power 3.03 ± 1.46D, and average endothelial count 1877 ± 375 cells/mm2. CONCLUSION: In severe ocular surface diseases, big-bubble technique frequently failed to separate predescemtic plane; however, it effectively created air-filled stroma which was easier to remove. Although BCVA was suboptimal due to ocular surface disorders, graft survival and clarity rate is high, justifying the application of DALK in these cases.

Corneal Neovascularization as a Target for Nucleotide-Based Therapies

The cornea is a transparent tissue with avascular characteristics (corneal avascularity). It can be compromised by imbalances of angiogenic factors due to chemical injuries, infections, or autoimmune diseases such as Stevens- Johnson’s syndrome, which can lead to corneal blindness. Clinically, the etiologies of corneal neovascularization (NV), such as inflammation, immune rejection, limbal stem cell deficiency, or hypoxia, are usually long-lasting; therefore, conventional treatment modalities, including antiangiogenic medications, laser, or surgeries have only a suboptimal effect, and the prognosis is even worse with multiple recurrences. In contrast, novel treatment modalities, such as gene therapy (enhanced intracellular expression of antiangiogenic factors) and nucleotide-based antiangiogenic therapy (antisense oligonucleotides, silence-RNA, and micro-RNA) have been accomplished in animal models or clinical trials in recent years. Because of its specific and long-lasting effect, as well as the improvement and verification of the safety of expression vectors and carriers, the clinical value of nucleotide-based therapy has been increasingly appreciated. This review summarizes and updates relevant research, and provides a better understanding regarding the mechanism and treatment of corneal NV.