A method to preserve limbus during penetrating keratoplasty for a case of presumed PHACES syndrome with sclerocornea: A case report.

BACKGROUND: Sclerocornea, a congenital corneal pathology characterized by bilateral scleralization of the cornea, which can be found in few cases with posterior fossa malformationshemangiomas-arterial anomalies-cardiac defects-eye abnormalities-sternal cleft and supraumbilical raphe (PHACES) syndrome. Presence of vascularization in peripheral cornea and smaller diameter of recipient cornea correlate to poor outcome of penetrating keratoplasty (PKP) in sclerocornea. Here we report a method to preserve limbus during PKP for small, irregular, and scleralized cornea. METHODS: A 12-year-old boy with multiple congenital anomalies diagnosed as PHACES syndrome suffered from bilateral total sclerocornea and poor visual acuity. Due to the fact that the left eye cornea was small (6.5 mm × 10 mm), lamellar dissection and posterior recession of inferior limbus was first performed and followed by a 6 mm trephination and PKP with a 6.5 mm graft for left eye. At the same time, lens aspiration and release of peripheral anterior synechia were performed. RESULTS: After 6 years of follow-up, the cornea remained clear, and there has been no sign of inflammation and conjunctivalization. The patient maintained useful vision of 20/400 in left eye. CONCLUSION: The stabilization of corneal surface is possible after PKP for sclerocornea if the limbus can be preserved during the operation, and epithelium can remain corneal in phenotype preventing pannas growth.

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

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 ß1, and integrin-linked kinase (ILK). Nuclear ß-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 ß-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 ß1/ILK, which indirectly activated Wnt/ß-catenin and subsequently deltaNp63α. Crosstalk between the integrin ß1/ILK and Wnt/ß-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 ß1 to the Wnt/ß-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 ß1, followed by activation of the ILK/Wnt/ß-catenin/p63 cascade.

Epithelial phenotype in total sclerocornea.

PURPOSE: To understand whether the epithelial phenotype in total sclerocornea is corneal or conjunctival in origin. METHODS: Four cases of total sclerocornea (male:female = 1:3; mean age = 5.4 ± 4.3; 1-11 years old) who received penetrating keratoplasty (PKP) at our hospital between 2008 and 2011 were included. Corneal buttons obtained during PKP were used for transmission electron microscopy (TEM) as well as immunoconfocal microscopy for cytokeratins 3, 12, and 13, goblet cell mucin MUC5AC, connexin 43, stem cell markers p63 and ABCG2, laminin-5, and fibronectin. RESULTS: After a mean follow-up period of 38.8 ± 14.0 (12-54) months, the grafts remained clear in half of the patients. TEM examination revealed a markedly attenuated Bowman's layer in the scleralized corneas, with irregular and variably thinned collagen lamellar layers, and disorganization and random distribution of collagen fibrils, which were much larger in diameter compared with a normal cornea. Immunoconfocal microscopy showed that keratin 3 was expressed in all four patients, while p63, ABCG2, and MUC5AC were all absent. Cornea-specific keratin 12 was universally expressed in Patients 1 to 3, while mucosa (including conjunctiva)-specific keratin 13 was negative in these patients. Interestingly, keratin 12 and 13 were expressed in Patient 4 in a mutually exclusive manner. Linear expression of laminin-5 in the basement membrane zone and similar expression of fibronectin were observed. CONCLUSIONS: The epithelia in total sclerocornea are essentially corneal in phenotype, but in the event of massive corneal angiogenesis, invasion by the conjunctival epithelium is possible.

Up-regulation of heat shock protein 70-1 (Hsp70-1) in human limbo-corneal epithelial cells cultivated on amniotic membrane: A proteomic study.

Transplantation of cultivated human limbo-corneal epithelial (HLE) cells has been recognized as an effective stem cell (SC) therapy for treating corneal epithelial SC deficiency caused by burn or other diseases. With this technique, cryo-preserved human intact amniotic membrane (IAM) has been successfully used as a cell culture substrate and carrier, and is reported to preferentially preserve HLE stem/progenitor cells in vitro. However, little is known about what factors released by HLE cells are involved in the progenitor cell-preserving mechanism. Using proteomic method, we identified 13 proteins over-expressed by HLE cells cultured on IAM, which included heat shock protein 70-1 (Hsp70-1), Hsp-27, glutathione (GSH) S-transferase, annexin A2, galectin-7, and protein S100-A9. Increased Hsp70-1 expression was confirmed by Western blot and real-time PCR. The role of Hsp70-1 in promoting HLE cell survival was demonstrated by increased apoptosis index and increased cleaved poly ADP-ribose polymerase (CPARP) formation in hsp70-1-silenced, but not normal HLE cells after exposure to sublethal UVB irradiation or hydrogen peroxide. To understand the regulatory mechanism of Hsp70-1 expression in HLE cells, the role of transcription factor deltaNp63 (a well-recognized HLE stem cell; SC marker) was studied. We found that over-expression of deltaNp63α by plasmid vector induced a corresponding increase in Hsp70-1 protein production. Likewise, Hsp70-1 expression decreased in HLE cells after addition of deltaNp63α SiRNA. Immunoconfocal microscopy also revealed a paralleled expression of both proteins in corneal specimens. Thus, deltaNp63α-associated Hsp70-1 over-expression may promote HLE progenitor cell survival on IAM, possibly through the cytoprotective and anti-apoptotic effect of Hsp70-1.