Corneal keratin aggresome (CKAGG) formation and clearance by proteasome activation.

PURPOSE: To understand the mechanism of corneal keratin expression and clearance in corneal epithelium with Limbal Stem Cell Deficiency (LSCD). The hypothesis is that LSCD-induced proteasome dysfunction is a contributing factor to keratin aggregation, causing corneal keratin aggresome (CKAGG) formation. METHOD: LSCD was surgically induced in rabbit corneas. LSCD corneal epithelial cells (D-CEC) were collected to investigate keratin K4 and K13 expression and CKAGG formation. Oral mucosal epithelial cells (OMECS) were isolated and cultured to study K4 and K13 expression. Cultured cells were treated with proteasome inhibitor to induce CKAGG formation. RESULTS: K4 and K13 were strongly expressed in D-CEC, with additional higher molecular weight bands of K4 and K13, suggesting CKAGG formation. Double staining of K4/K13 and ubiquitin showed co-localization of these keratins with ubiquitin in D-CEC. Proteasome inhibition also showed K4/K13 modification and accumulation in cultured OMECS, similar to D-CEC. Proteasome activation was then performed in cultured OMEC. There was no accumulation of keratins, and levels of unmodified keratins were found significantly reduced. CONCLUSION: Results showed an abnormal expression of K4 and K13 after LSCD-induced proteasome dysfunction, which coalesce to form CKAGG in Corneal Epithelial Cells (CEC). We propose that CKAGG formation may be one of the causative factors of morphological alterations in the injured corneal epithelium, and that CKAGG could potentially be cleared by enhancing proteasome activity.

Proteasomes in corneal epithelial cells and cultured autologous oral mucosal epithelial cell sheet (CAOMECS) graft used for the ocular surface regeneration.

PURPOSE: This study focuses on characterizing proteasomes in corneal epithelial cells (CEC) and in cultured autologous oral mucosal epithelial cell sheets (CAOMECS) used to regenerate the ocular surface. METHODS: Limbal stem cell deficiency (LSCD) was surgically induced in rabbit corneas. CAOMECS was engineered and grafted onto corneas with LSCD to regenerate the ocular surface. RESULTS: LSCD caused an increase in inflammatory cells in the ocular surface, an increase in the formation of immunoproteasomes (IPR), and a decrease in the formation of constitutive proteasome (CPR). Specifically, LSCD-diseased CEC (D-CEC) showed a decrease in the CPR chymotrypsin-like, trypsin-like and caspase-like activities, while healthy CEC (H-CEC) and CAOMECS showed higher activities. Quantitative analysis of IPR inducible subunit (B5i, B2i, and B1i) were performed and compared to CPR subunit (B5, B2, and B1) levels. Results showed that ratios B5i/B5, B2i/B2 and B1i/B1 were higher in D-CEC, indicating that D-CEC had approximately a two-fold increase in the amount of IPR compared to CAOMECS and H-CEC. Histological analysis demonstrated that CAOMECS-grafted corneas had a re-epithelialized surface, positive staining for CPR subunits, and weak staining for IPR subunits. In addition, digital quantitative measurement of fluorescent intensity showed that the CPR B5 subunit was significantly more expressed in CAOMECS-grafted corneas compared to non-grafted corneas with LSCD. CONCLUSION: CAOMECS grafting successfully replaced the D-CEC with oral mucosal epithelial cells with higher levels of CPR. The increase in constitutive proteasome expression is possibly responsible for the recovery and improvement in CAOMECS-grafted corneas.

The Role of E-Cadherin in Maintaining the Barrier Function of Corneal Epithelium after Treatment with Cultured Autologous Oral Mucosa Epithelial Cell Sheet Grafts for Limbal Stem Deficiency.

The role of E-cadherin in epithelial barrier function of cultured autologous oral mucosa epithelial cell sheet (CAOMECS) grafts was examined. CAOMECS were cultured on a temperature-responsive surface and grafted onto rabbit corneas with Limbal Stem Cell Deficiency (LSCD). E-cadherin levels were significantly higher in CAOMECS compared to normal and LSCD epithelium. Beta-catenin colocalized with E-cadherin in CAOMECS cell membranes while phosphorylated beta-catenin was significantly increased. ZO-1, occludin, and Cnx43 were also strongly expressed in CAOMECS. E-cadherin and beta-catenin localization at the cell membrane was reduced in LSCD corneas, while CAOMECS-grafted corneas showed a restoration of E-cadherin and beta-catenin expression. LSCD corneas did not show continuous staining for ZO-1 or for Cnx43, while CAOMECS-grafted corneas showed a positive expression of ZO-1 and Cnx43. Cascade Blue® hydrazide did not pass through CAOMECS. Because E-cadherin interactions are calcium-dependent, EGTA was used to chelate calcium and disrupt cell adhesion. EGTA-treated CAOMECS completely detached from cell culture surface, and E-cadherin levels were significantly decreased. In conclusion, E cadherin high expression contributed to CAOMECS tight and gap junction protein recruitment at the cell membrane, thus promoting cellular adhesion and a functional barrier to protect the ocular surface.

Carrier-free cultured autologous oral mucosa epithelial cell sheet (CAOMECS) for corneal epithelium reconstruction: a histological study.

This study investigates the therapeutic effects of carrier-free cultured autologous oral mucosa epithelial cell sheet (CAOMECS) transplantation for experimentally induced severe rabbit limbal stem cell deficiency (LSCD). Buccal biopsies were performed and CAOMECS were cultured and transplanted onto diseased corneas. Six-month follow-up examinations indicated that three out of four corneas with CAOMECS grafts showed a decrease in superficial vascularization, while almost all the sham corneas did not show a similar decrease. H&E staining of corneas showed that CAOMECS transplantation reduced blood vessel invasion of central cornea, reduced lymphocyte infiltration and fibrotic tissue formation. DeltaNp63 stained markedly in the grafted cornea and to a lesser extent in the sham corneas. PCNA and Ki-67 staining were much greater in the sham corneas than in the grafted and normal corneas. K3 and K13 staining demonstrated that CAOMECS transplanted corneas had much more K3- and less K13- positive cells compared to the sham corneas. Muc5AC was decreased in the central region of grafted corneas. Very little alpha-smooth muscle actin (aSMA) staining was detected in grafted corneas, while there was a greater amount of aSMA staining in sham corneas. Staining for anti-angiogenic factor TIMP -3 was also increased, and pro-angiogenic factor MMP-3 was decreased in grafted corneas compared to sham corneas. Our results indicate that CAOMECS grafts resulted in improved epithelialization of the corneal surface and decreased vascularization and fibrosis of the diseased corneas.