A fine-tuned β-catenin regulation during proliferation of corneal endothelial cells revealed using proteomics analysis

Abstract

Corneal endothelial (CE) dysfunction is the main indication for corneal transplantation, an invasive procedure with several limitations. Developing novel strategies to reactivate CE regenerative capacity is, therefore, of fundamental importance. This goal has proved to be challenging as corneal endothelial cells (CEnC) are blocked in the G0/G1 phase of the cell cycle in vivo and, albeit retaining proliferative capacity in vitro, this is further hindered by endothelial-to-mesenchymal transition. Herein we investigated the mechanisms regulating CEnC proliferation in vitro. Comparing the proteome of non-proliferating (in vivo-G0/G1) and proliferating (in vitro-G2/M) rabbit CEnC (rCEnC), 77 proteins, out of 3,328 identified, were differentially expressed in the two groups (p < 0.005). Literature and Gene Ontology analysis revealed β-catenin and transforming growth factor (TGF-β) pathways to be correlated with the identified proteins. Treatment of rCEnC with a β-catenin activator and inhibitor showed that β-catenin activation was necessary during rCEnC proliferation, but not sufficient for its induction. Furthermore, both pro-proliferative activity of basic fibroblast growth factor and anti-proliferative effects of TGF-β were regulated through β-catenin. Overall, these results provide novel insights into the molecular basis underlying the proliferation process that CEnC reactivate in vitro, consolidating the role of β-catenin and TGF-β. The corneal endothelium (CE) is a monolayer of cells localised in the innermost segment of the cornea, regulating solutes transport from and to the aqueous humor (pump-leak hypothesis) 1. Corneal endothelial cells (CEnC) are generally considered non-dividing in vivo 2 and arrested in the G0/G1 phase of the cell cycle 3,4. The presence of anti-proliferative factors in the aqueous humor, the stress-induced premature senescence, and the contact inhibition between cells are the main events triggering the expression of cell cycle regulators that induce CEnC mitotic block 2. For this reason, as CEnC density decreases by 0.6% each year 5,6 , cell loss is compensated through migration and cell enlargement 2,7. When, as a consequence of pathologies or surgical treatments, endothelial cell density falls below 500 cells/mm 2 , cell enlargement is no longer able to compensate for the passive leaking 7. In this case, the impaired CE results in corneal swelling and, if untreated, in the subsequent severe loss of corneal transparency, ultimately leading to blindness. CEnC dysfunction is the underlying cause of about 40% of all corneal transplants performed worldwide 7. Corneal grafts, despite decisive advancements in the last decades, are still complex and invasive procedures, with some severe limitations, including immune graft rejection, graft failure, and a general scarcity of donor corneas 7. Alternative clinical procedures have been recently proposed in order to increase the number of patients that can be treated and to improve their quality of life. Currently, descemetorhexis is the only therap...

Fluctuations in Corneal Endothelial LAP2 Expression Levels Correlate with Passage Dependent Declines in Their Cell Proliferative Activity

“…A similar decrease either in ki67 or LAP2 protein expression was observed in rCEnCs treated with TGFβ and FGF simultaneously ( Figure 3 b,c). Whenever the cells were treated with FGF only, we detected an increase in ki67 expression, as expected [ 18 , 20 ]. A parallel decrease in LAP2 protein expression levels was observed in FGF treated rCEnCs ( Figure 3 b,c).…”

“…Similarly, Pekovic et al described a LAP2α upregulation whenever cells are entering G 1 phase and a downregulation when the cells are quiescent in G 0 [ 29 ] . This was further confirmed by a decreased LAP2 expression, both at RNA and protein level ( Figure 3 ), observed in CEnCs treated with TGFβ, which was proved to reduce proliferation ( Figure 3 b, low ki67) [ 18 , 30 , 31 ].…”

“…FGF (Thermo Fisher Scientific, Waltham, MA, USA) and TGFβ (Miltenyi Biotec, Bergisch Gladbach, Germany) were used at a final concentration of 20 ng/mL and 10 ng/mL, respectively, in the culture medium. All the treatments described were performed 3 h after plating and the cells harvested 24 h after the treatment, as described previously [ 18 ].…”

Regenerative Medicine of Epithelia: Lessons From the Past and Future Goals

“…These approaches include heterologous CEnCs expanded in vitro and injected into the patient's anterior chamber (Okumura et al, 2012;Kinoshita et al, 2018) or implanted through tissue-engineered endothelial keratoplasty (TE-EK) (Peh et al, 2017). A deep knowledge of the finely regulated molecular mechanisms involved in CE proliferation (Joyce, 2012;Frausto et al, 2020;Maurizi et al, 2020) is therefore fundamental to harness a proper approach for this peculiar epithelium, and more advancements are still needed to achieve a full control over its regeneration.…”

Altered gene expression in slc4a11−/− mouse cornea highlights SLC4A11 roles

“…The CEC defects observed in CHED and FECD compromise this pump, leading to stromal edema, corneal hazing and vision loss. A second feature of CHED and FECD is an increased rate of CEC loss, which cannot be compensated by cell proliferation as mature CEC are arrested in the G0-G1 phase of the cell cycle 20 .…”