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

Abstract

The corneal endothelium is the inner corneal mono-layered epithelium, fundamental for preserving corneal hydration and transparency. However, molecular mechanisms that regulate corneal endothelial cells (CEnCs), in particular regarding their proliferative capacity, have been only partially elucidated. CEnCs are quiescent in vivo and they easily undergo endothelial to mesenchymal transition (EnMT) in vitro. This study aims to analyze CEnCs behavior and expression in vitro, either in sub-confluent growing (S) or confluent (C) CEnCs cultures. Primary rabbit and human CEnCs were cultured and used for RT-PCR, immunofluorescence or western blot analysis. These methods allowed identifying a novel molecular marker, LAP2, that is upregulated in S while downregulated in C human or rabbit CEnCs. Those results were observed for several subsequent passages in culture and this, together with the correlation between ki67 and LAP2 expression, suggested LAP2 as a novel possible indicator for culture ageing. Finally, treatment with FGF and TGFβ in rCEnCs highlighted how LAP2 can vary as the cells regulate their proliferative state. In conclusion, we have identified a novel marker for CEnCs, LAP2, that regulates its expression depending on the cells sub/confluent state and that correlates with CEnCs proliferation.

Specular microscopy of the corneal endothelial cells of bovines: An ex vivo study

“…However, there are few ophthalmological studies related to endothelial parameters in bovine species ( Tofflemire et al , 2015 ). Several methods have been tested to evaluate the proliferative capacity of the corneal endothelium ( Katz et al , 1994 ; Maurizi et al , 2020 ; Pei et al , 2021 ; Maurizi et al , 2022 ). Faye and collaborators reviewed different aspects related to the cultivation of endothelial cells in vitro ( Faye et al , 2021 ).…”

Nanoneedles Induce Targeted siRNA Silencing of p16 in the Human Corneal Endothelium

“…[4] Moreover, increasing the number of HCEnCs through their expansion in eye bank corneas would be beneficial to reduce tissue wastage, [5] as the high cell death rate during storage, in particular following apoptosis in the corneal endothelium, leads to a decrease in HCEnCs density [5] and rejection of more than 35% of stored corneas. [2,6] A deeper understanding and modulation of the molecular mechanisms regulating HCEnCs proliferation is fundamental to increase their density; [7,8] gene therapy has been explored for this purpose in many ways, including by inhibiting apoptosis through overexpression of antiapoptotic genes (e.g., Bcl-xl), [9,10] by inducing cell proliferation through overexpression of transcription factors such (e.g., E2F2), [11] or by downregulation of cell cycle inhibitors (e.g., p21, p16, [12,13] p27, [14] SNAI1, and CDK2). [15] Yet, nucleotide delivery to the corneal endothelium is challenging, as the cells are postmitotic and thus hard to transfect.…”

“…A deeper understanding and modulation of the molecular mechanisms regulating HCEnCs proliferation is fundamental to increase their density; [ 7,8 ] gene therapy has been explored for this purpose in many ways, including by inhibiting apoptosis through overexpression of antiapoptotic genes (e.g., Bcl‐xl), [ 9,10 ] by inducing cell proliferation through overexpression of transcription factors such (e.g., E2F2), [ 11 ] or by downregulation of cell cycle inhibitors (e.g., p21, p16, [ 12,13 ] p27, [ 14 ] SNAI1, and CDK2). [ 15 ] Yet, nucleotide delivery to the corneal endothelium is challenging, as the cells are postmitotic and thus hard to transfect.…”

GSK3 inhibition reverts mesenchymal transition in human primary corneal endothelial cells

“…However, if EnMT is dysregulated, for instance during in vitro expansion when HCEnCs are induced to leave their quiescence (G0/G1) and become proliferative (G2), it may lead to an irreversible mesenchymal transformation 10 . EnMT transformed HCEnCs (positive for specific markers such as -SMA), arise not only after passaging 12 , but also consequently to the cell junctions disruption following EDTA treatment 13 , as well as upon growth factors stimulation [13][14][15][16][17] . It has been largely documented that epithelial and fibroblast growth factors (EGF and FGF, respectively) are able to promote cellular proliferation while preserving the cellular phenotype [18][19][20] , but only for few passages [13][14][15][16][17]21 , while transforming growth factor (TGF induces EnMT upon certain conditions 17 .…”