Abstract
Purpose :
To study the role of the third extracellular loop (EL3) of SLC4A11 in the adhesion of corneal endothelial cells (CECs) to Descemet membrane (DM).
Methods :
Proteins from native or post-confluent cultures of human CECs were harvested to study the expression of SLC4A11 using Western blots. Sub-confluent primary cultures of CECs were transfected using lipid vectors and a plasmid of EL3 for 40 hours. Efficiency of the transfection was verified using dot blots, Western blots and immunostainings. Transfected (or untransfected) cells were seeded on DMs previously decellularized using three freeze/thaw cycles, incubated for 2 hours, then rinsed. Adhered cells were photographed using a stereomicroscope and counted. In another set of experiments, functional anti-integrin antibodies (α1, α2β1 and β1) or blocking peptide RGD were used to inhibit integrin activity in the adhesion assays. In parallel, CECs were seeded on glass coverslips and cultured 7 days, then fixed and immunostained against integrins subunits (α1, α2, α3, α4, α5, α6, α10, α11, αV, αVβ3, αVβ5 and β1).
Results :
Compared to native cells, the protein expression of SLC4A11 decreased in vitro. Transfection of EL3 allowed to increase its expression, and was correctly cytolocalized to the membrane. No difference in cell adhesion to DM was observed between the EL3-transfected and the untransfected cells. Since the immunostaining experiments revealed that integrin subunits α1, α2, α3 and β1 were expressed in primary cultures of CECs, the adhesion assays were performed in the presence of functional anti-integrins or blocking peptide RGD. RGD blocking peptide inhibited 27% of CEC adhesion, while the use of functional antibodies didn’t show a difference in cell adhesion to DM.
Conclusions :
Transfection of EL3 in human CECs using lipid vectors allowed to increase its expression. Expressed integrin subunits identified in primary cultures of CECs interfered with the EL3 studies in cell adhesion to DM. This adhesion is partially mediated by RGD. Additional knowledge on how cells adhere to DM is needed in order to study EL3 and learn more about its role in CEC adhesion.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.