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Kim Santerre, Stephanie Proulx; Active TGF-β promotes intercellular junction formation for post-confluent corneal endothelial cells.. Invest. Ophthalmol. Vis. Sci. 2018;59(9):1363.
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Recently, we demonstrated that the addition of TGF-β1 to post-confluent cultures of corneal endothelial cells (CEC) preserved an endothelial phenotype. As aqueous humour contains all TGF-β isoforms (TGF-β1, -β2 and -β3), we investigated the effect of isoform 2 and 3 during CEC culture.
Human CEC (N=4) at passage 3 were cultured using a dual media approach. Cells were first cultured in a proliferation medium, then switched to a maturation medium supplemented (or not) with TGF-β1, -β2 or -β3 (5 ng/ml). Cells were maintained post-confluent for 7 days before analysis. Cell circularity (4π area/perimeter) was calculated from phase contrast images in order to compare cell morphology. The expression and cytolocalisation of intercellular junction-related proteins (ZO-1, β-catenin and N-cadherin) was assessed using immunofluorescence. Western blot (WB) against N-cadherin was also performed. Finally, transendothelial electric resistance (TER) and permeability to FITC-dextran (50kDa) were measured to evaluate CEC functionality.
Post-confluent CEC that matured without TGF-β demonstrated an elongated morphology with a circularity index of 0.56±0.16. Adding TGF-β to the maturation medium enhanced endothelial morphology, as seen with significantly higher circularity indexes (TGF-β1: 0.67±0.13; TGF-β2: 0.71±0.11; TGF-β3: 0.64±0.13). While N-cadherin was absent when TGF-β was omitted from the maturation medium, all 3 TGF-β isoforms induced its expression at the cell membrane. The upregulation of N-Cadherin was confirmed by WB. CEC cultured without TGF-β showed cytoplasmic ZO-1 and β-catenin. However, a cell membrane cytolocalisation of ZO-1 and β-catenin was observed for CEC cultured with TGF-β. TER and permeability measurements were similar regardless of the presence of TGF-β (TER: without TGF-β: 27.6±6.1 ohm-cm2; TGF-β1: 22.7±5.5 ohm-cm2; TGF-β2 30.6±6.4 ohm-cm2; TGF-β3: 28.2±3.8 ohm-cm2; permeability: without TGF-β: 20.1±16.4%; TGF-β1: 27.9±13.4%; TGF-β2: 16.1±6.8%; TGF-β3: 20.1±10.3%).
Our results suggest a role for TGF-β in the formation of intercellular junctions and endothelial phenotype preservation. As cell-cell junction and endothelial morphology are key features for CEC functionality, adding TGF-β during the maturation of cultured cells would confer an advantage for their clinical use.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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