Abstract
Purpose :
The inhibitor of dipeptidyl-peptidase-IV (DPP-IV) sitagliptin - often used to treat diabetes mellitus – impairs the barrier formed by immortalized bovine retinal endothelial cells (iBREC) starting about 30h after its addition at a concentration of 10nM, at which it specifically inhibits DPP-IV activity. Surprisingly, expression of tight-junction (TJ) protein claudin-1 is then significantly higher, that of TJ-protein claudin-5 and adherens junction (AJ) protein vascular endothelial cadherin (VEcadherin) remains unchanged, but that of integrin β1 is slightly lower. Here, we studied the nature of sitagliptin-induced barrier disturbance in more detail.
Methods :
Confluent iBREC were exposed to 10nM sitagliptin for 2d before subcellular protein fractions were prepared, resulting in proteins from the cytoplasm (fraction CP), from the membranes and organelles (fraction MO), and the cytoskeleton (fraction CS), subsequently analyzed by Western-blotting. Supernatants were tested by ELISA for presence of permeability-inducing growth factors or cytokines. To evaluate changes in para- and/or transcellular flow as well as adhesion of the cells during treatment with sitagliptin and 10nM tivozanib – inhibitor of VEGF receptor 2 –we continuously measured the cell index (CI) of iBREC grown on gold electrodes.
Results :
Inhibition of VEGF receptor 2 by tivozanib did not prevent sitagliptin-induced changes. Sitagliptin-exposed iBREC also did not secrete VEGF-A, IL-2, IL-6, IL-8, TNFα; low angiopoietin-2 levels (~200pg/ml) were not changed. The inhibitor induced a significant shift in subcellular localization of claudin-5 and adhesion protein CD9 from fraction CP to fraction MO, which also contained significantly more VEcadherin and integrin β1, but significantly less integrin α5. Unchallenged as well as sitagliptin-exposed iBREC did not express the regulator of transcellular flow plasma lemma vesicle assocated protein.
Conclusions :
Sitagliptin-induced impairment of the barrier formed by iBREC is not associated with signal transduction driven by VEGF-A; dysregulation of para- and transcellular flow is also prevented. However, by affecting subcellular localization of subunits integrin α5 and β1 of the fibronectin receptor in opposite ways, proper formation of this receptor is possibly affected. This confirms our hypothesis that sitagliptin interferes with iBREC’s interaction with the extracellular matrix.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.