Abstract
Purpose :
Previously, we have demonstrated the effect of Cathepsin D (CD) on the mechanical disruption of retinal endothelial cell junctions and increased vaso-permeability, and increased levels of cathepsin D in retinas of diabetic mice. Here we have further examined the effect of CD on the endothelial-pericyte interaction, and the effect of Dipeptidyl peptidase-4 (DPP-4) inhibitor on CD in the endothelial-pericyte interaction. The DPP-4 inhibitor has been shown to alter the function of the IGF2 receptor, which is the presumed binding site for CD on endothelial cells.
Methods :
Human retinal endothelial cells (HREC) and human retinal pericytes (HRP) were co-cultured and treated overnight with 25ug/ml of recombinant pro-CD along with 250nM DPP-4 inhibitor. Western blots were performed to check the levels of PDGFR-β, N-Cad, PKC-α and phosphor-PKC-α. Real-time PCR was used to measure angiopoietin-2 (Ang-2) levels in the treated co-cultured cells. GFP-HRP cells were co-cultured with HRECs to check the binding efficiency in treated conditions.
Results :
Co-cultured cells treated with pro-CD showed a significant decrease in the expression of PDGFR- β, a tyrosine kinase receptor required for pericyte cell survival and N-Cad, the key adherens junction protein between endothelium and pericytes, and also increase in the vessel destabilizing agent, Ang-2. The effect was reversed in the cells treated with DPP-4 inhibitor along with pro-CD. With pro-CD treatment, there was a significant increase in the downstream signaling protein PKC-α, which disrupts tight junction structure and function, and this was significantly reduced with DPP-4 inhibitor treatment. We observed significantly increased binding of endothelial cells and pericytes when cells were treated with the DPP-4 inhibitor and pro-CD.
Conclusions :
The cathepsin D decreases N-cad and PDGFR- β in the endothelium-pericyte alteration in the blood-retinal barrier, and the drug DPP-4 inhibitor can significantly diminish this effect. Thus, the DPP-4 inhibitor may be used as a potential adjuvant therapeutic strategy for treating diabetic macular edema.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.