Abstract
Purpose:
We have shown that monocytes/macrophages recruited into the retina in response to diabetes facilitate the breakdown of the blood-retinal barrier (BRB). We have identified a 30-100kDa fraction of macrophage-conditioned medium responsible for alteration of retinal permeability. In this study we further characterized this fraction and identified Cathepsin D and its role in endothelial barrier alteration and diabetic retinopathy.
Methods:
Phorbol 12-myristate 13-Acetate (PMA) stimulated U937 human macrophage conditioned media was fractioned by molecular weight and the effective fraction (30-100kDa) was further analysed by Liquid Chromatography-Mass Spectrometry (LC-MS). A role for cathepsin D in mediating transendothelial resistance in human retinal endothelial cells (HREC) was measured by electric cell-substrate Impedence Sensing (ECIS). Cathepsin D was immunoprecipitated with agarose beads from the 30-100kDa fraction and used to treat the HREC. The specificity of its role was confirmed by siRNA studies. Cathepsin D levels in retinas of four month streptozotocin induced diabetic mice was measured by western blot.
Results:
Mass spectroscopy results identified 30 proteins differentially regulated in macrophage conditioned 30-100kDa fraction in comparison with non stimulated cells, which included heat shock proteins, PAI-2, SOD and Cathepsin D. Cathepsin D was selected for further studies due to its known role in barrier alteration in cardiovascular disease and cancer. HREC treated with immunoprecipitated cathepsin D beads showed a significant alteration in barrier resistance. Treatement of U937 cells with cathepsin D siRNA blocked this effect. Additionally retinas from four month diabetic mice exhibited significantly increased levels of cathepsin D protein.
Conclusions:
In this study we have shown the role of cathepsin D as a potential therapeutic target in diabetic retinopathy due to its role in altering the BRB and its increased levels in diabetic animals.