Notch signaling functions in retinal pericytes survival

Abstract

Purpose: Pericytes, the cells that constitute the outer layer of capillaries, have been shown to play a crucial role in vascular development and stability. Loss of pericytes precedes endothelial cell dysfunction and vascular degeneration in diabetic retinopathy. Despite their obvious relevance, the cellular pathways controlling survival of retinal pericytes remain largely unknown. We examine the possible role of Notch signaling, a master regulator of cell fate decisions, in retinal pericytes using gain-of-function (GOF) and loss-of-function (LOF) approaches in a coculture system.

Methods: All experiments were performed with early passage (P 1-3), primary culture bovine retinal pericytes (BRP). For Notch GOF experiments, BRP’s were transfected with a Notch 3 constitutively active intracellular domain (N3ICD) fused to GFP. For Notch LOF experiments, BRP’s were exposed to compound E, a gamma-secretase inhibitor known to abrogate proteolytic activation of Notch receptors. Changes in protein expression were measured using chemiluminescent antibody arrays and by FACS. Coculture experiments were performed by culturing BRPs with Delta 1-like (Dll1) ligand-expressing mouse embryonic fibroblasts (MEFs). To examine the effect of more physiological levels of Notch activity on pericyte survival, BRPs were cocultured with DLL1 cells under light exposure conditions that have been previously reported to trigger cell death.

Results: Notch GOF experiments showed significant (P≤0.05) up regulation of five apoptosis-related proteins: Bcl-x, clusterin, cytochrome c, and SMAD/Diablo whereas Notch LOF experiments revealed down regulation of the same proteins (P≤0.05 for all except cytochrome c, which was not significant). In response to light exposure, BRPs cocultured with Dll1-expressing showed a significant decrease in cleaved caspase-3 expression compared to BRPs cocultured with control cells (P<0.05).

Conclusions: These results demonstrate a key role for Notch signaling in retinal pericyte survival, a key target of diabetic retinopathy. Manipulation of pericyte fates through Notch pathways may lead to novel therapeutic interventions for this defining complication of diabetes.

Keywords: 499 diabetic retinopathy • 426 apoptosis/cell death