Notch signaling functions in retinal pericyte survival

Abstract

Purpose: Pericytes, the vascular cells that constitute the outer layer of capillaries, have been shown to have a crucial role in vascular development and stability. Loss of pericytes precedes endothelial cell dysfunction and vascular degeneration in small-vessel diseases, including diabetic retinopathy. Despite their clinical relevance, the cellular pathways controlling survival of retinal pericytes remain largely uncharacterized. Therefore, we investigated the role of Notch signaling, a master regulator of cell fate decisions, in retinal pericyte survival.

Methods: A coculture system of ligand-dependent Notch signaling was developed using primary cultured retinal pericytes and a mesenchymal cell line derived from an inducible mouse model expressing the Delta-like 1 Notch ligand. This model was used to examine the effect of Notch activity on pericyte survival using quantitative PCR (qPCR) and a light-induced cell death assay. The effect of Notch gain- and loss-of-function was analyzed in monocultures of retinal pericytes using antibody arrays to interrogate the expression of apoptosis-related proteins.

Results: Primary cultured retinal pericytes differentially expressed key molecules of the Notch pathway and displayed strong expression of canonical Notch/RBPJK (recombination signal-binding protein 1 for J-kappa) downstream targets. A gene expression screen using gain- and loss-of-function approaches identified genes relevant to cell survival as downstream targets of Notch activity in retinal pericytes. Ligand-mediated Notch activity protected retinal pericytes from light-induced cell death.

Conclusions: Our results have identified signature genes downstream of Notch activity in retinal pericytes and suggest that tight regulation of Notch signaling is crucial for pericyte survival.

Keywords: coculture; diabetic retinopathy; notch signaling; pericyte; small-vessel; survival.

Figure 1

Delta-like 1–mediated activation of Notch signaling in retinal pericytes. (A) Western blot of lysates from ROSA26LSL-Dll1/CRE-ERT2 cells treated with 4-OHT or DMSO. (B) Bovine retinal pericytes were transfected with a TP1-luciferase Notch reporter before coculture with mesenchymal cells induced to express DLL1 or control cells for 48 hours in the presence of γ-secretase inhibitor Compound E or DMSO. (C) Bovine retinal pericytes were transfected with LFng or a control plasmid and TP1-Luciferase before coculture with DLL1 or control cells. Overexpression of LFng led to a significant enhancement of DLL1-mediated Notch signaling in BRPs. ***P < 0.005.

Figure 2

Principal component analysis was used to reduce the dimensionality of the data and to allow visualization and clustering of microarray gene expression from three datasets corresponding to Notch 3 LOF in cells/arteries from different tissues. (A, B) Percentage in each PC indicates the proportion of total variation represented by that component. The PCs are uncorrelated and ordered by descending magnitude. Note that in (B) most of the large ovals representing Notch 3 LOF are located in the coordinates corresponding to negative values. (C) Dot plot of Notch 3 expression across tissues.

Figure 3

Genes regulated by Notch LOF in retinal pericytes. (A) Bovine retinal pericytes were cocultured with PKH-labeled cells expressing DLL1 or control cells in the presence of Compound E (100 nM) or DMSO for 48 hours before separation by FACS. The Array column indicates if a gene has known function in the Notch pathway, apoptosis, or if it was selected for analysis in BRPs because it was misregulated in N3KO mice. Fold-change indicates gene expression differences in BRPs exposed to Compound E compared to controls as measured by qPCR. (B) Heat map represents changes in gene expression in BRP monocultures in the presence of increasing concentrations of Compound E (1 or 100 nM) for 48 hours before RNA harvest. P values of the differences between control and Compound E 100 nM are <0.001 in every case, except when listed as not significant (NS).

Figure 4

Apoptosis-related proteins regulated by Notch GOF in retinal pericytes. (A) Bovine retinal pericyte monocultures were transfected with N3ICD-GFP or GFP constructs and cultured for 24 hours before cell harvest. Duplicate dot blots indicate signal for each specific protein. Prominent dots at the edges of the arrays represent alignment controls. Coordinates for all the proteins are provided in Supplementary Table S4 (B) Shorter exposure of image show in (A).

Figure 5

Apoptosis-related proteins regulated by Notch LOF in retinal pericytes. (A) Bovine retinal pericyte monocultures were transfected with GFP and incubated with DMSO or 100 nM Compound E for 48 hours before cell harvest. Duplicate dot blots indicate signal for each specific protein. Prominent dots at the edges of the arrays represent alignment controls. Coordinates for all the proteins are provided in Supplementary Table S4 (B) Shorter exposure of image show in (A).

Figure 6

Tight regulation of Notch signaling is crucial for pericyte survival. (A) BRPs were cocultured with DLL1-expressing cells or control cells in light or dark conditions before labeling with an anticleaved caspase-3 antibody and FACS. Bovine retinal pericytes exposed to light had a 2.3-fold increase in cleaved caspase-3 signal compared to dark (*P < 0.01), which was significantly reduced by coculture with DLL1-expressing cells (**P < 0.02). (B) Our working model for the regulation of pericyte cell survival by Notch signaling. In this study, ligand-mediated Notch signaling has been shown to be protective for pericytes (as seen in [A]), whereas LOF and GOF result in misregulation of cell survival pathways.