The role of receptor MAS in microglia-driven retinal vascular development

Abstract

Objective: The receptor MAS, encoded by Mas1, is expressed in microglia and its activation has been linked to anti-inflammatory actions. However, microglia are involved in several different processes in the central nervous system, including the promotion of angiogenesis. We therefore hypothesized that the receptor MAS also plays a role in angiogenesis via microglia.

Approach and results: To assess the role of MAS on vascular network development, flat-mounted retinas from 3-day-old wild-type (WT) and Mas1^-/- mice were subjected to Isolectin B4 staining. The progression of the vascular front was reduced (- 24%, p < 0.0001) and vascular density decreased (- 38%, p < 0.001) in Mas1^-/- compared to WT mice with no change in the junction density. The number of filopodia and filopodia bursts were decreased in Mas1^-/- mice at the vascular front (- 21%, p < 0.05; - 29%, p < 0.0001, respectively). This was associated with a decreased number of vascular loops and decreased microglial density at the vascular front in Mas1^-/- mice (-32%, p < 0.001; - 26%, p < 0.05, respectively). As the front of the developing vasculature is characterized by reduced oxygen levels, we determined the expression of Mas1 following hypoxia in primary microglia from 3-day-old WT mice. Hypoxia induced a 14-fold increase of Mas1 mRNA expression (p < 0.01). Moreover, stimulation of primary microglia with a MAS agonist induced expression of Notch1 (+ 57%, p < 0.05), Dll4 (+ 220%, p < 0.001) and Jag1 (+ 137%, p < 0.001), genes previously described to mediate microglia/endothelial cell interaction during angiogenesis.

Conclusions: Our study demonstrates that the activation of MAS is important for microglia recruitment and vascular growth in the developing retina.

Keywords: Angiogenesis; Angiotensin receptors; CNS; Developmental biology; Endothelium; Macrophage; Renin angiotensin system; Vascular biology.

Fig. 1

Retinal vasculature in 3-day-old WT and Mas1^−/− mice (n = 8). Representative image of retinal vasculature stained with IB4 (green) (a, scale bar = 300 µm); Vascular front (b), vascular density (c) and junctions density (d); (n = 6–8). Representative pictures of vascular sprouts and filopodia at the vascular front (e; arrows: filopodia; yellow line: vascular front; white dots: filopodia bursts; stars: microglia; scale bars = 20 µm); Filopodia density (f), filopodia burst densities (g), number of sprouts (h), average sprout length (i) and number of vascular loops (j) at the vascular front of retinas from WT and Mas1^−/− mice. *p < 0.05 versus WT

Fig. 2

Retinal microglial densities. Vasculature (IB4) and microglia (Iba1) at the vascular front of retinas from 3-day-old WT and Mas1^−/− mice (a); microglial density in an avascular area (b) and at the vascular front (c); (n = 5–9). Il-6 and Mas1 mRNA expressions by primary microglia during hypoxia (d); Il-10, Notch1, Dll4 and Jag1 mRNA expressions in microglia treated with a MAS agonist (AVE0991, 1 µM) (e); (n = 2 experiments, triplicates). *p < 0.05 versus WT

Fig. 3

Impact of MAS stimulation on retinal endothelial cells in vitro. Tube formation assay with RF/6A cells (a–c) treated with PBS, VEGF (1 nM), Suramin (SUR, 10 µM) or a MAS receptor agonist (AVE0991, 1 µM). Representative pictures (a, scale bar = 200 µm); Quantification of total tube length (b) and junctions density (c) (n = 6–8); One-way ANOVA, followed by Tukey’s multiple comparisons post-test (*p < 0.05 vs. PBS; ^$p < 0.05 vs. VEGF). Mas1, Vegfa, Notch1 and Jag1 mRNA expressions in RF/6A cells treated with a MAS agonist (AVE0991, 1 µM) (d); (n = 2 experiments, triplicates)

Fig. 4

Schematic representation of the importance of Mas1 in retinal vascular development