Robo4 stabilizes the vascular network by inhibiting pathologic angiogenesis and endothelial hyperpermeability

Abstract

The angiogenic sprout has been compared to the growing axon, and indeed, many proteins direct pathfinding by both structures. The Roundabout (Robo) proteins are guidance receptors with well-established functions in the nervous system; however, their role in the mammalian vasculature remains ill defined. Here we show that an endothelial-specific Robo, Robo4, maintains vascular integrity. Activation of Robo4 by Slit2 inhibits vascular endothelial growth factor (VEGF)-165-induced migration, tube formation and permeability in vitro and VEGF-165-stimulated vascular leak in vivo by blocking Src family kinase activation. In mouse models of retinal and choroidal vascular disease, Slit2 inhibited angiogenesis and vascular leak, whereas deletion of Robo4 enhanced these pathologic processes. Our results define a previously unknown function for Robo receptors in stabilizing the vasculature and suggest that activating Robo4 may have broad therapeutic application in diseases characterized by excessive angiogenesis and/or vascular leak.

Figure 1

Robo4 expression is endothelial specific and stalk-cell centric. (a) Retinal flat mounts were prepared from P5 Robo4^+/AP mice and stained for endomucin (endothelial cell marker; green), NG2 (pericyte marker; red) and alkaline phosphatase (AP; Robo4; blue). The yellow arrow indicates a tip cell, and white arrows indicate pericytes (NG2-positive cells). T, tip cells. (b) Retinal flat mounts were prepared from adult Robo4^+/AP mice and stained for NG2 (pericytes) and AP (Robo4). (c) Quantitative RT-PCR was performed on the indicated samples using primers specific for Pecam1, Robo1 and Robo4. HAEC, human aortic endothelial cell; HASMC, human aortic smooth muscle cell; HMVEC, human microvascular endothelial cell. (d) Total cell lysates from HMVECs and HASMCs were probed with antibodies to Robo4, VE-cadherin, smooth-muscle actin and ERK1/2. Experiments were performed three times and error bars represent s.e.m.

Figure 2

Robo4 signaling inhibits VEGF-165–induced migration, tube formation, permeability and SFK activation. (a,b) Lung endothelial cells isolated from Robo4^+/+ and Robo4^AP/AP mice were used in migration (a), tube formation (b) and in vitro permeability assays (c). Robo4^+/+ and Robo4^AP/AP mice were used in the Miles assay (d) and retinal permeability assays (e). (f) Human microvascular endothelial cells were stimulated with VEGF-165 in the presence of Mock or Slit2 for 5 min, lysed and subjected to western blotting with antibodies to phospho-VEGFR2 (top) or phospho-Src (middle) or to Rac activation assays (bottom). (g) Robo4^+/+ and Robo4^AP/AP mice were subjected to retinal permeability assays after intravitreal injection of the SFK inhibitor PP2 or the inactive analog PP3. In all panels, Mock indicates a sham preparation of Slit2. *P < 0.05; **P < 0.005; ***P < 0.0005. NS, not significant. Error bars represent s.e.m. In a–c, experiments were repeated three times each in triplicate. In d, e and g, five mice were tested for each condition for each genotype.

Figure 3

Slit2 blocks oxygen-induced retinopathy in a Robo4-dependent manner. (a,b) Neonatal Robo4^+/+ (a) and Robo4^AP/AP (b) mice were subjected to OIR and perfused with fluorescein isothiocyanate (FITC)-dextran (green). Retinal flat mounts were prepared for each condition and analyzed by fluorescence microscopy. Top panels are low-magnification images (yellow arrows indicate areas of increased angiogenesis), and bottom panels are high-magnification images (white arrowheads indicate neovascular tufts). (c) Quantification of angiogenesis observed in a,b. Error bars represent s.e.m. **P < 0.005. A minimum of five mice were tested for each condition for each genotype.

Figure 4

Robo4 signaling inhibits pathologic angiogenesis. (a) Retinal flat mounts were prepared from neonatal Robo4^+/+ mice subjected to OIR, stained with fluorescent isolectin and analyzed by fluorescence microscopy. Top panels are low-magnification images (arrows indicate pathologic neovascular tufts), and bottom panels are high-magnification images. Arrowheads point to pathologic vascular tufts. (b) Quantification of pathologic neovascularization performed on a minimum of 12 mice for each condition for each genotype shown in a. Error bars represent s.e.m. (c) 2–3-month-old Robo4^+/+ and Robo4^AP/AP mice were subjected to laser-induced choroidal neovascularization. Choroidal flat mounts were prepared, stained with fluorescent isolectin and analyzed by confocal microscopy. (d) Quantification of pathologic angiogenesis performed on a minimum of ten mice for each condition for each genotype shown in c. In all panels, Mock indicates a sham preparation of Slit2. *P < 0.05. NS, not significant. Error bars represent s.e.m. (e,f) Slit-Robo signaling promotes vascular stability via inhibition of SFKs. In the retinal endothelium, the VEGF receptor (VEGFR2) is expressed in both the tip and stalk cells, whereas Robo4 is expressed predominantly in the stalk cells (e). Overproduction of VEGF-165 causes angiogenesis and hyperpermeability, leading to vascular dysfunction in the retina (top). Activation of Robo4 in the stalk cells by Slit proteins limits these pathologic processes (bottom). VEGF-165 stimulates angiogenesis and permeability by activating SFKs and Rac1 in a sequential fashion, (f). Slit-Robo4 signaling inhibits activation of SFKs and Rac1 to block these VEGF-165–driven processes.