Slit2 signaling through Robo1 and Robo2 is required for retinal neovascularization

Abstract

Ocular neovascular diseases are a leading cause of blindness. Vascular endothelial growth factor (VEGF) blockade improves vision, but not all individuals respond to anti-VEGF treatment, making additional means to prevent neovascularization necessary. Slit-family proteins (Slits) are ligands of Roundabout (Robo) receptors that repel developing axons in the nervous system. Robo1 expression is altered in ocular neovascular diseases, and previous in vitro studies have reported both pro- and anti-angiogenic effects of Slits. However, genetic evidence supporting a role for Slits in ocular neovascularization is lacking. Here we generated conditional knockout mice deficient in various Slit and Robo proteins and found that Slit2 potently and selectively promoted angiogenesis via Robo1 and Robo2 in mouse postnatal retina and in a model of ocular neovascular disease. Mechanistically, Slit2 acting through Robo1 and Robo2 promoted the migration of endothelial cells. These receptors are required for both Slit2- and VEGF-induced Rac1 activation and lamellipodia formation. Thus, Slit2 blockade could potentially be used therapeutically to inhibit angiogenesis in individuals with ocular neovascular disease.

Figure 1

Slit2 promotes retinal angiogenesis. (a) In situ hybridization for Slit1 mRNA on a P7 retina section (leftmost and rightmost panels) or flat-mounted retina (middle panel). The vasculature was stained with IB4 (green). The magenta pseudocolor is a negative image of the in situ signal. Arrows indicate horizontal cells (n = 3 retinas). (b) In situ hybridization for Slit2 mRNA on a P7 retina section (leftmost and rightmost panels) or flat-mounted retina (middle panel). The vasculature was stained with IB4 (green) (n = 2 retinas). (c) Retinal section from Slit2^+/− mice at P10 stained with anti-GFP (n = 2 retinas). (d) Binding of Slit2-AP and VEGF-AP to flat-mounted P5 retinas of wild-type mice. The data are representative of nine retinas per condition. v, vein; a, artery. (e) In situ hybridization (Slit2 exon 8–specific probe) on P7 retinas of Slit2^lox/loxSlit1^−/− (n = 6) and CAG:Slit2^lox/loxSlit1^−/− (n = 11) mice. For the Slit2^lox/loxSlit1^−/− and CAG: Slit2^lox/loxSlit1^−/− mice, tamoxifen was administered at P0. (f) Western blot quantification (one retina per lane) of Slit2 expression in P7 retinal extracts from mice injected with tamoxifen at P0 (n = 3 Slit2^lox/loxSlit1^−/− retinas; n = 11 CAG:Slit2^lox/loxSlit1^−/− retinas; *P = 0.02, Mann–Whitney test). (g,h) Flat-mounts of retinas from P7 Slit-knockout mice and control littermates stained with IB4. Branch point density (n = 24, 13, 32, 11, 15 and 19 retinas for Slit2^lox/lox, Slit2^lox/loxSlit1^−/−, CAG:Slit2^lox/lox, CAG:Slit2^lox/loxSlit1^−/−, Slit2^lox/loxSlit1^−/− and Slit2^ΔECSlit1^−/− mice, respectively) and the percentage of vascular coverage (n = 15, 15, 16, 11, 11 and 13 retinas for Slit2^lox/lox, Slit2^lox/loxSlit1^−/−, CAG:Slit2^lox/lox, CAG:Slit2^lox/loxSlit1^−/−, Slit2^lox/loxSlit1^−/− and Slit2^ΔECSlit1^−/− mice, respectively) were determined. Rightmost panels in g show sections stained with anti-calretinin (green) and anti-calbindin (CaBP) (magenta) (n = 2 Slit2^lox/loxSlit1^−/− retinas; n = 3 CAG:Slit2^lox/loxSlit1^−/− retinas). All results are presented as mean ± s.e.m. (also see Supplementary Table 1). *P < 0.05, ***P < 0.001, Student's t-test. Scale bars, 50 μm in a–c, e and rightmost images in g; 150 μm in d; and 500 μm in leftmost and middle images in g and all panels in h. INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; RGCL, retinal ganglion cell layer.

Figure 2

Abnormal retinal angiogenesis in Robo1- and Robo2-knockout mice. (a,b) Robo expression as assessed by microarray analysis of mouse retinal endothelial cells (a), qPCR analysis of human endothelial cell mRNA (b, left) and western blot of the corresponding human endothelial cells (b, right). Results shown are from three independent experiments. HUVEC, human umbilical vein endothelial cell; HUAEC, human umbilical artery endothelial cell; HDMEC, human dermal microvascular endothelial cell. (c–e) Robo expression after Robo knockdown in HUVECs as determined by qPCR (c; **P < 0.01, ***P < 0.001 compared to scrambled siRNA control (siCT)) and western blot (d) and in mouse lung endothelial cells (MLEC) isolated from Robo1Robo2–double-knockout mice as determined by western blot (e) (two experiments for each group in d and e). siRobo, siRNA targeting the indicated Robo protein. (f) Slit2-AP binding on P5 retinal flat-mounts from Robo-knockout mice (n = 7, n = 2 and n = 11 retinas for Robo1^−/−Robo2^lox/lox, Robo4^−/− and Robo1^−/−Robo2^ΔEC mice, respectively). v, vein; a, artery. (g) Retinal flat-mounts of P7 Robo1- and Robo2-knockout mice and control mice stained with IB4. Rightmost panels show sections stained with anti-calretinin (green) and anti-CaBP (magenta; n = 3 Robo 1^−/−Robo2^lox/lox retinas; n = 6 Robo1^−/−Robo2^ΔEC retinas). (h,i) Quantification of branch point density (n = 10, 23, 21 and 21 retinas for Robo2^lox/lox, Robo1^−/−Robo2^lox/lox, Robo2^ΔEC and Robo1^−/−Robo2^ΔEC mice, respectively (h); n = 12, 8, 8 and 6 retinas for Robo1^+/−Robo2^lox/loxRobo4^+/−, Robo1^−/−Robo2^lox/loxRobo4^−/−, Robo1^−/−Robo2^ΔECRobo4^+/+ and Robo1^−/−Robo2^ΔECRobo4^−/− mice, respectively (i)). (j) Representative flat-mount of retina from P7 CAG:Robo2^lox/loxRobo1^−/− mouse stained with IB4. Branch point density and the percentage of vascular coverage were quantified (n = 6 Robo2^lox/loxRobo1^−/− retinas; n = 20 CAG:Robo2^lox/loxRobo1^−/− retinas). All results are presented as mean ± s.e.m. ***P < 0.001, Student's t-test (h) or Mann–Whitney test (i,j). NS, not significant. Controls used for comparison were Robo2^lox/lox (h) and Robo1^+/−Robo2^lox/loxRobo4^+/− (i). Scale bars, 200 μm in f, 500 μm in leftmost and middle images in g and in j, and 50 μm in rightmost images in g.

Figure 3

Slit2 promotes sprouting angiogenesis through Robo1 and Robo2. (a) Endothelial cell proliferation at the vascular front in flat-mounted P7 retinas from the indicated strains. Red, Erg-1/2/3; green, EdU; blue, IB4. The percentage of proliferating endothelial cells was quantified (n = 13, 16, 9 and 21 retinas for Slit2^lox/loxSlit1^−/−, CAG:Slit2^lox/loxSlit1^−/−, Robo1^−/−Robo2^lox/lox and Robo1^−/−Robo2^ΔEC mice, respectively). (b,c) P7 retinas stained for Dll4 and Vegfr2 (n = 9, 2, 4 and 6 retinas for Slit2^lox/loxSlit1^−/−, CAG:Slit2^lox/loxSlit1^−/−, Robo1^−/−Robo2^lox/lox and Robo1^−/−Robo2^ΔEC mice, respectively, in b; n = 3, 13, 5 and 12 retinas for Slit2^lox/loxSlit1^−/−, CAG:Slit2^lox/loxSlit1^−/−, Robo1^−/−Robo2^lox/lox and Robo1^−/−Robo2^ΔEC mice, respectively, in c; also see Supplementary Table 1). WT, wild type. (d) Expression of Vegfr2 (Kdr), Nrp1 and Vegfa as determined by qPCR in endothelial cells isolated from Robo1^−/−Robo2^ΔEC mice (upper panel) and in HUVECs with siROBO1 and siROBO2 compared to scrambled siRNA controls (siCT, lower panel). (e) Tip cell marker gene expression analysis by qPCR of HUVECs treated for 20 h with Slit2 or VEGF-A (upper panel, compared to PBS control (CT)) and of VEGF-A–treated HUVECs after application of scrambled control siRNA or knockdown of ROBO1 and ROBO2 (lower panel, compared to PBS control). **P < 0.01, ***P < 0.001. (f) Expression of NOTCH signaling molecules in HUVECs with knockdown of ROBO1 and ROBO2 or of NOTCH1 determined by qPCR (upper panel) and after sDLL4 stimulation (lower panel). (g) HUVEC sprouting in 3D fibrin gels (left) and quantification (right). Cells were treated with siRNAs and then with recombinant proteins as indicated for 96 h. *P < 0.05, **P < 0.01, ***P < 0.001 compared to PBS-treated control. (h) HUVEC scratch-wound migration assay (left) and quantification (right). Cells were treated with siRNAs and then with recombinant proteins as indicated for 16 h. Dashed lines mark wound migration edges. ***P < 0.001 compared to PBS-treated control. All results are presented as mean ± s.e.m. as determined via Student's t-test (a) or two-way analysis of variance and Tukey's multiple-comparisons test (n = 3 independent experiments in d–h). NS, not significant. Scale bars, 50 μm in a–c, 180 μm in g and h.

Figure 4

ROBO1 and ROBO2 regulate Slit2- and VEGF-A–induced RAC1 activation. (a) Phalloidin (red) and DAPI (blue) staining of HUVECs at the edge (left-hand side of all images) of a scratch wound 16 h after wounding. Cells were treated with siRNAs and then with recombinant proteins as indicated. Cells treated with siRNAs targeting ROBO1 and ROBO2 did not form lamellipodia in response to Slit2 and VEGF, but formation did occur in response to bFGF. Scale bars, 45 μm. (b,c) RAC1-GTP loading induced by Slit2 and VEGF in HUVECs with control siRNA and with siRNA targeting ROBO1 and ROBO2 (5-min stimulation) (b). The results of three independent experiments were quantified (c). *P < 0.05 compared to PBS-treated control. (d,e) Effects of low-dose Slit2 and VEGF treatment on RAC1-GTP loading (5-min stimulation) (d). The results of three experiments were quantified (e). *P < 0.05 compared to PBS-treated control. (f,g) Effects of Slit2 treatment on PAK2 and AKT phosphorylation in siRNA control HUVECs and cells with knockdown of ROBO1 and ROBO2 (f). The results of three independent experiments were quantified (g). *P < 0.05, **P < 0.01 compared to PBS-treated control. (h,i) Effects of VEGF-A on phosphorylation of ERK and AKT in HUVECs with control siRNA and with siRNA targeting ROBO1 and ROBO2 (h). Quantification of p-AKT compared to total AKT and p-ERK compared to total ERK was performed for three independent experiments (i). All values are mean ± s.e.m. P values were calculated using Student's t-test.

Figure 5

Targeting Slit2–Robo signaling blocks angiogenesis in OIR. (a,c,e,g) IB4-stained flat-mounts of P17 retinas from Slit-knockout (a,c) and Robo-knockout (e) mice after OIR. Retinas in g are from mice injected with adenovirus encoding GFP (adCTL) or adenovirus encoding ROBO1-Fc (adROBO1-Fc). A higher-magnification image of neovessel sprouting and vascular tufts is shown in c; images show areas indicated by the arrows (upper panels) and arrowheads (lower panels) in a. (b) Quantification of the avascular area in Slit-knockout mice (n = 10, 22, 12 and 23 retinas for Slit2^lox/lox, Slit2^lox/loxSlit1^−/−, CAG:Slit2^lox/lox and CAG:Slit2^lox/loxSlit1^−/− mice, respectively). (d) Quantification of neovessel sprouting from veins. (f) Quantification of avascular area in Robo-knockout mice (n = 31, Robo2^lox/loxRobo1^−/− retinas; n = 22, CAG:Robo2^lox/loxRobo1^−/− retinas). (h) Quantification of the avascular area in mice injected with adenovirus encoding GFP (n = 10 retinas) or adenovirus encoding ROBO1-Fc (n = 8 retinas). All results are presented as mean ± s.e.m. ***P < 0.001, Student's t-test. NS, not significant. Scale bars, 500 μm1 in a, e and g; 100 μm in c.