To ascertain the functional significance of Robo4 in vivo
, we generated mice in which exons one through five of the Robo4
locus were replaced with the human placental alkaline phosphatase (ALPP
) reporter gene (Supplementary Fig. 1a online
). The resulting allele, Robo4AP
, lacks the exons encoding the immunoglobulin (IgG) repeats of the Robo4 ectodomain, which are predicted to be required for interaction with Slit proteins9,10
. We intercrossed Robo4+/AP
animals to generate mice that are homozygous for the targeted allele, and verified transmission of this allele by Southern blotting (Supplementary Fig. 1b
). Neither Robo4
mRNA nor Robo4 protein could be detected in Robo4AP/AP
animals, indicating that Robo4AP
is a null allele (Supplementary Fig. 1c and Supplementary Fig. 2a online
). We also found that expression of Robo1, Robo2
and of Slit1, Slit2
is unaffected by loss of Robo4 (Supplementary Fig. 2b
animals were viable and fertile (Supplementary Table 1 online
), and they showed normal patterning of the intersomitic and cephalic vessels during early embryogenesis (Supplementary Fig. 1d and Supplementary Fig. 3 online
) as well as stereotypical nerve-artery alignment and smooth muscle coverage in the limb skin at embryonic day (E) 15.5 (Supplementary Fig. 4 online
). These data indicated that Robo4 is not required for sprouting angiogenesis or peripheral nerve–mediated arteriogenesis in the developing mouse, and they suggested an alternate function for Robo signaling in the mammalian endothelium.
Using alkaline phosphatase activity as a marker of Robo4
expression, we confirmed that Robo4
is specifically transcribed in the endothelium of the developing embryo and in various vascular beds of the adult mouse (Supplementary Fig. 5 online
). One vascular bed that is amenable to the analysis of developmental and pathologic angiogenesis is in the neonatal mouse retina11–13
. High-resolution imaging of these blood vessels has demonstrated the existence of two discrete populations of endothelial cells, which possess unique structural and functional characteristics12
. One population, the tip cells, are non-lumenized structures at the sprouting front of the vascular plexus that use filopodial extensions to sense and respond to extracellular cues, such as VEGF-165 (refs. 12,14
). The second population, the stalk cells, form a lumenized, interconnected network that defines the remainder of the retinal vascular plexus12
. To determine whether Robo4
shows cell type–specific expression within the retinal vascular bed, we compared the expression of alkaline phosphatase to that of the pan-endothelial marker endomucin and the pericyte marker NG2. As expected, endomucin labeled the entire endothelium, and NG2 delimited the retinal vessels (). Unexpectedly, we found that Robo4
was highly transcribed in the endothelial cells that form the stalk of retinal blood vessels, and absent from many of the tip cells. We were unable to detect coincident expression of Robo4
and NG2 in postnatal day (P) 5 and adult retinas (); nor could we identify Robo4 in human aortic smooth muscle cells (HASMCs) by quantitative RT-PCR or western blotting (), suggesting that Robo4 functions in an endothelial cell–autonomous fashion. The observed stalk cell–centric Robo4
transcription was surprising given the involvement of Robo signaling in axon guidance2,3
. Indeed, our a priori
hypothesis had been that Robo4
would be strongly expressed in the tip cells, the endothelial analog of the axonal growth cone1
. This expression pattern suggested that Robo4 has a biological role that is unrelated to the archetypal guidance mechanisms regulating vascular patterning.
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). (more ...)
Endothelial stalk cells are similar to the differentiated and stabilized phenotype characteristic of a mature, lumenized vascular tube. We hypothesized, therefore, that Robo4 expression might maintain this phenotype by inhibiting processes that are stimulated by proangiogenic factors, such as VEGF-165. Three assays routinely used to investigate angiogenesis in vitro
assess endothelial cell proliferation, migration and tube formation, and we sought to determine the effect of Robo4 signaling on these processes. We isolated endothelial cells from the lungs of Robo4+/+
mice and confirmed their identity through immunocytochemistry and flow cytometry (Supplementary Fig. 6 online
). We then used these cells in VEGF-165–dependent proliferation, migration and tube formation assays. We found that Slit2 had no effect on VEGF-165’s mitogenic activity (data not shown) but was capable of inhibiting both migration and tube formation of Robo4+/+
endothelial cells (). The inhibitory activity of Slit2 was lost in Robo4AP/AP
endothelial cells, demonstrating that Slit2 inhibits endothelial cell migration and tube formation in a Robo4-dependent manner.
Figure 2 Robo4 signaling inhibits VEGF-165–induced migration, tube formation, permeability and SFK activation. (a,b) Lung endothelial cells isolated from Robo4+/+ and Robo4AP/AP mice were used in migration (a), tube formation (b) and in vitro permeability (more ...)
In a mature vascular bed, endothelial cells do not behave independently of one another; rather, they form a monolayer that prevents the movement of protein, fluid and cells from the endothelial lumen into the surrounding tissue. This barrier function can be modeled in vitro by using a Transwell assay to analyze the transport of a test macromolecule, such as horseradish peroxidase (HRP), across a confluent cell monolayer. Stimulation of Robo4+/+ and Robo4AP/AP endothelial cells with VEGF-165, a known permeability-inducing factor, enhanced the accumulation of HRP in the lower chamber of the Transwell. Pretreatment of cell monolayers with Slit2 prevented this effect in Robo4+/+, but not Robo4AP/AP, endothelial cells (). Next, we examined whether Slit2 had a similar influence on endothelial barrier function in vivo by performing a Miles assay. We injected Evans Blue into the tail vein of Robo4+/+ and Robo4AP/AP mice, and subsequently injected VEGF-165 in the absence and presence of Slit2 into the dermis. The results were analogous to those of the in vitro assay: VEGF-165–stimulated leak of Evans Blue into the dermis could be prevented by concomitant administration of Slit2 in Robo4+/+ but not Robo4AP/AP mice (). We extended these observations by assessing the ability of Slit2 to suppress hyperpermeability of the retinal endothelium induced by VEGF-165. Intravitreal injection of VEGF-165 induced leak of Evans Blue from retinal blood vessels of Robo4+/+ mice, which could be suppressed by co-injection of Slit2 (). We repeated this experiment in the retinas of Robo4AP/AP mice and found that they were refractory to Slit2 treatment. These data demonstrated that Robo4 mediates Slit2-dependent inhibition of VEGF-165–induced endothelial hyperpermeability in vitro and in vivo.
The ability of VEGF-165 to promote angiogenesis and permeability is dependent on the activation of VEGFR2, which occurs by autophosphorylation after ligand binding15
. Subsequently, a number of non-receptor tyrosine kinases, serine-threonine kinases and small GTPases are activated to execute VEGF-165 signaling in a spatially and temporally specific manner15
. To determine where Slit2-Robo4 signaling intersects the VEGF-165–VEGFR2 pathway, we first analyzed VEGFR2 phosphorylation after stimulation with VEGF-165 and Slit2. Slit2 had no effect on VEGF-165–induced VEGFR2 phosphorylation (), indicating that the Slit2-Robo4 pathway must intersect VEGF-165 signaling downstream of the receptor. Next, we focused our attention on the Src family of nonreceptor tyrosine kinases (SFKs)—Fyn, Yes and Src—because of their well-documented role in mediating VEGF-165–induced angiogenesis and permeability16,17
. Treatment of endothelial cells with Slit2 reduced VEGF-165–stimulated phosphorylation of the SFKs (). Recently, several reports have shown that Src-dependent activation of the Rho family small GTPase Rac1 is essential for VEGF-165–induced endothelial cell migration and permeability18,19
. Treatment of endothelial cell mono-layers with Slit2 prevented VEGF-165–dependent Rac1 activation (). Together, these biochemical experiments suggested that the Slit2-Robo4 pathway suppresses VEGF-165–induced endothelial migration and hyperpermeability through inhibition of an SFK-Rac1 signaling axis.
We noticed that VEGF-165–stimulated Robo4AP/AP retinas had a higher rate of Evans Blue extravasation than did similarly stimulated wild-type retinas (). This could result from either a basal increase in endothelial permeability or hyper-responsiveness to VEGF-165. To distinguish between these possibilities, we analyzed the extravasation of Evans Blue from Robo4+/+ and Robo4AP/AP retinal vessels in the absence of stimulation with permeability-promoting factors. Robo4AP/AP mice showed a significant increase in Evans Blue accumulation, demonstrating that the retinal endothelium of Robo4AP/AP mice is more permeable than that of wild-type animals under basal conditions ().
The ability of Robo4 signaling to block SFK activation () suggested that the basal permeability defect observed in Robo4AP/AP animals could result from excessive SFK signaling. If this were true, pharmacological suppression of SFK function in Robo4AP/AP animals should restore retinal permeability to wild-type levels. To test this idea, we injected the SFK inhibitor PP2 and its inactive analog PP3 into contralateral eyes of Robo4+/+ and Robo4AP/AP mice and analyzed retinal permeability. PP2 and PP3 had no significant effect on permeability in Robo4+/+ animals. However, PP2 returned the increased basal permeability in Robo4AP/AP mice to a level comparable to that observed in wild-type mice, whereas PP3 was unable to alter permeability in these animals ().
In addition to Slit2, the Slit family is composed of Slit1 and Slit3 (ref. 20
). All three Slit proteins interact with Robo receptors10,21
and mediate chemorepulsion of olfactory bulb explants in vitro22
. There-fore, we determined whether Slit3 had the same activity as Slit2 in the assays described herein. In functional assays, we found that Slit3 blocked VEGF-165–induced Evans Blue extravasation from the retinas of Robo4+/+
but not Robo4AP/AP
mice; and in biochemical experiments we found that Slit3 inhibited VEGF-165–dependent SFK activation (Supplementary Fig. 7 online
). Thus, several Slit family members can activate Robo4 to suppress VEGF-165–induced vascular leak.
Overexpression of Robo4 in endothelial cells, like Slit treatment, might prevent cell migration to angiogenic stimuli7
. To test this possibility, we used adenoviral infection to overexpress Robo4 in endothelial cells. Augmented expression of Robo4, but not GFP, prevented the stimulation of cell migration and SFK phosphorylation by VEGF-165 (Supplementary Fig. 8 online
). Additionally, cells infected with the Robo4 virus showed a marked reduction in basal migration and SFK phosphorylation when compared to GFP-infected cells, suggesting that tonic Robo4 signaling under basal conditions is antiangiogenic.
Vascular leak is a hallmark of the pathologic angiogenesis observed in age-related macular degeneration, retinopathy of prematurity and diabetic retinopathy. The important contribution of VEGF-165 to this process is underscored by the clinical success of antibodies to VEGF in the treatment of these diseases23,24
. The demonstration that Robo4 signaling inhibits VEGF-165–dependent endothelial migration, tube formation and permeability suggests that activation of the Robo4 pathway might have clinical utility in retinal vascular disease. We used a mouse model of oxygen-induced retinopathy (OIR) that mimics the ischemia-induced angiogenesis observed in both diabetic retinopathy and retinopathy of prematurity to investigate the effect of Robo4 signaling on retinal vascular disease. In this model, P7 mice are maintained in a 75% oxygen environment for 5 d and then returned to 25% oxygen for an additional 2 d11
. The perceived oxygen deficit initiates a rapid increase in VEGF-165 expression in the retina, leading to increased angiogenesis and vascular leak25,26
. Intravitreal administration of Slit2 markedly reduced FITC-dextran labeled retinal blood vessels in Robo4+/+
but not Robo4AP/AP
mice (; arrows indicate areas of angiogenesis). Furthermore, Robo4AP/AP
mice showed significantly more FITC-dextran–labeled retinal blood vessels than Robo4+/+
mice after exposure to hyperoxic conditions (compare top left panels of ). In addition to FITC-dextran perfusion, we also analyzed OIR using isolectin staining and found that Slit2 treatment reduced the number of intravitreal neovascular tufts (). Together, these data demonstrated the therapeutic potential of activating Robo4 signaling after an ischemic insult and suggested that Robo4 provides a tonic signal that stabilizes retinal blood vessels. Consistent with this notion, we found that perturbation of vascular stability in animal models of hereditary hemorrhagic telangiectasia4
and oxygen-induced retinopathy (Supplementary Fig. 9 online
) resulted in a compensatory increase in Robo4
Figure 3 Slit2 blocks oxygen-induced retinopathy in a Robo4-dependent manner. (a,b) Neonatal Robo4+/+ (a) and Robo4AP/AP (b) mice were subjected to OIR and perfused with fluorescein isothiocyanate (FITC)-dextran (green). Retinal flat mounts were prepared for each (more ...)
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 (more ...)
The increased susceptibility of Robo4AP/AP
mice to oxygen-induced retinopathy suggested that endogenous Slit protein normally maintains the integrity of the retinal endothelium by acting on Robo4. If this were true, we reasoned that Slit protein would be expressed in or around the vasculature. To examine the expression of Slit2
in the mouse retina, we performed anti-GFP immunohistochemistry on mice bearing a GFP
gene knocked into the Slit2
. GFP fluorescence was detected in cells near the retinal blood vessels of Slit2+/GFP
but not Slit2+/+
mice (Supplementary Fig. 10 online
), demonstrating that endogenous Slit2 is available to activate Robo4 on the endothelium.
In addition to oxygen-induced retinopathy, laser-induced choroidal neovascularization, which mimics age-related macular degeneration, is commonly used to study pathologic angiogenesis in the mouse28
. In this model, a laser is used to disrupt Bruch’s membrane, which allows the underlying choroidal vasculature to penetrate into the subretinal pigment epithelium. To discern the effect of Slit2-Robo4 signaling on this pathologic process, we injected Slit2 into the eyes of 8–12-week-old mice subjected to laser-induced choroidal neovascularization. Similar to oxygen-induced retinopathy, intravitreal administration of Slit2 reduced angiogenesis in Robo4+/+
mice but not Robo4AP/AP
mice (). Together, the oxygen-induced retinopathy and choroidal neovascularization models indicate that two vascular beds with distinct characteristics, one a tight blood-brain barrier and the other a fenestrated endothelium, are protected from pathologic insult by Slit2-dependent activation of Robo4 signaling.
In this report, we provide the first genetic evidence to our knowledge that Slit-Robo signaling regulates a critical function of the vasculature. We demonstrate that Robo4 is essential for Slit2-dependent inhibition of VEGF-165–induced endothelial cell migration, tube formation and hyperpermeability in vitro and for the moderation of angiogenesis and VEGF-165 driven hyperpermeability in vivo. Further, our mechanistic studies identify the SFKs as a crucial molecular target of the Slit2-Robo4 signaling axis. Collectively, these data imply that in addition to the classic paradigm of Robo receptors controlling guidance decisions in the nervous system, Slit2-dependent cues mediated by Robo4 control the integrity of the endothelium (). The clinical success of anti-VEGF therapies in restoring vascular integrity suggests that stabilization of the mature vascular bed by activation of the Robo4 pathway may have broad therapeutic potential.