Vascular Robo4 restricts proangiogenic VEGF signaling in breast

Abstract

Formation of the vascular system within organs requires the balanced action of numerous positive and negative factors secreted by stromal and epithelial cells. Here, we used a genetic approach to determine the role of SLITs in regulating the growth and organization of blood vessels in the mammary gland. We demonstrate that vascularization of the gland is not affected by loss of Slit expression in the epithelial compartment. Instead, we identify a stromal source of SLIT, mural cells encircling blood vessels, and show that loss of Slit in the stroma leads to elevated blood vessel density and complexity. We examine candidate SLIT receptors, Robo1 and Robo4, and find that increased vessel angiogenesis is phenocopied by loss of endothelial-specific Robo4, as long as it is combined with the presence of an angiogenic stimulus such as preneoplasia or pregnancy. In contrast, loss of Robo1 does not affect blood vessel growth. The enhanced growth of blood vessels in Robo4 −/− endothelium is due to activation of vascular endothelial growth factor (VEGF)-R2 signaling through the Src and FAK kinases. Thus, our studies present a genetic dissection of SLIT/ROBO signaling during organ development. We identify a stromal, rather than epithelial, source of SLITs that inhibits blood vessel growth by signaling through endothelial ROBO4 to down-regulate VEGF/ VEGFR2 signaling.angiogenesis | ROBO | SLIT | mammary gland | organogenesis

Heparan sulfate deficiency disrupts developmental angiogenesis and causes congenital diaphragmatic hernia

“…For example, ROBO4 promotes intersomitic vessel development in zebrafish (19), and soluble ROBO4 inhibits angiogenesis in mice (33). Whereas in breast, ROBO4 counteracts VEGF signaling to inhibit angiogenesis and to stabilize the vasculature in skin and retina (18,34,35), in our current study, ROBO4 exhibited a proangiogenic function in diaphragm. The literature suggests that the pro-or antiangiogenic function of ROBO4 is ligand dependent.…”

Molecular Mechanisms of Tumor Angiogenesis

“…138 Interestingly, inactivation of Robo4 in endothelial cells produces more VEGF, has higher levels of activated VEGFR-2, and has increased angiogenic capacity in a mammary fat pad transplant assay. 139 This study also identified smooth muscle cells as the source of Slit-2 and Slit-3 in the developing mammary fat pad, underscoring the relevance of the microenvironment in angiogenesis. Seeking to reconcile the different effects that Slit-2 has on Robo signaling, one group has presented evidence that suggests that ephrinA1 inhibits the normally proangiogenic effects of Slit-2-induced mTORC2-Akt-Rac signaling pathway by inhibiting Akt and Rac.…”

A Slit/miR-218/Robo regulatory loop is required during heart tube formation in zebrafish

“…Slit2 inhibits Vegf signaling in endothelial cells (Jones et al, 2008;Jones et al, 2009;Marlow et al, 2010) and vegfa knockdown induces profound pericardial edema by 48 hpf (Nasevicius et al, 2000). However, it is unclear whether this edema is exclusively attributable to defective angiogenesis from 22 hpf onward, or if Vegf also plays a primary role in cardiac morphogenesis.…”

“…However, SLIT2 inhibits VEGF signaling through a ROBO4-dependent pathway (Jones et al, 2008;Jones et al, 2009;Marlow et al, 2010), perhaps by binding to a ROBO4 coreceptor, such as ROBO1 or Syndecan (Sheldon et al, 2009). To determine whether ROBO1 promotes VEGF signaling directly or by antagonizing SLIT2/ROBO4 inhibition of VEGF signaling, we knocked down ROBO4 in HUVEC (Fig.…”