VE-PTP, the first known endothelial-specific receptor-type tyrosine phosphatase, associates with Tie-2 and VE-cadherin and is essential for vascular remodeling during embryonic development. In this study we show that VE-PTP controls vascular remodeling via regulating the ability of Tie-2 to drive endothelial cell proliferation. These mechanistic insights into the physiological function of VE-PTP were enabled by antibodies against VE-PTP, which selectively dissociate VE-PTP from Tie-2 but not from VE-cadherin. The antibodies stimulated Tie-2 activation, as documented by increased tyrosine phosphorylation of Tie-2 and activation of the downstream signaling target Erk1/2. In addition, we found that the anti–VE-PTP antibodies elicit enlargement of vascular structures in allantois explants and in 1–2-wk-old mice, accompanied by enhanced endothelial cell proliferation. Most importantly, enlargement of vascular structures by these antibodies was eliminated in the absence of Tie-2 and required activation of Erk1/2. Our results establish VE-PTP as an essential negative regulator of Tie-2 during blood vessel remodeling, providing a mechanism for how VE-PTP affects angiogenesis.
Precise balancing of positive and negative stimulation of Tie-2 is essential for vessel remodeling and angiogenesis. A lack of activation caused by deleting the Tie-2 gene itself or by disrupting the gene for the agonist Ang1 leads to embryonic vascular malformations and lethality (Dumont et al., 1994
; Sato et al., 1995
; Suri et al., 1996
), a similar phenotype that is observed upon overexpression of the antagonistic ligand Ang2 (Maisonpierre et al., 1997
). On the other hand, hyper-activation of Tie-2 via a missense mutation in the kinase domain leads to venous malformations in human patients (Vikkula et al., 1996
), and deletion of the gene for the antagonist Ang2 affects remodeling in hyaloid vasculature in the eye of newborn mice and lymphatic patterning (Gale et al., 2002
). Interestingly, defects in Ang2-deficient mice were limited to postnatal development, and no defects were found during embryonic vascular development in these mice. Thus, VE-PTP represents the first negative regulator of Tie-2 essential for embryonic angiogenesis. We can not, of course, rule out that VE-PTP gene disruption affects additional molecular mechanisms besides Tie-2 signaling, e.g., VE-cadherin or other still-unknown substrates. However, the vascular aberrations caused by our anti–VE-PTP antibodies in allantois explant cultures were strictly dependent on Tie-2 and therefore identify Tie-2 as an essential substrate for VE-PTP during embryonic vascular remodeling.
We assume that VE-PTP represents a negative feedback control mechanism that limits the activation of Tie-2. In agreement with this, stimulation of Tie-2 with Ang1 leads to increased association of Tie-2 and VE-PTP, enabling the agonist to trigger activation and at the same time to launch the negative mechanism that ensures that the signal is switched off again. Whether, in addition, a ligand for VE-PTP may exist that could induce uptake of VE-PTP and thereby indirectly could enhance Ang1-driven activation of Tie-2 is unknown, and may be an interesting hypothesis to test in the future.
The effects of our anti–VE-PTP antibodies on blood vessel enlargement in young mice are similar to the effects observed in mice either injected with Ang1 or COMP-Ang1 (Thurston et al., 2005
; Kim et al., 2007
) or overexpressing COMP-Ang1 via adenovirus vectors (Cho et al., 2005
). However, as these studies were based on exogenously added large doses of Ang1, it could not be determined whether physiological levels of Ang1 would indeed play a role in the normal regulation of vessel size during perinatal development. Because the anti–VE-PTP antibodies simply dissociate a negative regulator from Tie-2, our results suggest that Tie-2–stimulating ligands are indeed acting in newborns to determine vessel size.
It is interesting that antibodies against VE-PTP also stimulated Tie-1 activation and that this only occurred in the presence of Tie-2. This is in agreement with other studies demonstrating that Tie-1 activation by Ang1 depends on Tie-2 (Saharinen et al., 2005
; Yuan et al., 2007
Tie-2 activation triggers various signaling pathways and biological activities, such as survival and protection from apoptosis, migration, permeability, tube formation, and sprouting, as has been summarized in excellent reviews (Brindle et al., 2006
; Eklund and Olsen, 2006
). However, studies on the ability of Ang1 to stimulate proliferation of cultured endothelial cells are controversial, ranging from no effect (Davis et al., 1996
; Witzenbichler et al., 1998
; Fujikawa et al., 1999
) to mild effects (Koblizek et al., 1998
; Teichert-Kuliszewska et al., 2001
) to substantial effects (Kanda et al., 2005
). The in vivo studies with Ang1 and COMP-Ang1 in newborns (Cho et al., 2005
; Thurston et al., 2005
; Kim et al., 2007
) suggest that vessel enlargement was accompanied by endothelial cell proliferation, establishing that Tie-2 can stimulate proliferation of endothelial cells in vivo. Like them, we found that Tie-2–dependent proliferation was independent from sprout formation and occurred within endothelial cord structures.
Kanda et al. (2005)
have shown that blocking Erk can partially inhibit Ang1-stimulated endothelial cell proliferation of cultured endothelial cells. This is in good agreement with our finding that treatment with anti–VE-PTP antibodies feeds into the Tie-2–signaling pathway leading to Erk activation. Importantly, the fact that the Erk1/2 inhibitors PD98059 and U0126 blocked the anti–VE-PTP effect on endothelial cell proliferation and on enlargement of vascular structures in the allantois indicates that Tie-2 triggers endothelial proliferation in the allantois via Erk1/2 and that VE-PTP counteracts this pathway. Collectively, these results indicate that anti–VE-PTP antibodies induce enlargement of vascular structures in the allantois by stimulating endothelial cell proliferation via the Tie-2, Erk1/2 pathway.
It is intriguing that VE-PTP molecules associated with Tie-2 were selectively sensitive to anti–VE-PTP antibody-triggered dissociation and endocytosis, whereas VE-cadherin–associated VE-PTP molecules were not sensitive for this effect. Likewise, anti–VE-PTP antibodies selectively affected tyrosine phosphorylation of Tie-2, but not the phosphorylation pattern of the components of the VE-cadherin complex in endothelial adherens junctions. We assume that VE-PTP complexed with VE-cadherin may not be accessible for antibodies, possibly masked within VE-cadherin clusters at cell contacts. Indeed, incubation of living, intact endothelial cells or allantois explants with anti–VE-PTP antibodies did not allow to stain endothelial cell contacts, whereas fixing and permeabilizing the specimens rendered extracellular epitopes of VE-PTP accessible to antibody staining ( and Videos 5–7).
In conclusion, our results establish VE-PTP as an essential negative regulator of Tie-2, which controls Tie-2–driven endothelial cell proliferation, which in turn affects blood vessel remodeling during embryonic development and determines blood vessel size during perinatal growth. In light of the publications analyzing the function of the Tie-receptor system in tumor angiogenesis (Shim et al., 2007
) it will be interesting to test a potential role of VE-PTP in this pathological process. Furthermore, because VE-PTP is an endothelial-specific transmembrane protein and antibodies against its extracellular part affect endothelial cell proliferation and angiogenesis in vivo, VE-PTP is generally an easily accessible, interesting novel target for pro- or anti-angiogenic therapeutic interventions.