While the contribution of VEGF to the induction of vascular growth has long been accepted, our data indicate that the function of this growth factor expands its classical, effect as the main mediator of developmental and pathological angiogenesis. Here we show that in vivo autocrine VEGF signaling is required for endothelial cell survival under non-pathological conditions in a manner that is cell-autonomous.
One of the surprises reveled by these experiments was that removal of endothelial VEGF could not be sufficiently compensated for by VEGF secreted from adjacent cell types. Evaluation of circulating VEGF protein and transcripts levels from several organs did not show any significant difference between control and VEGFECKO
mice, indicating that endothelial VEGF does not sum to a detectable proportion of the total growth factor present in any given organ. Interestingly, while endothelial VEGF constitutes a minor proportion of the total VEGF, it holds high functional significance. A second important conclusion drawn from our studies is that autocrine VEGF does not contribute to the angiogenic response, as vascular density and patterning was virtually identical between wild-type and VEGFECKO
mice. Clearly, the majority of the experimental data published so far supports the notion that the progression of angiogenesis requires exogenous presentation of VEGF. Indeed, removal of VEGF from cells other than endothelial has repeatedly resulted in the reduction of vascular growth (Gerber et al., 1999
), a phenotype that is not shared by the VEGFECKO
mice. Vascular density and pattern was not affected by removal of endothelial VEGF. Also, reduction in size or developmental retardation as a consequence of general VEGF decrease was never noted in VEGFECKO
mice. The nature of the defects in our model appears to be restricted to endothelial viability. Together these findings indicate that cell-autonomous signaling triggers a response that does not fully overlap with the events initiated by paracrine activation. Thus, paracrine VEGF signaling is essential for the angiogenic cascade, proliferation, survival, permeability responses and endothelial differentiation. In contrast, autocrine VEGF signaling only conveys survival signals (). Interestingly, both paracrine and autocrine activation are mediated by the main initiating receptor (VEGFR2). It should be stressed that our data does not exclude paracrine VEGF signaling in survival functions. Instead, our findings indicate that provision of survival signals in a paracrine mode alone is insufficient.
The concept of an autocrine signaling loop for VEGF has been previously shown in bone-marrow endothelial cell progenitors (Gerber et al., 2002
). The experiments here would indicate that the functional significance of cell-autonomous signaling is broader than anticipated and it impacts fully differentiated, normal endothelial cells as part of a homeostatic program. Thus, we propose a model in which survival signals are required to support viability of the endothelium under normal conditions. These survival signals are likely triggered by stress situations, such as irradiation, hypoxia and reactive oxygen species. Activation of VEGFR2 by endogenous VEGF sources enables endothelial survival (). While there is much to be understood, as to the regulation of endothelial VEGF in vivo
, our findings would indicate that long-term ablation of VEGF signaling within the endothelial compartment or direct blockade of VEGFR2 might have a deleterious systemic impact in the vasculature of normal tissues.
VEGF is an integral component of tumor angiogenesis and its inhibition results in reduction of tumor burden subsequent to the regression of some tumor vessels and improved drug delivery (Inai et al., 2004
; Jain, 2001
). Consequently, many therapeutic strategies for agents that block VEGFsignaling either by neutralizing VEGF, or by inhibiting its receptors, have been pursued. Some of these agents have shown promise in clinical trials (Hurwitz et al., 2004
; Willett et al., 2004
). However, although reductions in tumor burden and survival benefit have been demonstrated, a small, but consistent number of side effects have been also observed, including hypertension and thromboembolism (Jain et al., 2006
; Kabbinavar et al., 2003
). Other pathologies, while not as consistent, include hemorrhage, proteinuria, intestinal perforations and congestive heart failure (Jain et al., 2006
; Kabbinavar et al., 2003
). Such findings indicate that VEGF participates in a number of functions within normal tissues that exceed its role as pro-angiogenic agent. The contribution of VEGF in the regulation of blood pressure remains enigmatic. Deletion of VEGF from the endothelium does not result in hypertension, or changes in eNOS levels/activation (data not shown). Nor did we observe proteinuria, at least prior to 30weeks of age (data not shown). However, VEGFECKO
mice showed hemorrhage, thrombosis, and intestinal perforations; we attribute most of these side-effects to a long-term interruption in VEGF-mediated survival in both a paracrine and an autocrine manner. While the consequence of long-term pharmacological ablation (or reduction) of VEGF signaling is not really known, our data would indicate that the homeostatic function of the VEGF-VEGFR2 within the endothelial compartment is of considerable biological significance.
Evaluation of signaling by endogenous VEGF in vitro
suggests that activation of VEGFR2 might occur intracellularly. The experimental evidence for this conclusion is that blockade with Avastin did not affect the degree of VEGFR2 phosphorylation, unlike the effect mediated by a small molecular inhibitor of VEGFR2 that can enter the cell freely and completely suppress activation of the receptor. In addition, we found that wild-type endothelial cells are unable to rescue VEGFECKO
endothelial cells in co-culture experiments. Previous examples of intracrine signaling have been presented in the literature. For example, the amino-terminal propeptide of type I collagen have been shown to modulate cell adhesion by triggering activation of a putative intracellular receptor in the secretory pathway (Oganesian et al., 2006). Intracrine activation of receptor tyrosine kinases have been predicted for PDGF and TGF-beta (Betsholtz et al., 1984) and the concept that ligand-receptor binding complexes are internalized to signal only in an endosomal compartment has been long demonstrated for EGF (Cohen and Fava, 1985
). More recently, endosomes were implicated in the maintenance of Dpp signaling during mitosis and in the even distribution of signaling bodies to daughter cells (Bokel et al., 2006
). It is also becoming apparent that internalized cell surface receptors might use specifically localized complexes to initiate signals that are distinct from those triggered at the cell surface (Childress et al., 2006
; Lin et al., 2006
; Seto and Bellen, 2006
). In this manner, compartmentalization of signaling could regulate signal transduction spatially and temporally (van der Goot and Gruenberg, 2006
). This would ultimately assign unique biological responses through the same ligand-receptor complex. While we have yet to explore compartmentalization of VEGF signaling, it has been recently shown that internalized VEGF receptors can signal from endosomes in a manner that is regulated by VE-Cadherin (Lampugnani et al., 2006
In conclusion, the studies communicated here reveal a previously unknown function for VEGF in vascular homeostasis in vivo. They also indicate that cell-autonomous VEGF signaling is likely triggered inside the cell. These signaling events differ from those initiated by exogenous / paracrine VEGF, as they are restricted to maintenance of endothelial viability.