VEC has a central role in the control of vascular permeability, and deregulation of its expression or function is associated with increased oedema and vascular fragility1
. While recent studies have begun to uncover the mechanisms through which VEC activity is regulated in vitro7
, few data11
are available on VEC functional modulation in vivo
. In starved endothelial cells in vitro,
VEC phosphorylation in tyrosine is extremely low but is increased by permeability-increasing agents such as vascular endothelial growth factor (VEGF) or leucocyte adhesion14
By in vivo staining we observed that VEC is phosphorylated in Y658 and Y685 in veins also in the absence of inflammatory agents or vascular leakage. Therefore, VEC phosphorylation at these two tyrosine residues in vivo is not sufficient per se to reduce endothelial barrier function. Nevertheless, as discussed below, VEC phosphorylation may act as a priming mechanism to sensitize veins to permeability-inducing agents.
An important question is how VEC phosphorylation is modulated in vivo. We report evidence that hemodynamic conditions modulate VEC phosphorylation through Src activity and that this mechanism acts mostly in veins but not in small arteries. We have shown that in veins there is a constitutive and prolonged activation of Src, which in turn may mediate VEC phosphorylation.
Activation of Src family kinases and the subsequent VEC phosphorylation has been described as a critical step in the induction of permeability by growth factors and inflammatory cytokines9
. In vivo
we observed that Src is constitutively activated in veins and this is necessary but not sufficient to induce vascular leakage. These observations are in agreement with Adam et al.46
who showed that phosphorylation of VEC by Src was not sufficient to decrease barrier function of cultured endothelial cells.
The reasons why the above phenomena do not occur in arteries are not clear, notwithstanding the multiple differences that exist between arterial and venous vessels. These differences include the pattern and degree of investment by smooth muscle cells and/or pericytes, electrical coupling, distribution of endothelial markers such as ephrins and receptors to inflammatory mediators, and others1
. The data reported here suggest that hemodynamic factors also may modulate Src activity and, as a consequence, VEC phosphorylation. As reported here VEC phosphorylation is influenced by shear stress in vitro
and exposure of venous endothelial cells to arterial blood flow in vivo
leads to a decrease in VEC phosphorylation.
Although further work is required to define this mechanism in detail, data are in favour of a shear-sensing mechanism that modulates VEC phosphorylation. An interesting candidate is the endothelial mechanosensory complex formed by VEC, PECAM-1 and VEGFR-2. PECAM-1 has been shown to bind directly to Src and the binding is required for Src activation under shear49
. We cannot, however, exclude that other hemodynamic factors such as hydrostatic pressure may contribute to low VEC phosphorylation in arteries.
We also made efforts to understand the functional meaning of the constitutive VEC phosphorylation in vivo. As most inflammatory mediators act selectively and rapidly on veins, a tempting speculation is that VEC phosphorylation is a prerequisite for a rapid and fully reversible opening of endothelial junctions.
As for other cadherins, VEC endocytosis reduces endothelial cells barrier function2
. We found that bradykinin induces a quick disappearance of pY658 and pY685 VEC from junctions which, from the data collected in vitro
, we interpret as internalization. When we used VEC point mutants Y658F and pY685F to inhibit phosphorylation we also blocked bradykinin-induced VEC internalization and increase in vascular permeability in vitro
. It seems, therefore, that phosphorylation of VEC at these two tyrosine residues is required for bradykinin-induced vascular leakage.
To further elucidate the pathway through which pYVEC contributes to its internalization and possibly degradation we considered that cell–cell contacts are sites for recruitment of the ubiquitination machinery30
. Tyrosine phosphorylation of E-cadherin attracts Hakai, an E3 ubiquitin-ligase, which mediates its ubiquitination and endocytosis50
. In agreement with this observation, we report here that activation by bradykinin leads to VEC ubiquitination and that this process requires tyrosine phosphorylation of Y658 and Y685.
The decrease in phosphorylation of Y658 and Y685 in VEC may also be due to association to phosphatases. As above, the best candidates that were previously found to associate to VEC are VE-PTP52
and Dep-1 (ref. 17
). However, the knockdown of these phosphatases did not prevent the decrease in VEC phosphorylation upon bradykinin activation. Moreover, proteomic analysis of lung extracts of mice treated with bradykinin did not detect phosphatases associated to VEC complex. Thus, we have been unable to find, at least in our experimental system, a clear evidence for the role of phosphatases in regulating VEC phosphorylation.
We did not perform a detailed analysis of the role of each tyrosine present in the cytoplasmic tail of VEC but focused on Y658 and Y685 instead. Both tyrosines are phosphorylated in venous endothelium, and in vitro
studies on VEC phosphomimetic mutants show that Y658 phosphorylation is important in VEC internalization18
. Phosphorylation of VEC at Y658 may reduce its association to p120 and induce its internalization18
. Under the different experimental conditions used here, however, we have been unable to detect a significant dissociation of p120 from internalized pYVEC. Others46
using endothelial cells transfected by a constitutive active src, which induced pY658 phosphorylation, could not observe detachment of p120. Our data are consistent with this last report. These observations do not exclude that p120 is important for the stability of VEC at the membrane. However, detachment of p120 is unlikely to be the only way through which cadherins are internalized.
Gavard et al.10
reported that VEGF-mediated VE cadherin phosphorylation in the highly conserved Ser-665 induces the recruitment of β-arrestin2, VEC internalization and increase in permeability in cultured cells. We do not know whether phospho Ser-665 also contributes to bradykinin-induced VEC internalization at this stage. However, we cannot exclude that VEC activation by permeability increasing agents may result in different steps of phosphorylation which, downstream, contribute to the internalization of the protein.
Lambeng et al.44
describe, in organ extracts, VEC tyrosine phosphorylation under both resting and hormone-activated conditions. Interestingly and consistently with our data, phosphorylation of VEC was observed also under resting conditions in some organs such as the lung. When angiogenesis was stimulated by treatment with hormones, total VEC phosphorylation increased but only in uterus and ovary. Using heart extracts, Weis et al.11
show increased VEC phosphorylation after VEGF treatment and inhibition of such an effect by Src inhibitors. The stimuli and the organs studied are different from those considered in our paper. Furthermore, the use of organ extracts does not allow a detailed analysis of positive or negative vessels. It is conceivable that angiogenic stimuli increase tyrosine phosphorylation in the growing vasculature and as final result, cause an increase in tyrosine phosphorylation of the whole organ extract.
In conclusion, the data presented here help to draw a picture of how VEC processing is regulated in vivo by inflammatory mediators such as bradykinin or histamine. We report that VEC may be phosphorylated in Y658 and Y685 in veins through the action of hemodynamic forces. Our data suggest that phosphorylation in these tyrosines sensitizes VEC to the action of bradykinin, which induces rapid pYVEC internalization and ubiquitination. pYVEC in veins may, therefore, be important to prime the protein into a dynamic state that promotes, upon activation, its rapid internalization, leading to the quick and reversible opening of endothelial cells junctions and plasma leakage. We speculate that any agent capable of inhibiting VEC phosphorylation may effectively reduce vascular permeability induced by inflammatory agents.