To determine whether leukocytes need to open endothelial cell contacts during extravasation, we decided to generate mice with strongly stabilized endothelial junctions. To this end, we replaced VE-cadherin genetically by a VE-cadherin-a-catenin fusion construct. Such mice were completely resistant to the induction of vascular leaks by VEGF or histamine. Neutrophil or lymphocyte recruitment into inflamed cremaster, lung and skin were strongly inhibited in these mice, documenting the importance of the junctional route in vivo. Surprisingly, lymphocyte homing into lymph nodes was not inhibited. VE-cadherin-a-catenin associated more intensely with the actin cytoskeleton as demonstrated by its membrane mobility and detergent extractability. Our results establish the junctional route as the main pathway for extravasating leukocytes in several, although not in all tissues. Furthermore, in these tissues, plasticity of the VE-cadherin-catenin complex is central for the leukocyte diapedesis mechanism.

von Willebrand factor (VWF) is an important player in hemostasis but has also been suggested to promote inflammatory processes. Gene ablation of VWF causes a simultaneous defect in P-selectin expression making it difficult to identify VWF-specific functions. Therefore, we analyzed whether blocking antibodies against VWF would be able to interfere with neutrophil extravasation. We found that these antibodies inhibited neutrophil recruitment into thioglycollate-inflamed peritoneum and KC-stimulated cremaster by approximately 50%. Whereas platelet-VWF was not involved, the contribution of VWF to granulocyte recruitment was strictly dependent on the presence of platelets and the accessibility of their VWF-receptor glycoprotein Ib. Surprisingly, platelet P-selectin was largely dispensable for leukocyte extravasation, in agreement with our observation that anti-VWF antibodies did not affect leukocyte rolling and adhesion. Searching for possible effects downstream of leukocyte capture, we found that anti-VWF antibodies significantly inhibited thioglycollateinduced vascular permeability. The increase of permeability was independent of circulating granulocytes, showing that it was not a side effect of neutrophil diapedesis. Collectively, our results demonstrate that VWF-associated platelets strongly support neutrophil extravasation at a step downstream of leukocyte docking to the vessel wall. This step could be related to leukocyte diapedesis facilitated by destabilization of the endothelial barrier. IntroductionLeukocyte interactions with the endothelium leading to extravasation into inflamed tissues have classically been assigned to the action of selectins, chemokine receptors, integrins, 1 and various membrane proteins at endothelial cell contacts. 2 In addition to these well-established mechanisms, platelets were reported to participate in leukocyte extravasation in various inflammation models. [3][4][5][6] Whether or to what extent von Willebrand factor (VWF), a major ligand for platelets, is involved in this process is currently unknown.VWF can be found in high quantities in Weibel-Palade bodies and ␣-granules of endothelial cells (ECs) and platelets, respectively. Upon activation, these cells release VWF and deposit it on the subendothelium of injured vessels where it serves as an anchoring substance for platelets by binding to platelet glycoprotein Ib (GPIb). 7 Thus, VWF initiates the plug formation by platelets leading to resealing of injured vessels. The importance of VWF for hemostasis is impressively illustrated by von Willebrand disease, a strong bleeding disorder caused by functionally defective VWF. 8 VWF is most active as ultra-large multimers but is efficiently controlled by a disintegrin-like and metalloproteinase with thrombospondin type I repeats 13 (ADAMTS13), which cleaves ultralarge VWF, thus down-regulating the hemostatic process. 9 VWF does not only bind to subendothelial layers but can also be found on the apical surface of activated cultured ECs, where it forms ultra-large VWF multimers that s...

Artificially stabilizing contacts between VE-cadherin and VE-PTP reduce vascular permeability and leukocyte extravasation in vivo.

Vascular endothelial protein tyrosine phosphatase (VE-PTP) is an endothelial-specific receptor-type tyrosine phosphatase that associates with Tie-2 and VE-cadherin. VE-PTP gene disruption leads to embryonic lethality, vascular remodeling defects, and enlargement of vascular structures in extraembryonic tissues. We show here that antibodies against the extracellular part of VE-PTP mimic the effects of VE-PTP gene disruption exemplified by vessel enlargement in allantois explants. These effects require the presence of the angiopoietin receptor Tie-2. Analyzing the mechanism we found that anti–VE-PTP antibodies trigger endocytosis and selectively affect Tie-2–associated, but not VE-cadherin–associated VE-PTP. Dissociation of VE-PTP triggers the activation of Tie-2, leading to enhanced endothelial cell proliferation and enlargement of vascular structures through activation of Erk1/2. Importantly, the antibody effect on vessel enlargement is also observed in newborn mice. We conclude that VE-PTP is required to balance Tie-2 activity and endothelial cell proliferation, thereby controlling blood vessel development and vessel size.

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