VE-cadherin is a major component of endothelial adherens junctions and pivotal to the regulation of vascular barrier integrity. Whereas two phosphorylation sites of VE-cadherin (Y685 and Y731) are known to be relevant for the regulation of endothelial junctions in vivo, several others were suggested to be relevant based on in vitro studies. Here, we analyze for two of these, serine 665 (S665) and tyrosine 658 (Y658), whether they are relevant for the induction of vascular permeability in vivo. To this end, we generated and characterized two point-mutated VE-cadherin knock-in mouse lines where either S665 was replaced by valine (S665V) or Y658 by phenylalanine (Y658F). We found that the induction of vascular permeability by histamine or VEGF in the skin was clearly reduced in S665V mice, whereas Y658F mice showed a normal increase of permeability. In line with this, we found that histamine-induced endocytosis was impaired for the VE-cadherin-S665V mutant, but not for the Y658F mutant. Comparing the regulation of VE-cadherin phosphorylation at S665, Y658 and Y685, we found that only phosphorylation of S665 and Y685 were strongly induced by inflammatory mediators, while phosphorylation of Y658 increased weakly. Interestingly, phosphorylation of S665 and Y685 occurred with different kinetics, but independent of each other. Collectively, our results demonstrate that Y658 is irrelevant for vascular leak formation in the context of several tested inflammatory mediators and establish S665 of VE-cadherin as an important phosphorylation site regulating the induction of endothelial permeability in vivo.

PURPOSE:Glaucoma is the leading cause of irreversible blindness worldwide and is associated with high intraocular pressure (IOP). Schlemm's canal (SC), a hybrid vessel present in the anterior part of the eye, is known to control IOP by draining aqueous humor into the systemic circulation. Formation and function of SC is supported by the tyrosine kinase receptor Tie2. Likewise, inhibition of the vascular endothelial protein tyrosine phosphatase (VE-PTP), which associates with Tie2 has similar effects. However, VE-PTP also targets VE-cadherin and several other substrates. Here, we analyzed whether Tie2 is indeed the major substrate which is responsible for the role of VE-PTP in SC function. In addition, we analyzed the function of VE-PTP in SC of the aged eye in mice. METHODOLOGY:We tested the effects of the VE-PTP inhibitor AKB9778 and of VE-PTP gene inactivation on SC area and IOP in WT and in Tie2iLEC/SC-KO and VE-cadherin-Y685F mutant mice. RESULTS:Pharmacologic inhibition of VE-PTP with AKB9778 increased SC area only in mice expressing Tie2. The VE-cadherin-Y685F mutation had neither an effect on SC area nor on the effects of AKB9778 on SC formation. Induced VE-PTP gene inactivation in adult mice had similar effects as AKB9778. Furthermore, we could show that AKB9778 improved SC function in aged mice as judged by increasing SC area and lowering of IOP. CONCLUSION:Interference with VE-PTP function improves SC function in a strictly Tie2 dependent way and pharmacologic inhibition of VE-PTP with AKB9778 is a promising approach for improving SC function in the aged eye.

Vascular endothelial protein tyrosine phosphatase (VE-PTP) influences endothelial barrier function by regulating the activation of tyrosine kinase receptor Tie2. We determined whether this action is linked to the development of atherosclerosis by examining the influence of arterial shear stress on VE-PTP, Tie2 activation, plasma leakage, and atherogenesis. We found that exposure to high average shear stress led to downstream polarization and endocytosis of VE-PTP accompanied by Tie2 activation at cell junctions. In aortic regions with disturbed flow, VE-PTP was not redistributed away from Tie2. Endothelial cells exposed to high shear stress had greater Tie2 activation and less macromolecular permeability than regions with disturbed flow. Deleting endothelial VE-PTP in VE-PTPiECKO mice increased Tie2 activation and reduced plasma leakage in atheroprone regions. ApoE-/- mice bred with VE-PTPiECKO mice had less plasma leakage and fewer atheromas on a high-fat diet. Pharmacologic inhibition of VE-PTP by AKB-9785 had similar anti-atherogenic effects. Together, the findings identify VE-PTP as a novel target for suppression of atherosclerosis.

Cadherin-mediated cell adhesion requires anchoring via the β-catenin-α-catenin complex to the actin cytoskeleton, yet, α-catenin only binds F-actin weakly. A covalent fusion of VE-cadherin to α-catenin enhances actin anchorage in endothelial cells and strongly stabilizes endothelial junctions in vivo, blocking inflammatory responses. Here, we have analyzed the underlying mechanism. We found that VE-cadherin-α-catenin constitutively recruits the actin adaptor vinculin. However, removal of the vinculin-binding region of α-catenin did not impair the ability of VE-cadherin-α-catenin to enhance junction integrity. Searching for an alternative explanation for the junction-stabilizing mechanism, we found that an antibody-defined epitope, normally buried in a short α1-helix of the actin-binding domain (ABD) of α-catenin, is openly displayed in junctional VE-cadherin-α-catenin chimera. We found that this epitope became exposed in normal α-catenin upon triggering thrombin-induced tension across the VE-cadherin complex. These results suggest that the VE-cadherin-α-catenin chimera stabilizes endothelial junctions due to conformational changes in the ABD of α-catenin that support constitutive strong binding to actin.

Purpose:Tyrosine kinase with immunoglobulin-like and EGF-like domains 2 (Tie2) activation in Schlemm's canal (SC) endothelium is required for the maintenance of IOP, making the angiopoietin/Tie2 pathway a target for new and potentially disease modifying glaucoma therapies. The goal of the present study was to examine the effects of a Tie2 activator, AKB-9778, on IOP and outflow function. Methods:AKB-9778 effects on IOP was evaluated in humans, rabbits, and mice. Localization studies of vascular endothelial protein tyrosine phosphatase (VE-PTP), the target of AKB-9778 and a negative regulator of Tie2, were performed in human and mouse eyes. Mechanistic studies were carried out in mice, monitoring AKB-9778 effects on outflow facility, Tie2 phosphorylation, and filtration area of SC. Results:AKB-9778 lowered IOP in patients treated subcutaneously for diabetic eye disease. In addition to efficacious, dose-dependent IOP lowering in rabbit eyes, topical ocular AKB-9778 increased Tie2 activation in SC endothelium, reduced IOP, and increased outflow facility in mouse eyes. VE-PTP was localized to SC endothelial cells in human and mouse eyes. Mechanistically, AKB-9778 increased the filtration area of SC for aqueous humor efflux in both wild type and in Tie2+/- mice. Conclusions:This is the first report of IOP lowering in humans with a Tie2 activator and functional demonstration of its action in remodeling SC to increase outflow facility and lower IOP in fully developed mice. Based on these studies, a phase II clinical trial is in progress to advance topical ocular AKB-9778 as a first in class, Tie2 activator for treatment for ocular hypertension and glaucoma.

Tyrosine phosphorylation of the adhesion molecule VE-cadherin is assumed to affect endothelial junction integrity. However, it remains unclear whether tyrosine residues of VE-cadherin are required for the induction of vascular permeability and the regulation of leukocyte extravasation in vivo. We found here that knock-in mice expressing a Y685F mutant of VE-cadherin had impaired induction of vascular permeability, but those expressing a Y731F mutant did not. In contrast, mice expressing the Y731F VE-cadherin mutant showed decreased neutrophil-extravasation in cremaster tissue, but those expressing the Y685F mutant did not. Whereas inflammatory mediators induced the phosphorylation of Tyr685 in vivo, Tyr731 showed high baseline phosphorylation. Leukocytes triggered dephosphorylation of Tyr731 via the tyrosine phosphatase SHP-2, which allowed the adaptin AP-2 to bind and initiate endocytosis of VE-cadherin. Thus, Tyr685 and Tyr731 of VE-cadherin distinctly and selectively regulate the induction of vascular permeability or leukocyte extravasation.

Leukocyte adhesion deficiency II (LAD II), also known as congenital disorder of glycosylation IIc (CDG-IIc), is a human disease in which a defective GDP-fucose transporter (SLC35C1) causes developmental defects and an immunodeficiency that is based on the lack of fucosylated selectin ligands. Since the study of in vivo leukocyte trafficking in patients with LAD II is experimentally limited, we analyzed this process in mice deficient for Slc35c1. We found that E-, L-, and P-selectin-dependent leukocyte rolling in cremaster muscle venules was virtually absent. This was accompanied by a strong but not complete decrease in firm leukocyte adhesion. Moreover, neutrophil migration to the inflamed peritoneum was strongly reduced by 89%. Previous reports showed surprisingly normal lymphocyte functions in LAD II, which indicated sufficient lymphocyte trafficking to secondary lymphoid organs. We now found that while lymphocyte homing to lymph nodes was reduced to 1% to 2% in Slc35c1(-/-) mice, trafficking to the spleen was completely normal. In accordance with this, we found a defect in the humoral response to a T cell-dependent antigen in lymph nodes but not in the spleen. Taken together, Slc35c1(-/-) mice show strongly defective leukocyte trafficking but normal lymphocyte homing to the spleen, which may explain normal lymphocyte functions in LAD II.

Kooperation ist alles: In Sialyl-Lewisx-Glycopeptiden und deren Mimetika im Fucose- und Sialinsäureteil (siehe Formel) sind Peptid- und Saccharidteil für die Funktion wesentlich; richtig kombiniert kann das Derivat bis zu einhundertmal stärker an E-Selektin binden als das Sialyl-Lewisx-Saccharid. Die Synthese lieferte die Sialyl-Lewisx-Aminosäuren in so säurestabiler Form, dass sie in üblichen Festphasensynthesen eingesetzt werden können.

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immature dendritic cells (DC) reside in tissues where they initiate immune responses by taking up foreign antigens. Since DC have a limited tissue half-life, the DC pool in tissues has to be replenished constantly. This implies that precursor/immature DC must be able to cross non-activated endothelium using as yet unknown mechanisms. Here we show that immature, but not mature bone marrow-derived murine DC migrate across resting endothelial monolayers in vitro. We find that endothelial intercellular adhesion molecule-2 (ICAM-2) is a major player in transendothelial migration (TEM) of immature DC, accounting for at least 41% of TEM. Surprisingly, the ICAM-2-mediated TEM was independent of beta(2)-integrins, the known ICAM-2 ligands, since neither blocking of beta(2)-integrins with antibodies nor the use of CD18-deficient DC affected the ICAM-2-specific TEM. In humans, the C-type lectin DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN) was shown to interact with ICAM-2, suggesting a similar role in mice. However, we find that none of the murine DC-SIGN homologues mDC-SIGN, murine DC-SIGN-related molecule-1 (mSIGN-R1) and mSIGN-R3 is expressed on the surface of bone marrow-derived mouse DC. Taken together, this study shows that ICAM-2 strongly supports transmigration of immature DC across resting endothelium by interacting with ligands that are distinct from beta(2)-integrins and DC-SIGN homologues.

Nucleotide sugar transporters play a central role in the process of glycosylation. They are responsible for the translocation of nucleotide sugars from the cytosol, their site of synthesis, into the Golgi apparatus where the activated sugars serve as substrates for a variety of glycosyltransferases. We and others have recently identified and cloned the first GDP-fucose transporters of H. sapiens and C. elegans. Based on sequence similarity, we could identify a putative homolog in Drosophila melanogaster showing about 45% identity on protein level. The gene (CG9620) encodes a highly hydrophobic, multi-transmembrane spanning protein of 38.1 kDa that is localized in the Golgi apparatus. In order to test whether this protein serves as a GDP-fucose transporter, we performed complementation studies with fibroblasts from a patient with LADII (leukocyte adhesion deficiency II) which exhibit a strong reduction of fucosylation due to a point mutation in the human GDP-fucose transporter gene. We show that transient transfection of these cells with the Drosophila CG9620 cDNA corrects the GDP-fucose transport defect and reestablishes fucosylation. This study gives experimental proof that the product of the in silico identified Drosophila gene CG9620 serves as a functional GDP-fucose transporter.