Src Cannot Break Barriers Alone♦ : Src-induced Tyrosine Phosphorylation of VE-cadherin Is Not Sufficient to Decrease Barrier Function of Endothelial Monolayers
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PROVIDER: S-EPMC2844230 | biostudies-literature | 2010 Mar
REPOSITORIES: biostudies-literature
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Project description:Activation of Src family kinases (SFK) and the subsequent phosphorylation of VE-cadherin have been proposed as major regulatory steps leading to increases in vascular permeability in response to inflammatory mediators and growth factors. To investigate Src signaling in the absence of parallel signaling pathways initiated by growth factors or inflammatory mediators, we activated Src and SFKs by expression of dominant negative Csk, expression of constitutively active Src, or knockdown of Csk. Activation of SFK by overexpression of dominant negative Csk induced VE-cadherin phosphorylation at tyrosines 658, 685, and 731. However, dominant negative Csk expression was unable to induce changes in the monolayer permeability. In contrast, expression of constitutively active Src decreased barrier function and promoted VE-cadherin phosphorylation on tyrosines 658 and 731, although the increase in VE-cadherin phosphorylation preceded the increase in permeability by 4-6 h. Csk knockdown induced VE-cadherin phosphorylation at sites 658 and 731 but did not induce a loss in barrier function. Co-immunoprecipitation and immunofluorescence studies suggest that phosphorylation of those sites did not impair VE-cadherin ability to bind p120 and beta-catenin or the ability of these proteins to localize at the plasma membrane. Taken together, our data show that Src-induced tyrosine phosphorylation of VE-cadherin is not sufficient to promote an increase in endothelial cell monolayer permeability and suggest that signaling leading to changes in vascular permeability in response to inflammatory mediators or growth factors may require VE-cadherin tyrosine phosphorylation concurrently with other signaling pathways to promote loss of barrier function.
| S-EPMC2844154 | biostudies-literature
Project description:In this study, we present results demonstrating that mechanotransduction by vascular endothelial cadherin (VE-cadherin, also known as CDH5) complexes in endothelial cells triggers local cytoskeletal remodeling, and also activates global signals that alter peripheral intercellular junctions and disrupt cell-cell contacts far from the site of force application. Prior studies have documented the impact of actomyosin contractile forces on adherens junction remodeling, but the role of VE-cadherin in force sensation and its ability to influence endothelial cell and tissue mechanics globally have not been demonstrated. Using mechanical manipulation of VE-cadherin bonds and confocal imaging, we demonstrate VE-cadherin-based mechanotransduction. We then demonstrate that it requires homophilic VE-cadherin ligation, an intact actomyosin cytoskeleton, Rho-associated protein kinase 1 (ROCK1) and phosphoinositide 3-kinase. VE-cadherin-mediated mechanotransduction triggered local actin and vinculin recruitment, as well as global signals that altered focal adhesions and disrupted peripheral intercellular junctions. Confocal imaging revealed that VE-cadherin-specific changes appear to propagate across cell junctions to disrupt distant inter-endothelial junctions. These results demonstrate the central role of VE-cadherin adhesions and the actomyosin cytoskeleton within an integrated, mechanosensitive network that both induces local cytoskeletal remodeling at the site of force application and regulates the global integrity of endothelial tissues.
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Project description:GSTpi is a Phase II metabolic enzyme which is originally considered as an important facilitator of cellular detoxification. Here, we found that GSTpi stabilized VE-cadherin in endothelial cell membrane through inhibiting VE-cadherin phosphorylation and VE-cadherin/catenin complex dissociation, and consequently maintained endothelial barrier function. Our findings demonstrated a novel mechanism that GSTpi inhibited VE-cadherin phosphorylation through suppressing the activation of Src/VE-cadherin pathway. Mass spectrometry analysis and molecular docking showed that GSTpi enhanced Src S-glutathionylation at Cys185, Cys245, and Cys400 of Src. More important, we found that GSTpi promoted S-glutathionylation of Src was essential for GSTpi to inhibit Src phosphorylation and activation. Furthermore, in vivo experiments indicated that AAV-GSTpi exerted the protective effect on pulmonary vessel permeability in the animal model of acute lung injury. This study revealed a novel regulatory effect of GSTpi on vascular endothelial barrier function and the importance of S-glutathionylation of Src induced by GSTpi in the activation of Src/VE-cadherin pathway.
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Project description:Infiltration of the central nervous system (CNS) by leukemia is a problematic disease manifestation of acute lymphoblastic leukemia (ALL). The mechanisms by which leukocytes interact with human brain-derived microvasculature endothelial cells (HBMEC) and enter the CNS are largely derived from models of inflammation. However, our data indicate that ALL cells do not elicit an inflammatory phenotype by HBMEC. Our current investigation focuses on the contribution of the unique coexpression of vascular endothelial (VE)-cadherin and platelet endothelial cell adhesion molecule-1 (PECAM-1) by ALL in mediating leukemic cell interactions with HBMEC as an in vitro model of the blood-brain barrier.Primary ALL and ALL cell lines were evaluated for VE-cadherin and PECAM-1 expression. Lentiviral-mediated transduction of VE-cadherin and PECAM-1 into REH cells and antibody neutralization of VE-cadherin and PECAM-1 in SUP-B15 cells was used to delineate the role of these two proteins in mediating ALL adhesion to, and migration through, HBMEC monolayers.Although cell line models indicate that VE-cadherin and PECAM-1 expression is found on the surface Philadelphia chromosome-positive ALL, evaluation of primary ALL demonstrates that VE-cadherin and PECAM-1 are expressed independent of Philadelphia status. Expression of VE-cadherin and PECAM-1 by ALL enhanced the adhesion of ALL to HBMEC, while expression of PECAM-1 enhanced ALL adhesion to, and migration through, HBMEC.Expression of VE-cadherin and PECAM-1 by ALL cells positions them to interact with HBMEC. By increasing our understanding of molecular mechanisms through which ALL cells gain entry into the CNS, new strategies may be designed to prevent leukemia cell entry into the CNS.
| S-EPMC2929642 | biostudies-other
Project description:Adhesions of cells to extracellular matrix and adjacent cells are mediated by integrins and VE-cadherin, respectively. Although these adhesion processes play crucial roles in vascular cell migration and angiogenesis, it remains unclear as to how they are coordinated to regulate cellular functions. We report here that integrin engagement by treating bovine endothelial aortic cell monolayers with beads coated with fibronectin (Fn) led to disruption of the VE-cadherin-containing adherens junctions. This disruption was accompanied by increases of tyrosine phosphorylation of beta-catenin, gamma-catenin, and p120ctn, as well as the dissociation of alpha-catenin and gamma-catenin from VE-cadherin. We applied a membrane-targeted Src reporter based on the fluorescence resonance energy transfer technique to visualize the dynamic Src activation at subcellular levels in live cells. The integrin engagement induced by Fn-coated beads caused the activation of Src around the beads and at adherens junctions, which are subsequently disrupted. The inhibition of Src with PP1 blocked the effects of integrin engagement on adherens junctions. Although Ras can also modulate adherens junctions, the resulting patterns of phosphorylation and association of junction proteins were distinct from those induced by integrin engagement. The inhibition of Ras by RasN17 did not rescue the disruption of adherens junctions induced by integrin engagement or by Src activation. Integrin engagement by Fn-coated beads also induced a significant alteration of cortical actin filaments at adherens junctions. The results indicate that integrin engagement disrupts VE-cadherin-containing adherens junctions via the activation of Src, but not Ras, possibly as a result of modulation of the actin network.
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Project description:Blood-brain barrier (BBB) disruption is a serious pathological consequence of traumatic brain injury (TBI), for which there are limited therapeutic strategies. Tissue inhibitor of metalloproteinase-2 (TIMP2), a molecule with dual functions of inhibiting MMP activity and displaying cytokine-like activity through receptor binding, has been reported to inhibit VEGF-induced vascular hyperpermeability. Here, we investigate the ability of TIMP2 to ameliorate BBB disruption in TBI and the underlying molecular mechanisms. Both TIMP2 and AlaTIMP2, a TIMP2 mutant without MMP-inhibiting activity, attenuated neurological deficits and BBB leakage in TBI mice; they also inhibited junctional protein degradation and translocation to reduce paracellular permeability in human brain microvascular endothelial cells (ECs) exposed to hypoxic plus inflammatory insult. Mechanistic studies revealed that TIMP2 interacted with α3β1 integrin on ECs, inhibiting Src activation-dependent VE-cadherin phosphorylation, VE-cadherin/catenin complex destabilization, and subsequent VE-cadherin internalization. Notably, localization of VE-cadherin on the membrane was critical for TIMP2-mediated EC barrier integrity. Furthermore, TIMP2-mediated increased membrane localization of VE-cadherin enhanced the level of active Rac1, thereby inhibiting stress fiber formation. All together, our studies have identified an MMP-independent mechanism by which TIMP2 regulates EC barrier integrity after TBI. TIMP2 may be a therapeutic agent for TBI and other neurological disorders involving BBB breakdown.
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Project description:Hypoxia and consequent production of vascular endothelial growth factor A (VEGFA) promote blood vessel leakiness and edema in ocular diseases. Anti-VEGFA therapeutics may aggravate hypoxia; therefore, therapy development is needed. Oxygen-induced retinopathy was used as a model to test the role of nitric oxide (NO) in pathological neovascularization and vessel permeability. Suppression of NO formation was achieved chemically using L-NMMA, or genetically, in endothelial NO synthase serine to alanine (S1176A) mutant mice. Suppression of NO formation resulted in reduced retinal neoangiogenesis. Remaining vascular tufts exhibited reduced vascular leakage through stabilized endothelial adherens junctions, manifested as reduced phosphorylation of vascular endothelial (VE)-cadherin Y685 in a c-Src-dependent manner. Treatment with a single dose of L-NMMA in established retinopathy restored the vascular barrier and prevented leakage. We conclude that NO destabilizes adheren junctions, resulting in vascular hyperpermeability, by converging with the VEGFA/VEGFR2/c-Src/VE-cadherin pathway. This study was supported by the Swedish Cancer foundation (19 0119 Pj ), the Swedish Research Council (2020-01349), the Knut and Alice Wallenberg foundation (KAW 2020.0057) and a Fondation Leducq Transatlantic Network of Excellence Grant in Neurovascular Disease (17 CVD 03). KAW also supported LCW with a Wallenberg Scholar grant (2015.0275). WCS was supported by Grants R35 HL139945, P01 HL1070205, AHA MERIT Award. DV was supported by grants from the Deutsche Forschungsgemeinschaft, SFB1450, B03, and CRU342, P2.
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Project description:The aim of the experiment was to compare a newly defined population VE-Cadherin+GFP+ to control populations, VE-Cadherin- GFP+ and VE-Cadherin+GFP-.
2019-04-20 | GSE113403 | GEO
Project description:Endothelial junctions provide blood and lymph vessel integrity and are essential for the formation of a vascular system. They control the extravasation of solutes, leukocytes and metastatic cells from blood vessels and the uptake of fluid and leukocytes into the lymphatic vascular system. A multitude of adhesion molecules mediate and control the integrity and permeability of endothelial junctions. VE-cadherin is arguably the most important adhesion molecule for the formation of vascular structures, and the stability of their junctions. Interestingly, despite this prominence, its elimination from junctions in the adult organism has different consequences in the vasculature of different organs, both for blood and lymph vessels. In addition, even in tissues where the lack of VE-cadherin leads to strong plasma leaks from venules, the physical integrity of endothelial junctions is preserved. Obviously, other adhesion molecules can compensate for a loss of VE-cadherin and this review will discuss which other adhesive mechanisms contribute to the stability and regulation of endothelial junctions and cooperate with VE-cadherin in intact vessels. In addition to adhesion molecules, endothelial receptors will be discussed, which stimulate signaling processes that provide junction stability by modulating the actomyosin system, which reinforces tension of circumferential actin and dampens pulling forces of radial stress fibers. Finally, we will highlight most recent reports about the formation and control of the specialized button-like junctions of initial lymphatics, which represent the entry sites for fluid and cells into the lymphatic vascular system.
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Project description:par3 is a multiple-PDZ-containing scaffold protein that is central to the organization of an evolutionarily conserved cell polarity complex consisting of par3, par6, and aPKC. The ability of par3 PDZ domains to target various adhesion molecules and enzymes at the plasma membrane leads to the controlled localization of par6 and aPKC, which has firmly established its role in epithelial cell polarity. Of the numerous PDZ ligands associated with par3, interaction of its third PDZ domain with the class II ligand found within the C-terminal tail of vascular endothelial cadherin (VE-Cad) suggests a role in endothelial cell polarity as well, but the molecular details of the interaction are unknown. Previously determined structures of par3-PDZ3 bound to the class I ligand found within the C-terminal tail of the phosphoinositide phosphatase PTEN revealed two discrete binding sites: a canonical PDZ-ligand interaction site and a distal site involving charge-charge complements. Currently, it is unclear if par3-PDZ3 employs both canonical and distal binding modes in its association with VE-Cad or if these modes are unique to the PTEN interaction, suggesting a possible mechanism for ligand specificity within the polarity network. The structure of par3-PDZ3 bound to the C-terminal tail of VE-Cad presented in this work shows that both canonical and distal interactions are utilized in binding. Biophysical measurements using fluorescence polarization and two-dimensional NMR implicate the intermolecular charge pairing of aspartic acid 777 (VE-Cad) and arginine 609 (par3-PDZ3) as a crucial modulator of complex formation. Phosphorylation of VE-Cad at serine 776 increases its affinity for par3, demonstrating that post-translational modifications outside of the canonical carboxylate binding site can enhance PDZ-ligand interactions. Comparison of the VE-Cad and PTEN complexes highlights how the unique molecular architecture of par3-PDZ3 can accommodate both canonical and distal interaction modes that allow dual-class specificity for these two ligand types.
| S-EPMC2819025 | biostudies-literature