Beatriz Marcos-Ramiro

Adherens junctions connect stress fibres between adjacent endothelial cells

BackgroundEndothelial cell-cell junctions maintain endothelial integrity and regulate vascular morphogenesis and homeostasis. Cell-cell junctions are usually depicted with a linear morphology along the boundaries between adjacent cells and in contact with cortical F-actin. However, in the endothelium, cell-cell junctions are highly dynamic and morphologically heterogeneous.ResultsWe report that endothelial cell-cell junctions can attach to the ends of stress fibres instead of to cortical F-actin, forming structures that we name discontinuous adherens junctions (AJ). Discontinuous AJ are highly dynamic and are increased in response to tumour necrosis factor (TNF)-α, correlating with the appearance of stress fibres. We show that vascular endothelial (VE)-cadherin/β-catenin/α-catenin complexes in discontinuous AJ are linked to stress fibres. Moreover, discontinuous AJ connect stress fibres from adjacent cells independently of focal adhesions, of which there are very few in confluent endothelial cells, even in TNF-α-stimulated cells. RNAi-mediated knockdown of VE-cadherin, but not zonula occludens-1, reduces the linkage of stress fibres to cell-cell junctions, increases focal adhesions, and dramatically alters the distribution of these actin cables in confluent endothelial cells.ConclusionsOur results indicate that stress fibres from neighbouring cells are physically connected through discontinuous AJ, and that stress fibres can be stabilized by AJ-associated multi-protein complexes distinct from focal adhesions.

TNF-induced endothelial barrier disruption: beyond actin and Rho

The decrease of endothelial barrier function is central to the long-term inflammatory response. A pathological alteration of the ability of endothelial cells to modulate the passage of cells and solutes across the vessel underlies the development of inflammatory diseases such as atherosclerosis and multiple sclerosis. The inflammatory cytokine tumour necrosis factor (TNF) mediates changes in the barrier properties of the endothelium. TNF activates different Rho GTPases, increases filamentous actin and remodels endothelial cell morphology. However, inhibition of actin-mediated remodelling is insufficient to prevent endothelial barrier disruption in response to TNF, suggesting that additional molecular mechanisms are involved. Here we discuss, first, the pivotal role of Rac-mediated generation of reactive oxygen species (ROS) to regulate the integrity of endothelial cell-cell junctions and, second, the ability of endothelial adhesion receptors such as ICAM-1, VCAM-1 and PECAM-1, involved in leukocyte transendothelial migration, to control endothelial permeability to small molecules, often through ROS generation. These adhesion receptors regulate endothelial barrier function in ways both dependent on and independent of their engagement by immune cells, and orchestrate the crosstalk between leukocyte transendothelial migration and endothelial permeability during inflammation.

Endothelial membrane reorganization during leukocyte extravasation

Leukocyte trafficking from the bloodstream to inflamed tissues across the endothelial barrier is an essential response in innate immunity. Leukocyte adhesion, locomotion, and diapedesis induce signaling in endothelial cells and this is accompanied by a profound reorganization of the endothelial cell surfaces that is only starting to be unveiled. Here we review the current knowledge on the leukocyte-mediated alterations of endothelial membrane dynamics and their role in promoting leukocyte extravasation. The formation of protein- and lipid-mediated cell adhesion nanodomains at the endothelial apical surface, the extension of micrometric apical membrane docking structures, which are derived from microvilli and embrace adhered leukocytes, as well as the vesicle-trafficking pathways that are required for efficient leukocyte diapedesis, are discussed. The coordination between these different endothelial membrane-remodeling events probably provides the road map for transmigrating leukocytes to find exit points in the vessel wall, in a context of severe mechanical and inflammatory stress. A better understanding of how vascular endothelial cells respond to immune cell adhesion should enable new therapeutic strategies to be developed that can abrogate uncontrolled leukocyte extravasation in inflammatory diseases.

Microparticles in multiple sclerosis and clinically isolated syndrome: effect on endothelial barrier function.

BackgroundCell-derived microparticles are secreted in response to cell damage or dysfunction. Endothelial and platelet dysfunction are thought to contribute to the development of multiple sclerosis (MS). Our aim here is, first, to compare the presence of microparticles of endothelial and platelet origin in plasma from patients with different clinical forms of MS and with clinically isolated syndrome. Second, to investigate the effect of microparticles on endothelial barrier function.ResultsPlatelet-poor plasma from 95 patients (12 with clinically isolated syndrome, 51 relapsing-remitting, 23 secondary progressive, 9 primary progressive) and 49 healthy controls were analyzed for the presence of platelet-derived and endothelium-derived microparticles by flow cytometry. The plasma concentration of platelet-derived and endothelium-derived microparticles increased in all clinical forms of MS and in clinically isolated syndrome versus controls. The response of endothelial barriers to purified microparticles was measured by electric cell-substrate impedance sensing. Microparticles from relapsing-remitting MS patients induced, at equivalent concentrations, a stronger disruption of endothelial barriers than those from healthy donors or from patients with clinically isolated syndrome. MS microparticles acted synergistically with the inflammatory mediator thrombin to disrupt the endothelial barrier function.ConclusionsPlasma microparticles should be considered not only as markers of early stages of MS, but also as pathological factors with the potential to increase endothelial permeability and leukocyte infiltration.

Crosstalk Between Reticular Adherens Junctions and Platelet Endothelial Cell Adhesion Molecule-1 Regulates Endothelial Barrier Function

Objective—Endothelial cells provide a barrier between the blood and tissues, which is reduced during inflammation to allow selective passage of molecules and cells. Adherens junctions (AJ) play a central role in regulating this barrier. We aim to investigate the role of a distinctive 3-dimensional reticular network of AJ found in the endothelium. Methods and Results—In endothelial AJ, vascular endothelial-cadherin recruits the cytoplasmic proteins &bgr;-catenin and p120-catenin. &bgr;-catenin binds to &agr;-catenin, which links AJ to actin filaments. AJ are usually described as linear structures along the actin-rich intercellular contacts. Here, we show that these AJ components can also be organized in reticular domains that contain low levels of actin. Reticular AJ are localized in areas where neighboring cells overlap and encompass the cell adhesion receptor platelet endothelial cell adhesion molecule-1 (PECAM-1). Superresolution microscopy revealed that PECAM-1 forms discrete structures distinct from and distributed along AJ, within the voids of reticular domains. Inflammatory tumor necrosis factor-&agr; increases permeability by mechanisms that are independent of actomyosin-mediated tension and remain incompletely understood. Reticular AJ, but not actin-rich linear AJ, were disorganized by tumor necrosis factor-&agr;. This correlated with PECAM-1 dispersal from cell borders. PECAM-1 inhibition with blocking antibodies or small interfering RNA specifically disrupted reticular AJ, leaving linear AJ intact. This disruption recapitulated typical tumor necrosis factor-&agr;–induced alterations of barrier function, including increased &bgr;-catenin phosphorylation, without altering the actomyosin cytoskeleton. Conclusion—We propose that reticular AJ act coordinately with PECAM-1 to maintain endothelial barrier function in regions of low actomyosin-mediated tension. Selective disruption of reticular AJ contributes to permeability increase in response to tumor necrosis factor-&agr;.

RhoB controls endothelial barrier recovery by inhibiting Rac1 trafficking to the cell border

Rho/ROCK signaling is essential to maintain the integrity of the endothelial barrier, but the contributions of specific Rho GTPase family members are unclear. Here, Marcos-Ramiro et al. show that RhoB specifically regulates intracellular trafficking of the Rho GTPase Rac1 and thereby controls endothelial barrier restoration during inflammation.

MYADM regulates Rac1 targeting to ordered membranes required for cell spreading and migration.

Rac1 requires compartmentalization into specialized, condensed membranes to mediate cell migration. We show that myeloid-associated differentiation marker (MYADM), a member of the MAL family of proteins with ubiquitous expression, regulates membrane condensation required for Rac1 targeting and, subsequently, cell spreading and migration.

Apicobasal polarity controls lymphocyte adhesion to hepatic epithelial cells

Loss of apicobasal polarity is a hallmark of epithelial pathologies. Leukocyte infiltration and crosstalk with dysfunctional epithelial barriers are crucial for the inflammatory response. Here, we show that apicobasal architecture regulates the adhesion between hepatic epithelial cells and lymphocytes. Polarized hepatocytes and epithelium from bile ducts segregate the intercellular adhesion molecule 1 (ICAM-1) adhesion receptor onto their apical, microvilli-rich membranes, which are less accessible by circulating immune cells. Upon cell depolarization, hepatic ICAM-1 becomes exposed and increases lymphocyte binding. Polarized hepatic cells prevent ICAM-1 exposure to lymphocytes by redirecting basolateral ICAM-1 to apical domains. Loss of ICAM-1 polarity occurs in human inflammatory liver diseases and can be induced by the inflammatory cytokine tumor necrosis factor alpha (TNF-α). We propose that adhesion receptor polarization is a parenchymal immune checkpoint that allows functional epithelium to hamper leukocyte binding. This contributes to the haptotactic guidance of leukocytes toward neighboring damaged or chronically inflamed epithelial cells that expose their adhesion machinery.

MYADM controls endothelial barrier function through ERM-dependent regulation of ICAM-1 expression

Myeloid-associated differentiation marker (MYADM) protein belongs to the MAL family and regulates raft domains in epithelial cells. Membrane rafts participate in inflammatory responses. This study shows that MYADM is expressed in endothelial cells and controls the endothelial barrier by regulating ICAM-1 expression through ezrin, radixin, and moesin proteins, connectors between plasma membrane domains and actin cytoskeleton.

Activation of Rac1 and RhoA Preserve Corneal Endothelial Barrier Function

Purpose The corneal endothelium is responsible for the correct hydration of the corneal stroma. Corneal endothelial cells have a low proliferative capacity, so preserving their barrier function under suboptimal conditions that cause osmotic imbalance, such as those arising from corneal pathologies, age, cryopreservation, and transplantation, is essential for maintaining corneal transparency. We have investigated the signaling induced by hyperosmotic shock that reversibly disrupts corneal endothelial barriers in human endothelial cells and in murine corneas. Methods Endothelial barrier properties were analyzed in vitro by electric cell substrate impedance sensing (ECIS) and confocal microscopy of the human endothelial cell line B4G12-HCEC, and, ex vivo, by confocal microscopy and stimulated emission-depletion (STED) super-resolution microscopy of murine corneas. Cell signaling in response to hyperosmotic stress, induced with an excess of sodium chloride, was investigated in B4G12-HCECs. Rho GTPase activity was detected by pulldown assays with recombinant GST proteins fused to the Rho binding domains of Rho effectors. Results Hyperosmotic stress increased actin polymerization and activated the Rho GTPases Rac1 and RhoA, but not Cdc42. Rac1- and RhoA-mediated pathway inhibition had a minor effect on barrier disruption but partially delayed barrier reformation after stress withdrawal. In contrast, Rac1 and RhoA activation enhanced constitutive endothelial barrier function and accelerated barrier repair. Conclusions Our results indicate that Rac1 and RhoA activation do not mediate stress-induced cell contraction but are endothelial responses that act to restore and maintain barrier homeostasis. Therefore, pharmacological activation of these two GTPases could be a therapeutic strategy for preserving corneal endothelial barrier function.