Src Family Kinases collaborate with distinct TNF‐alpha‐induced signaling pathways to regulate actin dynamics at cell‐cell junctions and barrier function in endothelial cells

The increase in endothelial permeability in response to inflammatory mediators such as alpha-thrombin and histamine is accompanied by cell rounding and interendothelial gap formation, implicating that the predominant transport pathway is a diffusive one [i.e., via cellular junctions (paracellular transport)]. However, the possible contribution by vesicle-mediated transport (i.e., via albumin binding protein gp60) to the overall permeability increase needs investigation. Regulation of paracellular transport in endothelial cells is associated with modulation of actin-based systems which anchor the cell to its neighbor or extracellular matrix, thus maintaining endothelial integrity. At the cell-cell junctions, actin is linked indirectly to the plasma membrane by linking proteins (e.g., vinculin, catenins, alpha-actinin) to cadherins, which function in homophilic intercellular adhesion. Cadherins may also play a role in regulating the formation of tight junctions, which also may be associated with actin. At endothelial focal contacts, the transmembrane receptors (integrins) for matrix proteins are linked to actin via linking proteins (i.e., vinculin, talin, alpha-actinin). In response to inflammatory mediators, second messengers signal two regulatory pathways which modulate the actin-based systems, which may lead to impairment of the endothelial barrier integrity. One pathway is based on protein kinase C (PKC) isozyme-specific phosphorylation of linking proteins at the cell-cell and cell-matrix junctions. The increased phosphorylation is associated with actin reorganization, cell rounding, and increased paracellular transport. The other is the activation of myosin light-chain kinase, (MLCK), which causes an actin-myosin-based contraction that may lead to a centripetal retraction of endothelial cells. Current research is in the identification of protein substrates of PKC isozymes, the specific role of their phosphorylation in barrier function, and determining the precise role of MLCK in modulation of endothelial barrier function.

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Nuclear SUN1 stabilizes endothelial cell junctions to regulate blood vessel formation

10.1101/2021.08.11.455980 ◽

2021 ◽

Author(s):

Danielle B Buglak ◽

Ariel L Gold ◽

Allison P Marvin ◽

Shea N Ricketts ◽

Morgan Oatley ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Blood Vessel ◽

Barrier Function ◽

Cell Junctions ◽

Cell Junction ◽

Peripheral Cell ◽

Blood Vessel Formation ◽

Vessel Formation ◽

Cell Cell

Endothelial cells line all blood vessels and coordinate blood vessel formation and the blood-tissue barrier via endothelial cell-cell junctions. The nucleus also regulates endothelial cell behaviors, but the mechanisms are poorly understood. Here we show that nuclear-localized SUN1, a LINC complex component that connects the nucleus to the cytoskeleton, regulates endothelial cell-cell junction communication and blood vessel formation. Loss of murine endothelial Sun1 impaired blood vessel formation and destabilized junctions. At the cellular level, SUN1 stabilized endothelial cell-cell junctions and promoted barrier function. Abnormal SUN1-depleted junctions resembled those seen with loss of microtubules, and they were accompanied by impaired microtubule dynamics and actomyosin hypercontractility. Angiogenic sprouts formed but retracted in SUN1-depleted endothelial cells, and vessels of zebrafish lacking SUN1 had abnormal extension and were defective in forming connections. Thus, endothelial SUN1 regulates peripheral cell-cell junctions from the nucleus, likely via microtubule-based interactions, and this long-range regulation is important for blood vessel formation and barrier function.

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Glycocalyx modulates the motility and proliferative response of vascular endothelium to fluid shear stress

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00162.2007 ◽

2007 ◽

Vol 293 (2) ◽

pp. H1023-H1030 ◽

Cited By ~ 119

Author(s):

Yu Yao ◽

Aleksandr Rabodzey ◽

C. Forbes Dewey

Keyword(s):

Endothelial Cells ◽

Vascular Endothelium ◽

Fluid Shear Stress ◽

Vascular Endothelial Cells ◽

Surface Profile ◽

Cell Junctions ◽

Vascular Endothelial ◽

Fluid Shear ◽

New Perspective ◽

Cell Cell

Flow-induced mechanotransduction in vascular endothelial cells has been studied over the years with a major focus on putative connections between disturbed flow and atherosclerosis. Recent studies have brought in a new perspective that the glycocalyx, a structure decorating the luminal surface of vascular endothelium, may play an important role in the mechanotransduction. This study reports that modifying the amount of the glycocalyx affects both short-term and long-term shear responses significantly. It is well established that after 24 h of laminar flow, endothelial cells align in the direction of flow and their proliferation is suppressed. We report here that by removing the glycocalyx by using the specific enzyme heparinase III, endothelial cells no longer align under flow after 24 h and they proliferate as if there were no flow present. In addition, confluent endothelial cells respond rapidly to flow by decreasing their migration speed by 40% and increasing the amount of vascular endothelial cadherin in the cell-cell junctions. These responses are not observed in the cells treated with heparinase III. Heparan sulfate proteoglycans (a major component of the glycocalyx) redistribute after 24 h of flow application from a uniform surface profile to a distinct peripheral pattern with most molecules detected above cell-cell junctions. We conclude that the presence of the glycocalyx is necessary for the endothelial cells to respond to fluid shear, and the glycocalyx itself is modulated by the flow. The redistribution of the glycocalyx also appears to serve as a cell-adaptive mechanism by reducing the shear gradients that the cell surface experiences.

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Spatial and temporal relationships between cadherins and PECAM-1 in cell-cell junctions of human endothelial cells.

The Journal of Cell Biology ◽

10.1083/jcb.126.1.247 ◽

1994 ◽

Vol 126 (1) ◽

pp. 247-258 ◽

Cited By ~ 132

Author(s):

O Ayalon ◽

H Sabanai ◽

M G Lampugnani ◽

E Dejana ◽

B Geiger

Keyword(s):

Endothelial Cells ◽

Plasma Membrane ◽

Vascular System ◽

Adherens Junctions ◽

Microscopic Analysis ◽

Membrane Receptors ◽

Temporal Organization ◽

Cell Junctions ◽

Short Treatment ◽

Cell Cell

The integrity of the endothelial layer, which lines the entire cavity of the vascular system, depends on tight adhesion of the cells to the underlying basement membrane as well as to each other. It has been previously shown that such interactions occur via membrane receptors that determine the specificity, topology, and mechanical properties of the surface adhesion. Cell-cell junctions between endothelial cells, in culture and in situ, involve both Ca(2+)-dependent and -independent mechanisms that are mediated by distinct adhesion molecules. Ca(2+)-dependent cell-cell adhesion occurs mostly via members of the cadherin family, which locally anchor the microfilament system to the plasma membrane, in adherens junctions. Ca(2+)-independent adhesions were reported to mainly involve members of the Ig superfamily. In this study, we performed three-dimensional microscopic analysis of the relative subcellular distributions of these two endothelial intercellular adhesion systems. We show that cadherins are located at adjacent (usually more apical), yet clearly distinct domains of the lateral plasma membrane, compared to PECAM-1. Moreover, cadherins were first organized in adherens junctions within 2 h after seeding of endothelial cells, forming multiple lateral patches which developed into an extensive belt-like structure over a period of 24 h. PECAM-1 became associated with surface adhesions significantly later and became progressively associated with the cadherin-containing adhesions. Cadherins and PECAM-1 also differed in their detergent extractability, reflecting differences in their mode of association with the cytoskeleton. Moreover, the two adhesion systems could be differentially modulated since short treatment with the Ca2+ chelator EGTA, disrupted the cadherin junctions leaving PECAM-1 apparently intact. These results confirm that endothelial cells possess distinct intercellular contact mechanisms that differ in their spatial and temporal organization as well as in their functional properties.

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Cell confluence regulates tyrosine phosphorylation of adherens junction components in endothelial cells

Journal of Cell Science ◽

10.1242/jcs.110.17.2065 ◽

1997 ◽

Vol 110 (17) ◽

pp. 2065-2077 ◽

Cited By ~ 13

Author(s):

M.G. Lampugnani ◽

M. Corada ◽

P. Andriopoulou ◽

S. Esser ◽

W. Risau ◽

...

Keyword(s):

Endothelial Cells ◽

Tyrosine Phosphorylation ◽

Adherens Junction ◽

Cell Junctions ◽

Molecular Organization ◽

Beta Catenin ◽

Cell Contacts ◽

Cell Type Specific ◽

Dynamic Structures ◽

Cell Cell

In src- and ras-transformed cells, tyrosine phosphorylation of adherens junction (AJ) components is related to impairment of cell-cell adhesion. In this paper we report that in human endothelial cells (EC), tyrosine phosphorylation of AJ can be a physiological process regulated by cell density. Immunofluorescence analysis revealed that a phosphotyrosine (P-tyr) antibody could stain cell-cell junctions only in sparse or loosely confluent EC, while the staining was markedly reduced in tightly confluent cultures. This process was reversible, since on artificial wounding of EC monolayers, the cells at the migrating front reacquired P-tyr labelling at cell contacts. In EC, the major cadherin at intercellular AJ is the cell-type-specific VE-cadherin. We therefore analyzed whether this molecule was at least in part responsible for the changes in P-tyr content at cell junctions. Tyrosine phosphorylation of VE-cadherin, beta-catenin and p120, occurred in looser AJ, i.e. in recently confluent cells, and was notably reduced in tightly confluent cultures. Changes in P-tyr content paralleled changes in the molecular organization of AJ. VE-cadherin was mostly associated with beta-catenin and p120 in loose EC monolayers, while in long-confluent cells, these two catenins were largely replaced by plakoglobin. Inhibition of P-tyr phosphatases (PTPases) by PV markedly augmented the P-tyr content of VE-cadherin, which bound p120 and beta-catenin more efficiently, but not plakoglobin. Transfection experiments in CHO cells showed that p120 could bind to a VE-cadherin cytoplasmic region different from that responsible for beta-catenin binding, and PV stabilized this association. Overall these data indicate that endothelial AJ are dynamic structures that can be affected by the state of confluence of the cells. Tyrosine phosphorylation of VE-cadherin and its association to p120 and beta-catenin characterizes early cell contacts, while the formation of mature and cytoskeleton-connected junctions is accompanied by dephosphorylation and plakoglobin association.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e11-11-0914 ◽

2013 ◽

Vol 24 (4) ◽

pp. 483-494 ◽

Cited By ~ 17

Author(s):

Juan F. Aranda ◽

Natalia Reglero-Real ◽

Beatriz Marcos-Ramiro ◽

Ana Ruiz-Sáenz ◽

Laura Fernández-Martín ◽

...

Keyword(s):

Endothelial Cells ◽

Plasma Membrane ◽

Inflammatory Response ◽

Barrier Function ◽

Leukocyte Adhesion ◽

Cell Junctions ◽

Membrane Domains ◽

Filamentous Actin ◽

Cytokine Tumor Necrosis Factor ◽

Factor Α

The endothelium maintains a barrier between blood and tissue that becomes more permeable during inflammation. Membrane rafts are ordered assemblies of cholesterol, glycolipids, and proteins that modulate proinflammatory cell signaling and barrier function. In epithelial cells, the MAL family members MAL, MAL2, and myeloid-associated differentiation marker (MYADM) regulate the function and dynamics of ordered membrane domains. We analyzed the expression of these three proteins in human endothelial cells and found that only MYADM is expressed. MYADM was confined in ordered domains at the plasma membrane, where it partially colocalized with filamentous actin and cell–cell junctions. Small interfering RNA (siRNA)-mediated MYADM knockdown increased permeability, ICAM-1 expression, and leukocyte adhesion, all of which are features of an inflammatory response. Barrier function decrease in MYADM-silenced cells was dependent on ICAM-1 expression. Membrane domains and the underlying actin cytoskeleton can regulate each other and are connected by ezrin, radixin, and moesin (ERM) proteins. In endothelial cells, MYADM knockdown induced ERM activation. Triple-ERM knockdown partially inhibited ICAM-1 increase induced by MYADM siRNA. Importantly, ERM knockdown also reduced ICAM-1 expression in response to the proinflammatory cytokine tumor necrosis factor-α. MYADM therefore regulates the connection between the plasma membrane and the cortical cytoskeleton and so can control the endothelial inflammatory response.

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PKM2 regulates endothelial cell junction dynamics and angiogenesis via ATP production

Scientific Reports ◽

10.1038/s41598-019-50866-x ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 4

Author(s):

Jesús Gómez-Escudero ◽

Cristina Clemente ◽

Diego García-Weber ◽

Rebeca Acín-Pérez ◽

Jaime Millán ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Inflammatory Disease ◽

Chronic Inflammatory Disease ◽

Cell Junctions ◽

Cell Junction ◽

Collective Migration ◽

Sprouting Angiogenesis ◽

Cell Cell

Abstract Angiogenesis, the formation of new blood vessels from pre-existing ones, occurs in pathophysiological contexts such as wound healing, cancer, and chronic inflammatory disease. During sprouting angiogenesis, endothelial tip and stalk cells coordinately remodel their cell-cell junctions to allow collective migration and extension of the sprout while maintaining barrier integrity. All these processes require energy, and the predominant ATP generation route in endothelial cells is glycolysis. However, it remains unclear how ATP reaches the plasma membrane and intercellular junctions. In this study, we demonstrate that the glycolytic enzyme pyruvate kinase 2 (PKM2) is required for sprouting angiogenesis in vitro and in vivo through the regulation of endothelial cell-junction dynamics and collective migration. We show that PKM2-silencing decreases ATP required for proper VE-cadherin internalization/traffic at endothelial cell-cell junctions. Our study provides fresh insight into the role of ATP subcellular compartmentalization in endothelial cells during angiogenesis. Since manipulation of EC glycolysis constitutes a potential therapeutic intervention route, particularly in tumors and chronic inflammatory disease, these findings may help to refine the targeting of endothelial glycolytic activity in disease.

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Flow mechanotransduction regulates traction forces, intercellular forces, and adherens junctions

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00975.2011 ◽

2012 ◽

Vol 302 (11) ◽

pp. H2220-H2229 ◽

Cited By ~ 48

Author(s):

Lucas H. Ting ◽

Jessica R. Jahn ◽

Joon I. Jung ◽

Benjamin R. Shuman ◽

Shirin Feghhi ◽

...

Keyword(s):

Endothelial Cells ◽

Laminar Flow ◽

Adherens Junctions ◽

Cell Junctions ◽

Flow Conditions ◽

Traction Forces ◽

Disturbed Flow ◽

Cytoskeletal Tension ◽

Static Conditions ◽

Cell Cell

Endothelial cells respond to fluid shear stress through mechanotransduction responses that affect their cytoskeleton and cell-cell contacts. Here, endothelial cells were grown as monolayers on arrays of microposts and exposed to laminar or disturbed flow to examine the relationship among traction forces, intercellular forces, and cell-cell junctions. Cells under laminar flow had traction forces that were higher than those under static conditions, whereas cells under disturbed flow had lower traction forces. The response in adhesion junction assembly matched closely with changes in traction forces since adherens junctions were larger in size for laminar flow and smaller for disturbed flow. Treating the cells with calyculin-A to increase myosin phosphorylation and traction forces caused an increase in adherens junction size, whereas Y-27362 cause a decrease in their size. Since tugging forces across cell-cell junctions can promote junctional assembly, we developed a novel approach to measure intercellular forces and found that these forces were higher for laminar flow than for static or disturbed flow. The size of adherens junctions and tight junctions matched closely with intercellular forces for these flow conditions. These results indicate that laminar flow can increase cytoskeletal tension while disturbed flow decreases cytoskeletal tension. Consequently, we found that changes in cytoskeletal tension in response to shear flow conditions can affect intercellular tension, which in turn regulates the assembly of cell-cell junctions.

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The Basic Helix-Loop-Helix TAL1/SCL and Its Partners E47 and LMO2 Act Upstream of the VE-Cadherin Gene in Endothelial Cells.

Blood ◽

10.1182/blood.v108.11.1795.1795 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1795-1795

Author(s):

Virginie Deleuze ◽

Elias Chalhoub ◽

Rawan El-Hajj ◽

Christiane Dohet ◽

Mikael Le Clech ◽

...

Keyword(s):

Endothelial Cells ◽

Ectopic Expression ◽

Cell Junctions ◽

Small Interfering Rnas ◽

Hek 293 ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

Cell Cell

Abstract The basic helix-loop-helix protein TAL-1/SCL, essential for the formation of the hematopoietic system, is also required for vascular development and more particularly for embryonic angiogenesis. We previously reported that TAL-1 acts as a positive factor for post-natal angiogenesis by stimulating endothelial morphogenesis. To understand how TAL-1 modulates angiogenesis, we investigated the functional consequences of TAL-1 silencing, mediated by small-interfering RNAs, in human primary endothelial cells (ECs). We found that TAL-1 knockdown impaired in vitro EC tubulomorphogenesis (in 2-D on Matrigel or 3-D in collagen I gel), with the notable absence of cell-cell contacts, a prerequisite for morphogenesis initiation. This cellular deficiency was associated with a dramatic reduction in the vascular-endothelial (VE)-cadherin at intercellular junctions, the major component of endothelial adherens junctions. In contrast, PECAM (or CD31) was present at cell-cell junctions at the same levels as control cells. Importantly, silencing of two known TAL-1-partners in hematopoietic cells, E47 or LMO2, produce the same effects as TAL-1. Accordingly, silencing of TAL-1, as well as E47 and LMO2, provoked down-regulation of VE-cadherin at both the mRNA and protein levels. Transient transfection experiments in HUVECs showed that TAL-1 and E47 regulate the VE-cadherin promoter through a specialized E-box element. Finally, endogenous VE-cadherin transcription could be directly activated in non-endothelial HEK-293 cells that neither express TAL-1 or LMO2, by the sole concomitant ectopic expression of TAL-1, E47 and LMO2. Overall, our data demonstrate that a multiprotein complex containing at least TAL-1, LMO2 and E47 act upstream of the VE-cadherin gene. We are currently performing chromatin immunoprecipitation (ChIP) to investigate whether the TAL-1-containing complex binds in vivo the VE-cadherin promoter. This study identifies VE-cadherin as an upstream TAL-1-target gene in the endothelial lineage, and provides a first clue in TAL-1 function in the control of angiogenesis.

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