scholarly journals PKM2 regulates endothelial cell junction dynamics and angiogenesis via ATP production

2019 ◽  
Vol 9 (1) ◽  
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.

scholarly journals Vinculin controls endothelial cell junction dynamics during vascular lumen formation

2021 ◽  
Author(s):  
Maria P. Kotini ◽  
Miesje M. van der Stoel ◽  
Mitchell K. Han ◽  
Bettina Kirchmaier ◽  
Johan de Rooij ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Cell Junctions ◽  
Cell Junction ◽  
Vascular Integrity ◽  
Lumen Formation ◽  
Cell Behaviors ◽  
Sprouting Angiogenesis ◽  
Dynamic Cell ◽  
External Forces ◽  
Cell Cell

AbstractBlood vessel morphogenesis is driven by coordinated endothelial cell behaviors, which depend on dynamic cell-cell interactions. Remodeling of endothelial cell-cell junctions promote morphogenetic cellular events while preserving vascular integrity. Here, we have analyzed the dynamics of endothelial cell-cell junctions during lumen formation in angiogenic sprouts. By live-imaging of the formation of intersegmental blood vessels in zebrafish, we demonstrate that lumen expansion is accompanied by the formation of transient finger-shaped junctions. Formation and maintenance of these junctional fingers are positively regulated by blood pressure whereas inhibition of blood flow prevents their formation. Using fluorescent reporters, we show that the tension-sensor Vinculin localizes to junctional fingers. Furthermore, loss of vinculin function, in vinculin a and -b double knockouts, prevents junctional finger formation in angiogenic sprouts, whereas endothelial expression of a vinculin transgene is sufficient to restore junctional fingers. Taken together, our findings suggest a mechanism in which lumen expansion during angiogenesis leads to an increase in junctional tension, which triggers recruitment of vinculin and formation of junctional fingers. We propose that endothelial cells may employ force-dependent junctional remodeling to react to changes in external forces to protect cell-cell contacts and to maintain vascular integrity during sprouting angiogenesis.

scholarly journals Excess centrosomes disrupt vascular lumenization and endothelial cell adherens junctions

Angiogenesis ◽  
2020 ◽  
Vol 23 (4) ◽  
pp. 567-575
Author(s):  
Danielle B. Buglak ◽  
Erich J. Kushner ◽  
Allison P. Marvin ◽  
Katy L. Davis ◽  
Victoria L. Bautch
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Polarity ◽  
Adherens Junctions ◽  
Cell Junctions ◽  
Microtubule Organizing Center ◽  
Sprouting Angiogenesis ◽  
Organizing Center ◽  
Important Regulator ◽  
Cell Cell

Abstract Proper blood vessel formation requires coordinated changes in endothelial cell polarity and rearrangement of cell–cell junctions to form a functional lumen. One important regulator of cell polarity is the centrosome, which acts as a microtubule organizing center. Excess centrosomes perturb aspects of endothelial cell polarity linked to migration, but whether centrosome number influences apical–basal polarity and cell–cell junctions is unknown. Here, we show that excess centrosomes alter the apical–basal polarity of endothelial cells in angiogenic sprouts and disrupt endothelial cell–cell adherens junctions. Endothelial cells with excess centrosomes had narrower lumens in a 3D sprouting angiogenesis model, and zebrafish intersegmental vessels had reduced perfusion following centrosome overduplication. These results indicate that endothelial cell centrosome number regulates proper lumenization downstream of effects on apical–basal polarity and cell–cell junctions. Endothelial cells with excess centrosomes are prevalent in tumor vessels, suggesting how centrosomes may contribute to tumor vessel dysfunction.

scholarly journals Septin Roles and Mechanisms in Organization of Endothelial Cell Junctions

2020 ◽  
Author(s):  
Joanna Kim ◽  
John A. Cooper
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Adherens Junctions ◽  
Cell Junctions ◽  
Cell Junction ◽  
Cell Contact ◽  
Junction Molecules ◽  
Tnf Α ◽  
Multiple Cell ◽  
Cell Cell

AbstractSeptins play an important role in regulating the barrier function of the endothelial monolayer of the microvasculature. Depletion of septin 2 protein alters the organization of vascular endothelial (VE)-cadherin at cell-cell adherens junctions as well as the dynamics of membrane protrusions at endothelial cell-cell contact sites. Here, we report the discovery that localization of septin 2 at endothelial cell junctions is important for the distribution of a number of other junctional molecules. We also found that treatment of microvascular endothelial cells with the inflammatory mediator TNF-α led to sequestration of septin 2 away from cell junctions and into the cytoplasm, without an effect on the overall level of septin 2 protein. Interestingly, TNF-α treatment of endothelial monolayers produced effects similar to those of depletion of septin 2 on various molecular components of adherens junctions (AJs) and tight junctions (TJs). Immunofluorescence staining revealed disruption of the integrity of AJs and TJs at cell-cell junctions without significant changes in protein expression except for VE-cadherin and nectin-2. To investigate the mechanism of junctional localization of septin 2, we mutated the polybasic motif of septin 2, which is proposed to interact with PIP2 in the plasma membrane. Overexpression of PIP2-binding mutant (PIP2BM) septin 2 led to loss of septin 2 from cell junctions with accumulation in the cytoplasm. This redistribution of septin 2 away from the membrane led to effects on cell junction molecules similar to those observed for depletion of septin 2. We conclude that septin localization to the membrane is essential for function and that septins support the localization of multiple cell junction molecules in endothelial cells.

scholarly journals Formation of a PKCζ/β-catenin complex in endothelial cells promotes angiopoietin-1–induced collective directional migration and angiogenic sprouting

Blood ◽  
2012 ◽  
Vol 120 (16) ◽  
pp. 3371-3381 ◽  
Author(s):  
Malika Oubaha ◽  
Michelle I. Lin ◽  
Yoran Margaron ◽  
Dominic Filion ◽  
Emily N. Price ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Leading Edge ◽  
Cell Junctions ◽  
Endothelial Cell Migration ◽  
Directional Migration ◽  
Cell Contacts ◽  
Angiopoietin 1 ◽  
Cell Cell

Abstract Angiogenic sprouting requires that cell-cell contacts be maintained during migration of endothelial cells. Angiopoietin-1 (Ang-1) and vascular endothelial growth factor act oppositely on endothelial cell junctions. We found that Ang-1 promotes collective and directional migration and, in contrast to VEGF, induces the formation of a complex formed of atypical protein kinase C (PKC)-ζ and β-catenin at cell-cell junctions and at the leading edge of migrating endothelial cells. This complex brings Par3, Par6, and adherens junction proteins at the front of migrating cells to locally activate Rac1 in response to Ang-1. The colocalization of PKCζ and β-catenin at leading edge along with PKCζ-dependent stabilization of cell-cell contacts promotes directed and collective endothelial cell migration. Consistent with these results, down-regulation of PKCζ in endothelial cells alters Ang-1–induced sprouting in vitro and knockdown in developing zebrafish results in intersegmental vessel defects caused by a perturbed directionality of tip cells and by loss of cell contacts between tip and stalk cells. These results reveal that PKCζ and β-catenin function in a complex at adherens junctions and at the leading edge of migrating endothelial cells to modulate collective and directional migration during angiogenesis.

scholarly journals Excess Centrosomes Disrupt Vascular Lumenization and Endothelial Cell Adherens Junctions

2019 ◽  
Author(s):  
Danielle B Buglak ◽  
Erich J Kushner ◽  
Allison P Marvin ◽  
Katy L Davis ◽  
Victoria L Bautch
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Polarity ◽  
Adherens Junctions ◽  
Cell Junctions ◽  
Microtubule Organizing Center ◽  
Sprouting Angiogenesis ◽  
Organizing Center ◽  
Important Regulator ◽  
Cell Cell

ABSTRACTProper blood vessel formation requires coordinated changes in endothelial cell polarity and rearrangement of cell-cell junctions to form a functional lumen. One important regulator of cell polarity is the centrosome, which acts as a microtubule organizing center. Excess centrosomes perturb aspects of endothelial cell polarity linked to migration, but whether centrosome number influences apical-basal polarity and cell-cell junctions is unknown. Here, we show that excess centrosomes alter the apical-basal polarity of endothelial cells in angiogenic sprouts and disrupt endothelial cell-cell adherens junctions. Endothelial cells with excess centrosomes had narrower lumens in a 3D sprouting angiogenesis model, and zebrafish intersegmental vessels had reduced perfusion following centrosome overduplication. These results indicate that endothelial cell centrosome number regulates proper lumenization downstream of effects on apical-basal polarity and cell-cell junctions. Endothelial cells with excess centrosomes are prevalent in tumor vessels, suggesting how centrosomes may contribute to tumor vessel dysfunction.

scholarly journals Nuclear SUN1 stabilizes endothelial cell junctions to regulate blood vessel formation

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.

scholarly journals Shear-induced endothelial cell-cell junction inclination

2010 ◽  
Vol 299 (3) ◽  
pp. C621-C629 ◽  
Author(s):  
Benoît Melchior ◽  
John A. Frangos
Keyword(s):  
Endothelial Cell ◽  
Flow Direction ◽  
Cell Junction ◽  
Structural Basis ◽  
Retrograde Flow ◽  
Arterial Circulation ◽  
Mouse Aorta ◽  
Cell Cell

Atheroprone regions of the arterial circulation are characterized by time-varying, reversing, and oscillatory wall shear stress. Several in vivo and in vitro studies have demonstrated that flow reversal (retrograde flow) is atherogenic and proinflammatory. The molecular and structural basis for the sensitivity of the endothelium to flow direction, however, has yet to be determined. It has been hypothesized that the ability to sense flow direction is dependent on the direction of inclination of the interendothelial junction. Immunostaining of the mouse aorta revealed an inclination of the cell-cell junction by 13° in direction of flow in the descending aorta where flow is unidirectional. In contrast, polygonal cells of the inner curvature where flow is disturbed did not have any preferential inclination. Using a membrane specific dye, the angle of inclination of the junction was dynamically monitored using live cell confocal microscopy in confluent human endothelial cell monolayers. Upon application of shear the junctions began inclining within minutes to a final angle of 10° in direction of flow. Retrograde flow led to a reversal of junctional inclination. Flow-induced junctional inclination was shown to be independent of the cytoskeleton or glycocalyx. Additionally, within seconds, retrograde flow led to significantly higher intracellular calcium responses than orthograde flow. Together, these results show for the first time that the endothelial intercellular junction inclination is dynamically responsive to flow direction and confers the ability to endothelial cells to rapidly sense and adapt to flow direction.

scholarly journals A mechanism of Rap1-induced stabilization of endothelial cell–cell junctions

2011 ◽  
Vol 22 (14) ◽  
pp. 2509-2519 ◽  
Author(s):  
Jian J. Liu ◽  
Rebecca A. Stockton ◽  
Alexandre R. Gingras ◽  
Ararat J. Ablooglu ◽  
Jaewon Han ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Small Gtpases ◽  
Cell Junctions ◽  
Physical Interaction ◽  
Dependent Manner ◽  
Cardiovascular Development ◽  
Cavernous Malformations ◽  
Cell Cell

Activation of Rap1 small GTPases stabilizes cell–cell junctions, and this activity requires Krev Interaction Trapped gene 1 (KRIT1). Loss of KRIT1 disrupts cardiovascular development and causes autosomal dominant familial cerebral cavernous malformations. Here we report that native KRIT1 protein binds the effector loop of Rap1A but not H-Ras in a GTP-dependent manner, establishing that it is an authentic Rap1-specific effector. By modeling the KRIT1–Rap1 interface we designed a well-folded KRIT1 mutant that exhibited a ∼40-fold-reduced affinity for Rap1A and maintained other KRIT1-binding functions. Direct binding of KRIT1 to Rap1 stabilized endothelial cell–cell junctions in vitro and was required for cardiovascular development in vivo. Mechanistically, Rap1 binding released KRIT1 from microtubules, enabling it to locate to cell–cell junctions, where it suppressed Rho kinase signaling and stabilized the junctions. These studies establish that the direct physical interaction of Rap1 with KRIT1 enables the translocation of microtubule-sequestered KRIT1 to junctions, thereby supporting junctional integrity and cardiovascular development.

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

Blood ◽  
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.

scholarly journals Regulation of Endothelial Cell Motility by Complexes of Tetraspan Molecules CD81/TAPA-1 and CD151/PETA-3 with α3β1 Integrin Localized at Endothelial Lateral Junctions

1998 ◽  
Vol 141 (3) ◽  
pp. 791-804 ◽  
Author(s):  
María Yáñez-Mó ◽  
Arántzazu Alfranca ◽  
Carlos Cabañas ◽  
Mónica Marazuela ◽  
Reyes Tejedor ◽  
...  
Keyword(s):  
Wound Healing ◽  
Endothelial Cell ◽  
Cell Motility ◽  
Cell Polarization ◽  
Cell Junctions ◽  
Cell Junction ◽  
Cell Contact ◽  
Collagen Gels ◽  
Α3β1 Integrin ◽  
Cell Cell

Cell-to-cell junction structures play a key role in cell growth rate control and cell polarization. In endothelial cells (EC), these structures are also involved in regulation of vascular permeability and leukocyte extravasation. To identify novel components in EC intercellular junctions, mAbs against these cells were produced and selected using a morphological screening by immunofluorescence microscopy. Two novel mAbs, LIA1/1 and VJ1/16, specifically recognized a 25-kD protein that was selectively localized at cell–cell junctions of EC, both in the primary formation of cell monolayers and when EC reorganized in the process of wound healing. This antigen corresponded to the recently cloned platelet-endothelial tetraspan antigen CD151/PETA-3 (platelet-endothelial tetraspan antigen-3), and was consistently detected at EC cell–cell contact sites. In addition to CD151/PETA-3, two other members of the tetraspan superfamily, CD9 and CD81/ TAPA-1 (target of antiproliferative antibody-1), localized at endothelial cell-to-cell junctions. Biochemical analysis demonstrated molecular associations among tetraspan molecules themselves and those of CD151/ PETA-3 and CD9 with α3β1 integrin. Interestingly, mAbs directed to both CD151/PETA-3 and CD81/ TAPA-1 as well as mAb specific for α3 integrin, were able to inhibit the migration of ECs in the process of wound healing. The engagement of CD151/PETA-3 and CD81/TAPA-1 inhibited the movement of individual ECs, as determined by quantitative time-lapse video microscopy studies. Furthermore, mAbs against the CD151/PETA-3 molecule diminished the rate of EC invasion into collagen gels. In addition, these mAbs were able to increase the adhesion of EC to extracellular matrix proteins. Together these results indicate that CD81/TAPA-1 and CD151/PETA-3 tetraspan molecules are components of the endothelial lateral junctions implicated in the regulation of cell motility, either directly or by modulation of the function of the associated integrin heterodimers.

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