real-time-imaging-endothelial-cell-cell-junctions-during-neutrophil

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

Sanquin Research and Landsteiner Laboratory, AMC at University of Amsterdam

During transendothelial migration, leukocytes use adhesion molecules, such as ICAM-1, to adhere to the endothelium. ICAM-1 is a dynamic molecule that is localized in the apical membrane of the endothelium and clusters upon binding to leukocytes. However, not much is known about the regulation of ICAM-1 clustering and whether membrane dynamics are linked to the ability of ICAM-1 to cluster and bind leukocyte integrins. Therefore, we studied the dynamics of endothelial ICAM-1 under non-clustered and clustered conditions.

Inside-out regulation of ICAM-1 dynamics in TNF-alpha-activated endothelium.

The guanine-nucleotide exchange factor Trio encodes two DH-PH domains that catalyze nucleotide exchange on Rac1, RhoG and RhoA. The N-terminal DH-PH domain is known to activate Rac1 and RhoG, whereas the C-terminal DH-PH domain can activate RhoA. The current study shows that the N-terminal DH-PH domain, upon expression in HeLa cells, activates Rac1 and RhoG independently from each other. In addition, we show that the flanking SH3 domain binds to the proline-rich region of the C-terminus of Rac1, but not of RhoG. However, this SH3 domain is not required for Rac1 or RhoG GDP-GTP exchange. Rescue experiments in Trio-shRNA-expressing cells showed that the N-terminal DH-PH domain of Trio, but not the C-terminal DH-PH domain, restored fibronectin-mediated cell spreading and migration defects that are observed in Trio-silenced cells. Kymograph analysis revealed that the N-terminal DH-PH domain, independent of its SH3 domain, controls the dynamics of lamellipodia. Using siRNA against Rac1 or RhoG, we found that Trio-D1-induced lamellipodia formation required Rac1 but not RhoG expression. Together, we conclude that the GEF Trio is responsible for lamellipodia formation through its N-terminal DH-PH domain in a Rac1-dependent manner during fibronectin-mediated spreading and migration.

The N-terminal DH-PH domain of Trio induces cell spreading and migration by regulating lamellipodia dynamics in a Rac1-dependent fashion.

Leukocyte transendothelial migration involves the active participation of the endothelium through the formation of apical membrane protrusions that embrace adherent leukocytes, termed docking structures. Using live-cell imaging, we find that prior to transmigration, endothelial docking structures form around 80% of all neutrophils. Previously we showed that endothelial RhoG and SGEF control leukocyte transmigration. In this study, our data reveal that both full-length Trio and the first DH-PH (TrioD1) domain of Trio, which can activate Rac1 and RhoG, interact with ICAM-1 and are recruited to leukocyte adhesion sites. Moreover, upon clustering of ICAM-1, the Rho-guanine nucleotide exchange factor Trio activates Rac1, prior to activating RhoG, in a filamin-dependent manner. We further show that docking structure formation is initiated by ICAM-1 clustering into ring-like structures, which is followed by apical membrane protrusion. Interestingly, we find that Rac1 is required for ICAM-1 clustering, whereas RhoG controls membrane protrusion formation. Finally, silencing endothelial Trio expression or reducing TrioD1 activity without affecting SGEF impairs both docking structure formation and leukocyte transmigration. We conclude that Trio promotes leukocyte transendothelial migration by inducing endothelial docking structure formation in a filamin-dependent manner through the activation of Rac1 and RhoG.

The Rho-guanine nucleotide exchange factor Trio controls leukocyte transendothelial migration by promoting docking structure formation.

Atherosclerosis preferentially develops at sites of disturbed blood flow. We tested the hypothesis that transglutaminase activity plays a role in plaque development at these locations.

Transglutaminase activity regulates atherosclerotic plaque composition at locations exposed to oscillatory shear stress.

Impaired endothelial barrier function results in a persistent increase in endothelial permeability and vascular leakage. Repair of a dysfunctional endothelial barrier requires controlled restoration of adherens junctions, comprising vascular endothelial (VE)-cadherin and associated β-, γ-, α-, and p120-catenins. Little is known about the mechanisms by which recovery of VE-cadherin-mediated cell-cell junctions is regulated. Using the inflammatory mediator thrombin, we demonstrate an important role for the Src homology 2-domain containing tyrosine phosphatase (SHP2) in mediating recovery of the VE-cadherin-controlled endothelial barrier. Using SHP2 substrate-trapping mutants and an in vitro phosphatase activity assay, we validate β-catenin as a bona fide SHP2 substrate. SHP2 silencing and SHP2 inhibition both result in delayed recovery of endothelial barrier function after thrombin stimulation. Moreover, on thrombin challenge, we find prolonged elevation in tyrosine phosphorylation levels of VE-cadherin-associated β-catenin in SHP2-depleted cells. No disassembly of the VE-cadherin complex is observed throughout the thrombin response. Using fluorescence recovery after photobleaching, we show that loss of SHP2 reduces the mobility of VE-cadherin at recovered cell-cell junctions. In conclusion, our data show that the SHP2 phosphatase plays an important role in the recovery of disrupted endothelial cell-cell junctions by dephosphorylating VE-cadherin-associated β-catenin and promoting the mobility of VE-cadherin at the plasma membrane.

The tyrosine phosphatase SHP2 regulates recovery of endothelial adherens junctions through control of β-catenin phosphorylation.

Inflammation is characterized by endothelium that highly expresses numerous adhesion molecules to trigger leukocyte extravasation. Central to this event is increased gene transcription. Small Rho-GTPases not only control the actin cytoskeleton, but are also implicated in gene regulation. However, in inflammation, it is not clear how this is regulated. Here, we show that the guanine-nucleotide exchange factor Trio expression is increased upon inflammatory stimuli in endothelium. Additionally, increased Trio expression was found in the vessel wall of rheumatoid arthritis patients. Trio silencing impaired VCAM-1 expression. Finally, we excluded that Trio-controlled VCAM-1 expression used the classical NFκB or MAP-kinase pathways, but rather acts on the transcriptional level by increasing phosphorylation and nuclear translocalization of Ets2. These data implicate Trio in regulating inflammation and provide novel targets for therapeutic purposes to treat inflammatory diseases such as rheumatoid arthritis.

The Rho-GEF Trio regulates a novel pro-inflammatory pathway through the transcription factor Ets2.

In aortic aneurysms the arterial vessel wall is dilated because of destruction of its integrity, which may lead to lethal vessel rupture. Chronic infiltration of inflammatory cells into the vessel wall is fundamental to aneurysm pathology. We aim to limit aneurysm growth by inhibition of inflammation and reducing endothelial cell (EC) activation with immunosuppressive drug azathioprine (Aza).

Immunosuppressive drug azathioprine reduces aneurysm progression through inhibition of Rac1 and c-Jun-terminal-N-kinase in endothelial cells.

Azathioprine and its metabolite 6-mercaptopurine (6-MP) are well established immunosuppressive drugs. Common understanding of their immunosuppressive properties is largely limited to immune cells. However, in this study, the mechanism underlying the protective role of 6-MP in endothelial cell activation is investigated. Because 6-MP and its derivative 6-thioguanosine-5'-triphosphate (6-T-GTP) were shown to block activation of GTPase Rac1 in T lymphocytes, we focused on Rac1-mediated processes in endothelial cells. Indeed, 6-MP and 6-T-GTP decreased Rac1 activation in endothelial cells. As a result, the compounds inhibited TNF-α-induced downstream signaling via JNK and reduced activation of transcription factors c-Jun, activating transcription factor-2 and, in addition, NF κ-light-chain-enhancer of activated B cells (NF-κB), which led to decreased transcription of proinflammatory cytokines. Moreover, 6-MP and 6-T-GTP selectively decreased TNF-α-induced VCAM-1 but not ICAM-1 protein levels. Rac1-mediated generation of cell membrane protrusions, which form docking structures to capture leukocytes, also was reduced by 6-MP/6-T-GTP. Consequently, leukocyte transmigration was inhibited after 6-MP/6-T-GTP treatment. These data underscore the anti-inflammatory effect of 6-MP and 6-T-GTP on endothelial cells by blocking Rac1 activation. Our data provide mechanistic insight that supports development of novel Rac1-specific therapeutic approaches against chronic inflammatory diseases.

Inhibition of GTPase Rac1 in endothelium by 6-mercaptopurine results in immunosuppression in nonimmune cells: new target for an old drug.

Chronic vascular inflammation is driven by interactions between activated leukocytes and the endothelium. Leukocyte β2-integrins bind to endothelial ICAM-1 (InterCellular Adhesion Molecule-1), which allows leukocyte spreading, crawling and transendothelial migration. Leukocytes scan the vascular endothelium for permissive sites to transmigrate suggestive for apical membrane heterogeneity within the endothelium. However, the molecular basis for this heterogeneity is unknown. Leukocyte adhesion induces ICAM-1 clustering which promotes its association to the actin-binding proteins FilaminB, α-Actinin-4 and Cortactin. We show that these endothelial proteins differentially control adhesion, spreading and transmigration of neutrophils. Loss of FilaminB, α-Actinin-4 and Cortactin revealed adapter-specific effects on a nuclear-to-peripheral gradient of endothelial cell stiffness. Conversely, increasing endothelial cell stiffness stimulates ICAM-1 function. We identify endothelial α-Actinin-4 as a key regulator of endothelial cell stiffness and of ICAM-1-mediated neutrophil transmigration. Finally, we found that the endothelial lining of human and murine atherosclerotic plaques shows elevated levels of α-Actinin-4. These results identify endothelial cell stiffness as an important regulator of endothelial surface heterogeneity and of ICAM-1 function which in turn controls adhesion and transmigration of neutrophils.

Article	Year
Inside-out regulation of ICAM-1 dynamics in TNF-alpha-activated endothelium. PloS one\| PubMed ID: 20596527	2010
The N-terminal DH-PH domain of Trio induces cell spreading and migration by regulating lamellipodia dynamics in a Rac1-dependent fashion. PloS one\| PubMed ID: 22238672	2012
The Rho-guanine nucleotide exchange factor Trio controls leukocyte transendothelial migration by promoting docking structure formation. Molecular biology of the cell\| PubMed ID: 22696684	2012
Transglutaminase activity regulates atherosclerotic plaque composition at locations exposed to oscillatory shear stress. Atherosclerosis\| PubMed ID: 22921425	2012
The tyrosine phosphatase SHP2 regulates recovery of endothelial adherens junctions through control of β-catenin phosphorylation. Molecular biology of the cell\| PubMed ID: 22956765	2012
The Rho-GEF Trio regulates a novel pro-inflammatory pathway through the transcription factor Ets2. Biology open\| PubMed ID: 23789107	2013
Immunosuppressive drug azathioprine reduces aneurysm progression through inhibition of Rac1 and c-Jun-terminal-N-kinase in endothelial cells. Arteriosclerosis, thrombosis, and vascular biology\| PubMed ID: 23950142	2013
Inhibition of GTPase Rac1 in endothelium by 6-mercaptopurine results in immunosuppression in nonimmune cells: new target for an old drug. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 24670805	2014
Actin-binding proteins differentially regulate endothelial cell stiffness, ICAM-1 function and neutrophil transmigration. Journal of cell science\| PubMed ID: 25107367	2014

Mark Hoogenboezem

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