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Filling the gaps
The endothelium in regulating vascular leakage and leukocyte extravasation
Schimmel, L.
Publication date
2018
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Citation for published version (APA):
Schimmel, L. (2018). Filling the gaps: The endothelium in regulating vascular leakage and
leukocyte extravasation.
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Department of Plasma Proteins, Molecular Cell Biology Lab, Sanquin Research and Landsteiner La-boratory, Academic Medical Center, University of Amsterdam, Plesmanlaan 125, Amsterdam 1066 CX,
The Netherlands. 2Department of Medical Biochemistry, Amsterdam Cardiovascular Sciences, Academic
Medical Center, Amsterdam, The Netherlands.
Manuscript submitted
Lilian Schimmel
1, Ivar Noordstra
1,
Aafke de Ligt
1, Jos van Rijssel
1, Jaap D.
van Buul
1& Vivian de Waard
2Rac1 activation by the
endothelial GEF Tiam1
marks sites for leukocyte
diapedesis through guiding
junctional membrane
ruffles
Abstract
During inflammation and immune surveillance, leukocytes cross the endo-thelial cell (EC) barrier in order to reach the site of infection. However, the specific location in the EC monolayer that leukocytes choose for diapedesis may not be determined by coincidence, but rather represent favored sites: transmigration ‘hotspots’. The exact components of such hotspots remain unclear, but it is suggested that ECs display cues such as chemokines and/ or adhesion molecules to attract leukocytes towards the hotspot. T-lympho-ma invasion and metastasis (Tiam1) is identified as a Rac1 specific guani-ne nucleotide exchange factor (GEF) that induces formation of membraguani-ne ruffles in fibroblast and NIH3T3 cells. Rac1 activity also drives formation of membrane ruffles in ECs, which are until now only studied in EC motility during angiogenesis, but never in leukocyte transendothelial cell migration (TEM).
In this study, we show that the inflammation-sensitive Rac1-GEF Tiam1 is able to locally induce endothelial membrane ruffles at the junctions. The formation of these ruffles is specific for Rac1 activation, but not restricted to Tiam1, since the Rac1-GEF TrioN also induces local EC membrane ruf-fles. These Rac1-driven junctional endothelial membrane ruffles functionally guide crawling leukocytes toward the preferred site of diapedesis and can therefore considered an essential part of the TEM hotspot.
Introduction
During inflammation and immune surveillance, leukocytes pass the endot-helial cell (EC) barrier in order to reach the site of infection by transendo-thelial migration (TEM). TEM is a process in which leukocytes follow the well-defined consecutive steps of rolling, firm adhesion and crawling, and finally diapedesis 1,2. Where previous studies focused on how these different
steps occur, little is known about the specific site on the endothelium that leukocytes choose for diapedesis. The favored location leukocytes take to exit the vasculature is presumably not random, but rather a preferred site, the so-called transmigration ‘hotspot’. The exact components that determine these hotspots remain unclear, but it is proposed that leukocytes use the cra-wling stage in order to find these hotspots 3. So far, several key determinants
of directed migration have been identified that appear to drive leukocytes to a preferred site of diapedesis: these comprise attraction towards an optimal concentration of chemokines (chemotaxis), density of adhesion molecules (haptotaxis), cellular stiffness (durotaxis) and the path of least resistance (tenertaxis) 3,4.
All of the above mentioned directionality components not only regula-te movement of the leukocyregula-tes, but some of them also affect the morphology of the ECs, such as stiffness 5. Over the last decades, it has become
well-es-tablished that small Rho GTPases are the major players in transducing ex-tracellular signals towards an inex-tracellular response of the actin cytoskeleton, thereby regulating morphology by means of cell adhesion, polarity, membra-ne protrusions and motility 6. Where activation of the GTPases Cdc42 and
Rac1 result in the formation of filopodia or lamellipodia, the GTPase RhoA is mainly involved in stress fiber formation and focal contacts 7–10. Because
the family of Rho GTPases affect major cellular processes, tight regulation of their activity by Rho-Guanine nuclueotide Exchange Factors (Rho-GEFs) and Rho GTPase Activating Proteins (Rho-GAPs) is necessary.
T-lymphoma invasion and metastasis (Tiam1) was identified as pro-oncogene that drives invasiveness of T-lymphoma cells 11. Later, Tiam1
was recognized to be a Rac1-specific GEF that induces formation of mem-brane ruffles in fibroblasts and NIH3T3 cells 12. Induction of polarized
mem-brane protrusions in astrocytes, through Tiam1-induced Rac1 activity, have a function in the process of wound healing 13.
For the activation of Rac1 by Tiam1, translocation of Tiam1 towards the plasma membrane by the N-terminal pleckstrin homology (PH) domain, and not the C-terminal PH domain or Discs-large homology region (DHR) domain, is essential 14.
So far, membrane ruffles in ECs are described to be involved in cell migration during development and angiogenesis. Formation of membrane
ruffles in ECs can be induced by several external stimulators like S1P, Ep-hrin-B1, or vascular endothelial growth factor (VEGF), resulting in phosp-horylation of the adaptor protein Crk for S1P and Ephrin-B1 or the GEF Vav2 for VEGF, and finally resulting in activation of their downstream target Rac1
15–17. Secondly, EC membrane ruffles surrounding leukocytes after firm
adhe-sion, are called docking structures or transmigratory cups (see 3 for a
detai-led overview of the different names for these structures).
A possible new role for membrane ruffles on ECs in the process of leukocyte TEM is investigated in this study. Using flow chambers, we reveal that constitutively active Tiam1-induced Rac1 activation results in junctional endothelial membrane ruffles, which guide leukocytes to the preferred site for diapedesis, as shown by high resolution immunofluorescent microscopy.
Results
Tiam1 is present in endothelial cells
Immunohistochemical staining for Tiam1 on ex vivo human aortic tissue reve-aled expression of Tiam1 in endothelial cells lining the large vessels like the aorta, and the microvascular arterioles (vasa vasorum externa) and venules (venous vasa vasorae) in the surrounding adventitia (Figure 1A). Immunop-recipitation (IP) of endogenous Tiam1 from cultured human umbilical vein endothelial cells (HUVECs) showed specific enrichment for Tiam1 compared to control (empty beads and IgG control) by Western blot analysis (Figure 1B) with a different Tiam1 antibody used for detection than for the IP. Ana-lysis of the Tiam1 IP was also performed using protein silver-staining to ex-clude any cross-reactivity between the two Tiam1 antibodies. In line with the previous results, the silver-stain analysis showed specific protein enrichment at the correct molecular weight of Tiam1 (160 kDa) that is absent in control IPs (Figure 1C). In order to prove specificity of the used Tiam1 antibody, HU-VECs were transfected with a HA-tagged constitutively active mutant Tiam1 construct containing the C-terminal 1199 amino acids (Tiam1-C1199-HA or C1199). Simultaneous detection of Tiam1 by anti-Tiam1 (green) and anti-HA (red) antibodies on Odyssey resulted in detection of exactly the same band, as shown by colocalisation (orange) in the merged picture (Figure 1D). Thus, Tiam1 is expressed in human ECs and can be specifically detected with available antibodies.
Endothelial expression of Tiam1 is inflammation sensitive
One of the hallmarks of chronic inflammation is increased extracellular ma-trix stiffness. To study the effects of substrate stiffness on Tiam1 expression in ECs, HUVECs and human aortic endothelial cells (HAECs) were cultured on different stiffness substrates and stimulated overnight with 10 ng/ml tu-mor necrosis factor-α (TNFα) or left untreated. Western blot analysis showed increased expression levels of Tiam1 upon increasing substrate stiffness. In addition, TNFα stimulation increased Tiam1 expression even when ECs were cultured on soft substrates, which under non-treated conditions sho-wed reduced Tiam1 expression (Figure 2A). In order to study the specificity of Tiam1 expression, regulated by stiffness or inflammatory factors such as TNFα or lipopolysaccharide (LPS), HUVECs were cultured on different stif-fness substrates, treated overnight with either 1 or 5 µg/ml LPS or 10 ng/ml TNFα. Western blot analysis for Tiam1 and intracellular adhesion molecule-1 (ICAM-1), known to be strongly upregulated by TNFα, showed that Tiam1 expression behaved similarly and was also increased by TNFα and not by LPS (Figure 2B). TNFα-mediated responses are regulated by the well-known inflammation regulating transcription factor Nuclear Factor kappa B (NFκB).
Figure 1
A
B
C
D
Empty beads Tiam1 IP TCL IP 250 Tiam1 Actin anti-HA anti-TIAM1IgG heavy chain
IgG light chain kDa 130 100 70 55 35 25 15 Tiam1 TCL Actin TCL IgG control beads
Empty beadsanti-T iam1 beads
anti-T iam1 beads
IgG control beadsEmpty beadsanti-T iam1 beads
IgG control beads
UntransfectedTiam1-C1 199-HA + - - -- - -+ + + + + 50 μm Aorta Tiam1 Tiam1 Zoom Arteriole Venule anti-TIAM1/anti-HA
Figure 1. Tiam1 in endothelial cells
(A) IHC staining for Tiam1 on ex vivo human aorta showing presence of Tiam1 in
ECs lining the large vessel (aorta) and also in the microvascular ECs in the adven-titia (arterioles and venules). (B) Immunoprecipitation of endogenous Tiam1 from
HUVECs with empty beads, anti-Tiam1 antibody (Santa Cruz) coated beads, and IgG control coated beads, signals detected with anti-Tiam1-DH antibody 33. (C)
Im-munoprecipitation of endogenous Tiam1 from HUVECs with empty beads, anti-Ti-am1 coated beads, and IgG control coated beads signal detected with Silverstain.
(D) Westernblot detection of Tiam1 protein in untransfected or Tiam1-C1199-HA
transfected HUVECs using Odyssey for simultaneous detection of HA-tag (red) and Tiam1 (green).
To investigate if Tiam1 expression is under control of NFκB , HUVECs were cultured on different stiffness substrates and treated with the NFκB signa-ling inhibitor SC514 in combination with TNFα stimulation. SC514 clearly
reduced ICAM-1, showing effectiveness of the NFκB inhibitor (Figure 2C). Moreover, TNFα-induced upregulation of Tiam1 at 25kPa stiffness is redu-ced in the presence of SC514 (Figure 2C). This indicates that TNFα-induredu-ced Tiam1 expression in ECs is under control of the NFκB pathway. Since incre-asing substrate stiffness can induce temporary NFκB signaling 18, this pa-thway could induce the stiffness-dependent expression of Tiam1. However, SC514 did not show any inhibitory effect on the stiffness-dependent Tiam1 expression. Depletion of the substrate stiffness-sensitive transcription factor yes-associated protein 1 (Yap) in HUVECs should reveal if Yap is involved in substrate stiffness-mediated Tiam1 expression. Yap was highly expressed in ECs cultured on plastic as compared to the 25kPa and 2 kPa substrates, thus indeed positively correlating with stiffness. However, silencing of Yap with siRNA was effective in decreasing Yap, but had no inhibitory effect on Tiam1 expression when compared to siCTRL transfected HUVECs (Figure 2D).
We conclude that expression of endothelial Tiam1 is up-regulated upon stimulation with TNFα via the NFκB pathway, but the stiffness-induced increase in Tiam1 expression is not regulated by the stiffness-sensitive tran-scription factor Yap or the NFκB signaling pathway.
Active Tiam1 results in a typical Rac1 phenotype of ECs
Overexpression of a constitutively active mutant Tiam1 construct containing the C-terminal 1199 amino acids (Tiam1-C1199-HA or C1199) in ECs resul-ted in the induction of ECs with an increase in cell size and linear vascular endothelial cadherin (VE-cadherin)-based cell-cell junctions (Figure 2E,F). Formation of linear VE-cadherin adherens junctions increases the transen-dothelial electrical monolayer resistance which relies on Rac1 activation by the GEF Trio 19. Moreover, staining for paxillin in C1199-positive ECs show
abundant presence of focal adhesions (Figure S1A). Quantification revealed a significant increase in the number of focal adhesions per cell, focal adhe-sion density and focal adheadhe-sion size in ECs overexpressing Tiam1-C1199-HA (Figure S1B-D). Both the increase in cell size and the increase in focal adhesions are typical phenotypes of enhanced Rac1 activation 20. Indeed,
the Rac1-activating capacity of the Tiam1-C1199-HA mutant is confirmed by Rac1 pulldown using biotin-tagged CRIB as a bait for active Rac1.
ECs expressing C1199 showed increased levels of active Rac1 (Figure 2G), in line with the observed EC morphology.
Thus, increased Tiam1 activity results in increased cell size, incre-ased focal adhesions and induction of linear VE-cadherin-bincre-ased cell-cell junctions, due to strong Rac1 activation by Tiam1.
Rac1 activity results in decreased PMN adhesion upon inflammation
Since endothelial Tiam1 expression is inflammation sensitive, and Rac1
Figure 2 A C E F G B D TNFα TNFα SC514 HUVEC Tiam1 Tiam1 Tiam1 Tiam1 ICAM1 Actin Actin HA VE-cadherin ICAM1 Actin Actin Actin TCL Rac1 TCL Active Rac1 Tiam1 TCL Yap HAEC Plastic Plastic + TNFαPlastic 25 kPa 25 kPa + TNFα25 kPa 2 kPa 2 kPa Plastic unstimulated1µg LPS
siCTRL siYAP siCTRL siYAP siCTRL
Untransfected
siYAP siCTRL siYAP siCTRL siYAP
5µg LPS10ng TNF unstimulated1µg LPS5µg LPS10ng TNF unstimulated1µg LPS5µg LPS10ng TNF
25 kPa 2 kPa Plastic 25 kPa 2 kPa
- + - + - + - + - + - +
Plastic 25 kPa 2 kPa
+ -- +- ++ -- +- +- ++ -- +- +- ++ TIAM1 C1199-HA TIAM1 C1199-HA 75μm Untransfected cell size (μ m 2) Untransfected 6000 *** 4000 2000 0 Cell size Tiam1-C1 199-HA
Figure 2. Inflammatory sensitive expression of Tiam1 in ECs
(A) Expression levels of Tiam1 in HUVEC and HAEC cultured on plastic, 25kPa or
2 kPa stiffness substrates, with and without overnight TNFα stimulation. (B)
Expres-sion levels of Tiam1 in HUVECs cultured on plastic, 25 kPa or 2 kPa stiffness sub-strates. Cells were unstimulated or stimulated overnight with 1µg/ml LPS, 5µg/ml LPS or 10 ng/ml TNFα. (C) Tiam1expression levels in HUVEC cultured on plastic,
25kPa or 2 kPa stiffness substrates, treated with NFκB signalling inhibitor SC514 with or without overnight TNFα strimulation. (D) Expression levels of Tiam1 in
HUVECs transfected with either control or Yap siRNA, cultured on plastic, 25 kPa or 2 kPa stiffness substrates and unstimulated or stimulated overnight with TNFα.
(E) Transfection of Tiam1-C1199-HA in HUVECs display increased cell size of HA
positive cells. (F) Quantification of cell size of untransfected and Tiam1-C1199-HA
transfected HUVECs. (G) Overall Rac1 activity upon Tiam1-C1199-HA expression
in HUVECs determined by a Rac1 pulldown assay with biotin-tagged CRIB as a bait. Western blot shows Tiam1 expression level, active and total Rac1.
plays a dominant role in inflammation-mediated leukocyte TEM 21–24, we
stu-died the effect of Tiam1-driven Rac1 activation on leukocyte TEM. Freshly isolated and activated polymorphonuclear leukocytes (PMNs) were perfused over a monolayer of control or C1199-transfected ECs with a speed of 0.8
Figure 3
A B
C
D
HA E-selectin ICAM1 PECAM E-selectin Zoom ICAM1 Zoom Merge +HA
Tiam1 C1 199-HA Control 75μm Control Tiam1-C1 199-HA Tiam1 HA E-selectin ICAM1 VCAM Actin Control Tiam1-C1 199 *** ICAM1 filopodia / μ m 2 0.00 0.05 0.10 0.15 ICAM1 filopodia 20 Tiam1-C1199 Adhesive PMNs Time (min) 0 0 5 10 15 5 10 15 20 Control 25
Figure 3. Effects of active Tiam1 on PMN adhesion
(A) Number of adhesive PMNs over time on control (black line) and on
Tiam1-C1199-HA (grey line)-transfected ECs under physiological flow rates of 0.8 dyn/ cm2. (B) Western blot of control HUVECs or transfected with Tiam1-C1199-HA
treated overnight with TNFα, showing expression of the construct by HA and Tiam1 detection. Total expression levels of the adhesion receptors E-selectin, ICAM1 and VCAM1 are decreased in Tiam1-C1199-HA expressing ECs. (C)
Immunofluores-cent staining on control or Tiam1-C1199-HA expressing HUVECs after overnight stimulation with TNFα for HA (blue) and adhesion molecules; E-selectin (green), ICAM1 (red) and PECAM (white), including zooms of E-selectin and ICAM1 fi-lopodia. Images are a maximum intensity Z-projection. (D) Quantification of
ICAM-1 filopodia/µm2 present on control and TiamICAM-1-CICAM-1ICAM-199-HA transfected ECs treated overnight with TNFα.
dyn/cm2. Interestingly, quantification of the number of adhesive PMNs on the apical surface of ECs showed a significant decrease upon expression of active Tiam1 (Figure 3A). The decrease in PMN adhesion is explained by a reduction in adhesion receptor expression levels, as was revealed by Wes-tern blot analysis of total cell lysates from non-transfected control ECs and C1199-overexpressing ECs. Upon overexpression of active Tiam1, expres-sion levels of the adheexpres-sion molecules E-selectin, ICAM-1 and VCAM-1 were all reduced (Figure 3B). Immunofluorescent staining for Tiam1, E-selectin, ICAM-1 and PECAM-1 on non-transfected control ECs or
Figure 4 Tiam1-C1199 Control Control 1 A B C H F G E Tiam1 C1 199-HA
Actin HA Zoom Actin Zoom
HA VE-cadherin Control Ctrl-Ctrl % TEM cells Ctrl-C1 199 C1199-C1 199 80 60 40 20 0 Transmigration 60 µm 80 µm + + + + + + - - -- -1 2 3 4 5 6
1 3:55 min 7:30 min 11:20 min 19:00 min
D
10 µm
Fluorescence intensity actin
0.2 0.0 0.4 0.6 0.8 1.0
Line scan position Control Tiam1-C1199 Fluorescence intensity TIam1-C1 199 0.2 0.0 0.4 0.6 0.8 1.0
Line scan position Tiam1-C1199 Fluorescent intensity 0.0 0.2 0.4 0.6 0.8 *** ** *** Junctional Actin Ctrl-Ctrl Ctrl-C1 199 C1199-C1 199
Figure 4. Effect of active Tiam1 on leukocyte diapedesis step
(A) Still from PMN TEM under physiological flow on ECs expressing
Tiam1-C1199-HA. White dashed line marks the border between control and Tiam-C1199-HA ex-pressing cells. Red stars indicate sites of PMN TEM, all at the border of control and Tiam1-C1199 cells. (B) Quantification of the percentage of PMNs that undergo TEM
through the junction of two control cells (ctrl-ctrl), a control and a Tiam1-C1199 cell (ctrl-C1199) or two Tiam1-C1199 cells (C1199-C1199). (C) Zoomed still from
the flow assay showing the migration tracks of PMNs after TEM underneath the ECs. White dashed line indicates Tiam-C1199 expressing cells. (D) Zooms from
transmigrating PMNs at the moment of breaching the EC layer. White dashed line indicates the border between control (-) and Tiam1-C1199 (+) cells. Red dashed line indicated site of PMN pseudopod formation always in the direction underneath the Tiam-C1199 expressing ECs, and is followed by PMN migration towards direction of the pseudopod. (E) Immunofluorescent staining for HA (green), F-actin (red) and
VE-cadherin (white) on control ECs and Tiam1-C1199 transfected ECs. White box indicates area of zoom and images are a maximum intensity Z-projection. White dashed line indicates site of line scan for measuring fluorescence intensity of (F)
Tiam1-C1199-HA and (G) actin in which the red dashed line indicates VE-cadherin
staining and that peaks at the cell-cell junction. (H) Junctional F-actin fluorescence
intensity quantified within 7.2 µm from the VE-cadherin positive cell-cell junctions. ted ECs showed a clear decrease of total expression levels of the adhesi-on receptors E-selectin and ICAM-1 in the C1199-positive cells (Figure 3C). Quantification of the number of ICAM-1-positive filopodia/µm2 (zooms of Fi-gure 3A), showed that not only total ICAM-1 expression is decreased, but also the formation of ICAM-1-rich filopodia is significantly diminished upon expression of active Tiam1 (Figure 3D). Taken together, Rac1 activation by Tiam1 results in decreased expression of endothelial E-selectin, ICAM-1 and VCAM-1, with the functional consequence of decreased adhesion of PMNs to the apical surface of these enlarged ECs under inflammatory conditions.
Rac1 activation directs PMNs to site of diapedesis
Although the number of adhesive PMNs on the apical surface of C1199 ex-pressing cells is decreased, PMN are still able to cross the ECs and PMNs showed specific preference for diapedesis at certain sites, namely the juncti-ons between control and C1199 cells (Figure 4A; red stars).
In a mixed monolayer of control and C1199 transfected ECs, 60% of PMNs breached the EC layer at a control-C1199 junction, while the other 40% is equally distributed over control-control and C1199-C1199 junctions (Figure 4B). Migration tracks of PMNs after diapedesis (starting at the junc-tion between control-C1199 ECs) show that the PMNs are retained under-neath the enlarged C1199-positive ECs (Figure 4C). Taking a closer look at the diapedesis stage at the control-C1199 junction, the probing podosome of PMNs underneath the EC layer (Figure 4D, red dotted line) is always pointed towards the C1199-positive EC, and determines the direction of migration underneath the ECs. Taken together, this indicates that local and junctional increased endothelial Rac1 activity attracts PMNs and therefore may act as a guide for PMNs to transmigrate.
Immunofluorescent staining of Tiam1 (C1199), F-actin and VE-cad-herin showed that Rac1 activation via C1199 overexpression induced the formation of thick cortical actin bundles compared to control cells (Figure 4E). C1199 mainly localized at the cell-cell junctions, as determined by a per-pendicular line scan across the cell-cell junction indicated by VE-cadherin. Clearly, Tiam1 fluorescence intensity peaked at the same site as VE-cad-herin (red dotted line) (Figure 4F). Similar line scans were made for F-actin fluorescence intensity, revealing increased presence of F-actin in C1199 ECs compared to control ECs (Figure 4G), particularly at the cell-cell junction, indicated by VE-cadherin (red dotted line). Quantification of junctional actin at the cell-cell borders, showed that upon Rac1 activation, junctional actin
Figure 5 B A D C 0:00 10 μm 0:00
Nuclei Tiam1-C1199-HA Actin VE-cadherin
0:50 2:00 2:20 1:00 3:00 3:10 Membrane protrusions No Yes % of tranmigrating PMNs 0 20 30 40 50 0.0 μm Tiam1-C1 199-HA Actin VE-cadherin Actin 30 μm 0.5 μm 1.0 μm 1.5 μm 2.0 μm 2.5 μm X Y Z
Figure 5. Endothelial junc-tional membrane ruffling (A) Immunofluorescent
stai-ning for HA (green), F-actin (red), VE-cadherin (white) and DNA (blue) on HUVECs transfected with Tiam1-C1199-HA after overnight TNFα stimulation. Filled white arrowheads indica-te the F-actin present at the cell-cell junction between a Tiam1-C1199-HA expressing cell and a control cell, where the open white arrowheads indicate the F-actin present at the junction between two control cells. Panel shows Z-stack from basal (0.0 µm) towards apical (2.5 µm) from left to right, respectively. Arrowheads indicate pre-sence of F-actin rich membrane ruffles in the different focal planes. (B) Stills from
PMN TEM under flow showing presence of endothelial membrane ruffles, indicated by white arrowheads. The membrane ruffles are present at the site of diapedesis already before PMN adhesion and indicate the spot where the PMN will breach the EC layer. (C) Quantification of the number of TEM events that showed endothelial
membrane ruffles prior to PMN TEM at the site of diapedesis. (D) 3D projection of
nuclei (blue), Tiam1-C1199-HA (green), F-actin (red) and VE-cadherin (white) on the junction of a control and Tiam1-C1199-HA expressing EC showing the enrich-ment and protrusion towards the apical site of F-actin.
significantly, and correlated to Rac1 activity, increases (Figure 4H). Taken together, the amount of junctional actin does not directly correlates with the TEM hotspots, but rather the differences on junctions between normal and enhanced Rac1-activated ECs creates an attractive TEM hotspot.
Rac1 activation by Tiam1 results in formation of junctional endothelial membrane ruffles
The increase of junctional actin is not only due to formation of parallel F-ac-tin bundles lining the cell-cell junctions. Z-stack confocal images showed that, especially at the junctions between control and C1199 ECs, the actin cytoskeleton of the C1199 EC is projected towards the apical surface (Figu-re 5A, from left to right going from basal to apical plane). These endothelial membrane ruffles extended into the luminal space up to 2.5 µm above the basal EC focal plane at sites of control-C1199 cellular junctions (filled white arrowheads).
Compared to the border of two control cells, which showed paral-lel actin bundles instead of membrane ruffles at the cell-cell junctions, that protrude only up to 1.0 µm above the basal EC focal plane (open white ar-rowheads). Thus, enhanced Rac1 activation by Tiam1 at the junctional sites induced membrane ruffling.
In order to address involvement of these junctional membrane ruf-fles in the process of leukocyte TEM, physiological flow assays of control HUVECs were scored for the presence of membrane ruffles at the site of and prior to PMN diapedesis. By means of DIC microscopy, stills showed endothelial membrane ruffles (white arrowheads) present prior to and at sites of PMN diapedesis, illustrated by two representative examples (Figure 5B). Quantification of multiple TEM events showed an increased trend of PMN TEM at sites where endothelial membrane ruffles are present, but presence of membrane ruffles is not indispensable for leukocyte TEM.
To visualize the effects of Rac1 activation by Tiam1 on the formation of endothelial membrane ruffles at junctions, a 3D projection of a detailed Z-stack was made. Here, red represents the F-actin which is more abundant and protrudes higher on the junctions between a control and C1199 (green) cell, forming membrane ruffles (Figure 5D, filled arrow). White represents VE-cadherin, showing the cell-cell junctions and visualizing the lack of thick and protruding F-actin membrane ruffles on the border of two control cells (Figure 5D, open arrow).
Figure 6 A B Transmigration D Transmigration 0.0 μm Control EHT Tiam1-C1 199-HA Actin VE-cadherin Tiam1-C1 199-HA Actin VE-cadherin Actin Actin 0.5 μm 1.0 μm 1.5 μm 2.0 μm 2.5 μm Control EHT TEM cells 20 15 10 5 0 % TEM cells Adhesive PMNs 0 20 40 60 80 Ctrl-Ctrl Ctrl-TrioN TrioN-T rioN Adhesion C 0 0 5 10 15 20 20 40 60 80 100 Time (min) TrioN Control E Inflammation Inflammation Rac1 Inflammation Rac1 Rac1
Figure 6. Specific Rac1 activity is essential for ruffle formation
(A) Immunofluorescent staining for HA (green), F-actin (red), VE-cadherin
(whi-te) and DNA (blue) on HUVECs transfected with Tiam1-C1199-HA after overnight TNFα stimulation and treated for 30 minutes with control H20 (top panel) or 50 µM EHT Rac1 inhibitor (bottom panel). Filled white arrowheads indicate the F-actin present at the cell-cell junction between a Tiam1-C1199-HA expressing cell and a control cell, where the open white arrowheads indicate the F-actin present at the junction between two control cells. Panel shows Z-stack from basal (0.0 µm)
to-wards apical (2.5 µm) from left to right, respectively where treatment with EHT reduces the formation of extending membrane ruffles. (B) Quantification of the
num-ber of PMN that have transmigrated under physiological flow through ECs trans-fected with Tiam1-C1199-HA and pre-treated with EHT Rac1 inhibitor or not as control. (C) Number of adhesive PMNs over time on control (black line) and on
Tri-oN-GFP (grey line)-transfected ECs under physiological flow rates of 0.8 dyn/cm2.
(D) Quantification of the percentage of PMNs that undergo TEM through the
juncti-on of two cjuncti-ontrol cells (ctrl-ctrl), a cjuncti-ontrol and a TrioN cell (ctrl-TrioN) or two TrioN cells (TrioN-TrioN) in a physiological flow PMN TEM assay with TrioN transduced ECs. (E) Scheme showing the effects of inflammation (shading underneath ECs) on
ECs and subsequently leukocyte TEM. Red lines represent cortical actin (left panel). Under non-inflamed conditions, ECs do not form strong cortical actin bundles and membrane ruffles, leaving a rather smooth surface for crawling leukocytes (second panel). Upon local inflammation, ECs form high Rac1-driven membrane ruffles on junctions between high- and low-Rac1 cells, forming a physical barrier for crawling leukocytes which enhances diapedesis (third panel). However, on junctions between two high-Rac1 cells, actin branches also induce formation of membrane ruffles, but these types of ruffles, consisting from two cells, do not provide leukocytes with the physical benefit for diapedesis (fourth panel).
Specific activation of Rac1 and no other GTPases is necessary for for-mation of junctional endothelial membrane ruffles
In order to confirm that solely Rac1 activation and no other GTPases upon C1199 expression is responsible for formation of the junctional membrane ruffles, confocal microscopy on HUVECs transfected with C1199, and trea-ted with the Rac1 inhibitor EHT was performed. Immunofluorescent staining for Tiam1, F-actin and VE-cadherin showed reduced membrane ruffles pre-sent in EHT-treated ECs (bottom panel) compared to control treated (top panel) C1199-expressing ECs (Figure 6A). Where control C1199 ECs again extended F-actin positive membrane ruffles up to 2.5 µm into the apical lu-men at cell-cell junctions between control and C1199 cells (indicated by filled white arrowheads), Rac1 inhibition by EHT completely abrogated the forma-tion of membrane ruffles (indicated by open white arrowheads).
To study if Rac1 inhibition would affect PMN transmigration, C1199-transfected HUVECs pre-treated with EHT to reduce formation of junctional membrane ruffles were used for PMN TEM under physiological flow. Upon reduction of Rac1 activity by EHT, there is a clear inhibition in the total number of TEM events (Figure 6B). However, it should be kept in mind that Rac1 activation by another GEF, Trio, enhances PMN TEM via increased ICAM-1 clustering upon leukocyte adhesion 24. Both pathways of
membrane ruffle formation and ICAM-1 clustering require Rac1 and both will be blocked by EHT, indicating that de strong reduction of PMN TEM upon
EHT treatment is presumably a combination of reduced membrane ruffling and reduced ICAM-1 clustering.
Since Rac1 activation enhances EC membrane ruffling, the questi-on remains if this is a Tiam1 specific feature. As previously indicated, the GEF Trio is another major Rac1 activator in ECs. The effect of enhanced Trio activity on junctional endothelial membrane ruffling was determined by using overexpression of TrioN. ECs transduced with TrioN displayed a simi-lar Rac1 phenotype as was observed for C1199-transfected ECs: increased cell size and linearized VE-cadherin cell-cell-junctions and Rac1 activation (data not shown) 19,25. Adhesion of PMNs on TrioN expressing ECs is not
reduced as compared to control ECs (Figure 6C). Because TrioN overex-pression does not affect exoverex-pression levels of adhesion molecules like ICAM-1 25, this shows that elevated Rac1 activation itself is not directly involved in
regulating adhesion molecule expression levels like what was observed with C1199 overexpression. Quantification of leukocyte diapedesis sites, reve-aled that PMNs transmigrate preferentially at sites of control-TrioN cellular junctions (75%) (Figure 6D). The remaining TEM events occurred for 20% at TrioN-TrioN cellular junctions and the remaining 5% of PMNs crossed the EC layer through control-control cellular junctions. Taken together, activa-tion of endothelial Rac1 either by Tiam1 or TrioN results in the formaactiva-tion of local membrane ruffles at the junctions extending into the apical lumen. The difference in junctional membrane ruffling between cells seems to guide transmigrating PMNs towards the preferred site for diapedesis, the so-called transmigration hotspot (Figure 6E).
Discussion
Here we show that the Rac1 specific GEF Tiam1 is expressed in the vascu-lature, especially in ECs. Tiam1 protein expression is upregulated by pro-in-flammatory stimuli, such as increased matrix stiffness and TNFα, via different signalling pathways. The TNFα -induced Tiam1 upregulation depends on the NFκB signalling pathway, whereas the stiffness-dependent upregulation of Tiam1 does not involve the stiffness-sensitive transcription factor Yap, and needs to be further investigated.
The key finding of this study is that differences Rac1 activity of adja-cent ECs result in diverse junctional membrane ruffles that facilitate leuko-cyte TEM. Actually, it seems that Rac1-driven junctional membrane ruffles mark the site for leukocyte diapedesis, making it tempting to speculate that these membrane ruffles are part of the so-called transmigration hotspot. In-terestingly, most leukocytes transmigrated in between ECs with high and low Rac1 activation, as opposed to cells with equally low or equally high Rac1 activation. We propose that the inequality of EC membrane ruffles on one cell and no ruffles on the adjacent cell creates a physical benefit for PMNs to facilitate diapedesis (Figure 6E). However, the concept of Rac1-driven membrane ruffles is not specific for Tiam1, since TrioN showed the same phenomenon, revealing that high levels of active Rac1 are essential in the formation of junctional endothelial membrane ruffles. The question remains if such heterogeneity in ECs activation also exists in vivo.
A difference between Tiam1- and Trio-mediated Rac1 activation in ECs, is that Tiam1 reduced ICAM-1 expression at the cell surface and the-reby negatively influences PMN adhesion under flow conditions, while Tri-oN-induced Rac1 activation is known to enhance ICAM-1 clustering upon leukocyte binding 24, and has no effects on expression levels of adhesion
molecules 25. This demonstrates that elevated Rac1 activation itself is not
directly involved in regulating adhesion molecules expression levels and that different Rac1-GEFs are only partially redundant. Moreover, the amount of adhesion molecules, such as ICAM-1, present on ECs did not determine the amount of membrane ruffles that are formed. However, how Tiam1 activity results in reduced ICAM-1 surface expression remains to be elucidated, but determination of ICAM-1 internalization rates upon Tiam1-C1199 expression could be a first step to answer this question.
Formation of cell-cell junctions normally inhibit the formation of membrane ruffles, due to contact inhibition and physical restriction of cells to spread 26. Yet, in epithelial cells, engagement of E-cadherin at cell-cell
junctions results in increased Rac1 and Cdc42 activity to promote junction assembly 27–29. Although ECs do not express E-cadherin, it has been shown
that non-junctional VE-cadherin is able to induce Cdc42-driven membrane
protrusions, to restore EC junction barrier 30. This suggests that even though
contact inhibition normally reduces the formation of membrane ruffles neces-sary for cell spreading, engagement of cadherins could induce Rac1 activity specifically at the cell border in order to form junctional membrane ruffles, to promote leukocyte TEM.
Although the presence of Rac1-induced membrane ruffles in ECs has been shown before during angiogenesis 15,16, their function in leukocyte TEM
at cell-cell junction regions is completely novel. To what extent endothelial junctional membrane ruffles play a role in guiding leukocytes towards the right site for diapedesis in vivo remains to be further elucidated. However, possible heterogeneity of EC activation at early onset of disease is to be expected and may be supported by an inflammatory disease of the arterial vessel wall, namely atherosclerosis. During development of atherosclerotic plaques, macrophage influx is mainly observed in the shoulder region of the atherosclerotic plaque, as opposed to on top of the plaque 31,32. The shoulder
region is the border between the non-inflamed and inflamed (EC) area and there is a possible higher heterogeneity of ECs and formation of asymmetri-cal junctional membrane ruffles in this region to promote leukocyte extrava-sation.
In conclusion, we show that the inflammatory sensitive Rac-GEF Tiam1 is able to induce Rac1-driven junctional endothelial membrane ruffles. These Rac1-driven membrane ruffles mark the preferred diapedesis site and thereby attract crawling leukocytes towards this site and can therefore be considered to be part of the transmigration hotspot. Using the presence of these membrane ruffles to predict where leukocytes will cross the junction will assist in further unravelling the composition of the transmigration hot-spot.
Experimental Procedures
DNA and RNA constructsTiam1-C1199-HA constitutively active mutant was kind gift of John Collard and was microporated into HUVECs for expression using Neon Transfecti-on System (ThermoFisher) according to manufacturer’s protocol. TrioN-GFP adenovirus was produced and used as described in 25. siRNA for human
Yap1 was purchased from Sigma Aldrich Mission esiRNA (Cat#EHU113021).
Antibodies
Monoclonal mouse antibodies against actin (Cat#A3853) (1:1000 for WB), and against HA (Cat#H3663) (1:1000 for WB), and polyclonal rabbit anti-body against HA (Cat#H6908) (1:300 for IF) were purchased from Sigma. Polyclonal rabbit antibodies against ICAM-1 (Cat#SC-7891) (1:1000 for WB) and against Tiam1 (Cat#SC-872) (1:1000 for WB), and polyclonal goat antibody against VCAM1 (Cat#SC-1504) (1:1000 for WB) were purchased from Santa Cruz. Monoclonal mouse antibodies against VE-cadherin AF647 (Cat#561567) (1:200 for IF), against PECAM (Cat#561654) (1:500 for IF), and against Rac1 (Cat#610651) (1:1000 for WB) were purchased from BD Biosciences. Monoclonal mouse antibody against ICAM-1 FITC BA20) (1:200 for IF), and polyclonal goat antibody against E-selectin (Cat#B-BA18) (1:100 for IF and 1:1000 for WB) were purchased from R&D systems. Polyclonal rabbit antibody against Yap1 (Cat#GTX129151) (1:1000 for WB) was purchased from GeneTex. Polyclonal rabbit antibody against Tiam1-DH (1:100 for WB) was a kind gift of John Collard 33. Alexa Fluor 555
Phalloi-din (Cat#PHDH1) (1:500 for IF) was purchased from Cytoskeleton. Hoechst 33342 (Cat#H-1399) (1:25000 for IF) was purchased from Molecular probes. Secondary HRP-conjugated goat anti-mouse (Cat#P0447) (1:5000 for WB), swine anti-rabbit (Cat#P0399) (1:5000 for WB) antibodies were purchased from Dako. Secondary donkey antibody for Odyssey against Rabbit-IR800 (Cat#926-32213) and Mouse-IR800 (Cat#926-32212) were both from LI-COR Biosciences. All antibodies were used according to manufacturer’s protocol.
Cell cultures and treatments.
Pooled human umbilical vein endothelial cells (HUVECs) perchased from Lonza (P1012, Cat # C2419A), were cultured until passage 7 on fibronec-tin (FN)-coated dishes in EGM-2 medium, supplemented with singlequots (Lonza). All cells were cultured at 37°C and 5% CO2. Rac1 inhibition was obtained by treatment with 50 µM EHT (Sigma) for < 30 minutes prior to experiment.
PMN isolation
Polymorphonuclear cells (PMNs) were isolated from whole blood derived from healthy donors in sodium heparin tubes. Whole blood was diluted (1:1) with 10% (v/v) TNC in PBS. Diluted whole blood was pipetted carefully on 12,5 ml Percoll (room temperature) 1.076 g/ml. Tubes were centrifuged (Ro-tanta 96R) at 2000 rpm, slow start, low brake for 20 minutes. After erythrocy-te lysis in an ice-cold isotonic lysis buffer (155 mM NH4CL, 10 mM KHCO3, 0.1 mM EDTA, pH7.4 in Milli-Q (Millipore)), PMNs were centrifuged at 1500 rpm for five minutes at 4°C, incubated once with lysis buffer for 5 minutes on ice, centrifuged again at 1500 rpm for five minutes at 4°C, washed once with PBS, centrifuged again at 1500 rpm for five minutes at 4°C and resuspended in N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES)-buffer pH7.4 (20 mM HEPES, 132 mM NaCl, 6 mM KCL, 1 mM CaCL2, 1 mM MgSO4, 1.2 mM K2HPO4, 5 mM glucose (all from Sigma-Aldrich), and 0.4 % (w/v) human serum albumin (Sanquin Reagents) and kept at room tempe-rature for not longer than four hours until use. PMN counts were determined by cell counter (Casey).
PMN TEM under physiological flow
150.000 HUVECs transfected as indicated, were cultured per channel in a FN-coated Ibidi µ-slide VI0.4 (Ibidi) the day before the experiment was exe-cuted and stimulated overnight with 10 ng/ml TNFα (Peprotech). Freshly iso-lated PMNs were resuspended at 1*106 cells/ml in HEPES medium pH7.4 and were activated for 30 minutes at 37ºC. Ibidi flow chambers were con-nected to a perfusion system and exposed to 0.5 ml/minute HEPES medium pH7.4 shear flow for 5 minutes (0.8 dyne/cm2). PMNs were subsequently injected into the perfusion system and PMN-endothelial interactions were recorded for 20 minutes with an interval of 5 seconds by a Zeiss Observer Z1 microscope. All live imaging was performed at 37°C in the presence of 5% CO2.
Western blotting
For total cell lysates, cells were washed once with PBS+/+ (1mM CaCl and 0.5 mM MgCl), and lysed with 95°C SDS-sample buffer containing 4% β-me-capto-ethanol. Samples were boiled at 95°C for 5-10 minutes to denature proteins. Proteins were separated on 10% SDS running gel in running buffer (200 mM Glycine, 25 mM Tris, 0.1% SDS (pH8.6)), transferred to nitrocellu-lose membrane (Thermo Scientific Cat#26619) in blot buffer (48 nM Tris, 39 nM Glycine, 0.04% SDS, 20% MeOH) and subsequently blocked with 5% (w/v) milk (Campina) in Tris-buffered saline with Tween 20 (TBST) for 30 mi-nutes. The immunoblots were analyzed using primary antibodies incubated overnight at 4°C and secondary antibodies linked to horseradish peroxidase (HRP) (Dako, Aligent Tochnologies) or IR680/800 dye (LI-COR Biosciences),
after each step immunoblots were washed 4x with TBST. HRP signals were visualized by enhanced chemiluminescence (ECL) (Thermo Scientific) and light sensitive films (Fujifilm), IR signals were visualized with Odyssey.
Confocal laser scanning microscopy and image analysis
Immunofluorescent staining was in general performed on HUVECS cultu-red on 12 mm glass coverslips coated with 5 µg/ml FN and treated with or without o/n TNFα (10ng/ml) (Peprotech), washed with PBS+/+(1mM CaCl2 and 0.5mM MgCl2), fixated in 4% PFA (Merck), blocked for 30 min with 2% BSA (Affimetrix) and mounted in Mowiol4-88/DABCO solution. Z-stack ima-ge acquisition was performed on a confocal laser scanning microscope (Lei-ca SP8) using a 63x NA 1.4 oil immersion objective. 3D reconstruction of Z-stack was made with LasX software (Leica). ICAM1 positive filopodia and Paxillin positive focal adhesions where quantified using ImageJ 1.51p. Maxi-mum projections of fluorescent images were subjected to Gaussian blur and Unsharp filtering, followed by automated ‘Max entropy’ thresholding. Next, a watershed-based segmentation was applied and particles were detected using particle analysis with a minimal size cut-off of 8 pixels (0,18 µm/pixel). Junctional actin enrichment was quantified using ImageJ 1.51p. VE-cadherin labeling was used to visualize cell-cell junctions. 30 pixel (0,18 µm/pixel) width line was drawn on junction of interest and cumulative fluorescent actin signal was measured and normalized by total area.
Statistics
Data in graphs are represented as mean ± SEM. IHC and immunofluores-cent images are representatives. Comparisons between the indicated con-ditions were made in Prism Graph-Pad using unpaired T-test. P values for results are: n.s. P>0.05, * P≤ 0.05, ** P≤0.01, *** P≤0.001.
Acknowledgements
We kindly thank Bram Piersma, Matrix Research Group, Department of Pa-thology and Medical Biology, University Medical Center Groningen, Univer-sity of Groningen, Groningen, Netherlands for the gift of Yap antibody and Yap siRNA. We sincerely thank Prof. Dr. Peter Hordijk for critically reading the manuscript.
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Figure S1
HA Actin Paxillin Actin Zoom Paxillin
TIAM1 C1 199-HA Untransfected 75μm A B C D TIAM1 C1199-HA Untransfected TIAM1 C1199-HA Untransfected TIAM1 C1199-HA Untransfected 150 *** *** *** 100 50 # Focal Adhesions / cell # Focal Adhesions / μm 2 Focal Adhesion size ( μm 2)
Focal Adhesions per cell Focal Adhesions per μm2 Focal Adhesion size
0 0.000 0.0 0.5 1.0 1.5 0.005 0.010 0.015 0.020 0.025
Supplemental figures
Figure S1. Matrix adhesion of ECs upon Tiam1 activation
(A) Immunofluorescent staining for HA (green), F-actin (red), Paxillin (white) and
DNA (blue) on untransfected or Tiam1-C1199-HA transfected HUVECs after over-night TNFα stimulation. Quantification of the focal adhesions based on Paxillin stai-ning showing (B) the number of focal adhesions present per EC, (C) the density of
focal adhesions per µm2 and (D) the average size of the focal adhesion on
untrans-duced control ECs compared to Tiam1-C1199-HA expressing ECs.
