Filling the gaps: The endothelium in regulating vascular leakage and leukocyte extravasation - Thesis (complete)

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Filling the gaps

The endothelium in regulating vascular leakage and leukocyte extravasation

Schimmel, L.

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2018

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Final published version

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Schimmel, L. (2018). Filling the gaps: The endothelium in regulating vascular leakage and

leukocyte extravasation.

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Filling the gaps

The endothelium in regulating

vascular leakage and leukocyte

extravasation

Lilian Schimmel

Filling the gaps: The endothelium in regulating vascular leakage and leukocyte extravasation Lilian Schimmel

Uitnodiging

voor het bijwonen van de

openbare verdediging van

het proefschrift

Filling the gaps: The

endothelium in regulating

vascular leakage and

leukocyte extravasation

door

Lilian Schimmel

Vrijdag 15 juni 2018

om 14:00 uur

Agnietenkapel

Oudezijds Voorburgwal

231, Amsterdam

Na afloop bent u van

harte uitgenodigd voor de

receptie ter plaatse

Lilian Schimmel

paranimfen

Mark Hoogenboezem

Simon Tol

Filling the gaps

The endothelium in regulating

vascular leakage and leukocyte

extravasation

ISBN: 978-94-6233-962-0

The studies performed in this thesis were performed at the Department of Plasma Proteins, Molecular Cell Biology Lab, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of

Amsterdam, The Netherlands and financially supported by the Rembrandt Institute of Cardiovascular Science.

Printing of this thesis was financially supported by Sanquin Research and Vasculitis Stichting.

Photo cover by Willem Schimmel Layout by Ivar Noordstra

Printed by Gildeprint © 2018 by Lilian Schimmel All rights reserved

Filling the gaps

The endothelium in regulating vascular leakage and leukocyte extravasation

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam op gezag van de Rector Magnificus

prof. dr. ir. K.I.J. Maex

ten overstaan van een door het College voor Promoties ingestelde commissie, in het openbaar te verdedigen in de Agnietenkapel

op vrijdag 15 juni 2018, te 14.00 uur

door

Lilian Schimmel

Promotiecommissie:

Promotor: prof. dr. P.L. Hordijk Universiteit van Amsterdam

Copromotores: dr. J.D. van Buul Universiteit van Amsterdam

dr. V. de Waard AMC-UvA

Overige leden: prof. dr. T.W.J. Gadella Universiteit van Amsterdam prof. dr. C.J.M de Vries Universiteit van Amsterdam

prof. dr. E. Lutgens Universiteit van Amsterdam

dr. J.C. Sluimer Maastricht University

prof. dr. A. Sonnenberg Leiden Academic Centre for

Drug Research

“All we know is still infinitely less than all that remains unknown”

Table of contents

Leukocyte transendothelial migration: A local affair F-actin rich contractile endothelial pores prevent vascular leakage during leukocyte diapedesis through local RhoA signaling

Endothelial RhoB and RhoC are dispensable for leukocyte diapedesis and for maintaining vascular integrity during diapedesis

The Rho-GEFs FGD5 and Tuba differentially regulate the small GTPase Cdc42 to control leukocyte

extravasation and vascular permeability

Stiffness-induced endothelial DLC-1 expression forces leukocyte spreading through stabilization of the ICAM-1 adhesome

Rac1 activation by the endothelial GEF Tiam1 marks sites for leukocyte diapedesis through guiding junctional membrane ruffles

Summary and concluding remarks Nederlandse samenvatting Curriculum Vitae Portfolio Publication list Dankwoord Chapter 1 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Chapter 7 Chapter 8 Addendum

1_{Depart of Plasma Proteins, Molecular Cell Biology Lab, Sanquin Research and Landsteiner} Labora-tory, Academic Medical Center, University of Amsterdam, Plesmanlaan 125, Amsterdam 1066 CX, The

Netherlands *Authors contributed equally

Modified from: Rho GTPases: Molecular Biology

in Health and Disease, Chapter 10 Endothelial

specific GTPase signaling during leukocyte

ex-travasation, 2018. ISBN: 978-981-3228-78-8

Lilian Schimmel

*, Sofia Morsing

*, Jos

van Rijssel

* & Jaap D. van Buul

*

Chapter 1

10

General introduction

William Harvey was the first to discover the existence of blood circulation through a closed network of arteries and veins composing the vascular system. He published his findings in 1628, in his book entitled “Exercitatio Anatomica de Motu Cordis et Sanguinis in Animalibus”, which is translated as “An anatomical exercise on the motion of the heart and blood in living beings”. This new insight rocked the worlds general perspective of blood flow through the human body.

Harvey roughly measured the amount of blood the heart could pump out into the body and discovered that with each beat about 56.8 ml (2 oun-ces) of blood left the heart. When this was extrapolated, under resting condi-tions of 72 beats per minute, the heart would pump 245 liters (540 pounds) of blood into the body every hour. This large volume of blood indicated that the blood must be circulating, since it would be impossible for a body to conti-nuously generate such large volume of new blood every hour. The presence of aortic valves between the heart and aorta, which only functioned under one-way flow direction, prompted Harvey to conclude that the blood goes out of the heart to all parts of the body via the arteries and it returns to the heart via the veins, making blood circulation a closed circuit (Figure 1) However, Harvey was never able to physically see the connection between arteries and veins by the so-called capillaries. It was only three years after Harvey’s death, that Marcello Malpighi proved the missing link in Harvey’s model of blood circulation in 1660.

Figure 1

A B Figure 1. Development of models

of the cardiovascular system. (A) One of the first models of the

cardiovascular system according to Erasistratus (315-240 BC), in which the arteries are filled with air, and all the blood, derived from the liver and is distributed throughout the body by the veins. Finally, the blood evaporates via the skin after it reached its destination organs.

(B) William Harvey’s (1578-1657)

closed circulatory model, in which not only the pulmonary circuit is a closed loop. Also the arteries and veins are connected with each other, after his discovery that the quantity of blood

General introduction

11 After Harvey’s new perception of the vascular system, the main compo-nent of blood, the red blood cell was described about the same time by Jan Swammerdam in 1658. However, it took about 2 centuries before Gabriel Andral and William Addison discovered the existence of white blood cells or leukocytes in 1843. Leukocytes are part of the immune system and defend the body against pathogens and foreign materials.

Inflammation and leukocyte transendothelial migration

Nowadays, we know that the blood circulation exists of a dense and complex network of connected blood vessels throughout the entire human body, for-ming the vascular system. This organ permits transport of nutrients, oxygen, hormones and red and white blood cells in order to maintain body homeos-tasis, which is essential for human health. In addition, the vascular system governs guidance to leukocytes that traffic through the blood vessels provi-ding protective immune functions and keeping our body free of pathogens,

cancer and foreign materials 2,3_.

Inflammation is part of the complex protective response of tissues

to harmful stimuli, such as pathogens or damaged cells 4 _{in which}

leukocy-tes, blood vessels, and molecular mediators are all involved to eliminate, clear and repair tissue damage. An impaired inflammatory response leads to progressive tissue damage due to harmful stimuli (e.g., bacteria), which compromises the survival of the organism. On the other hand, excessive or chronic inflammatory responses may lead to a plethora of diseases like hay fever, atherosclerosis or rheumatoid arthritis. Therefore, tight regulation of the inflammatory process is crucial.

During inflammation or immune surveillance, the interaction between leukocytes and the inner lining of blood vessels, the endothelial cells (ECs) intensifies, which leads to leukocyte adhesion and subsequent transendothe-lial migration (TEM). TEM, also referred to as extravasation, is a well-defined multistep process in which leukocytes exit the blood vessels following the successive steps of rolling, firm adhesion and crawling and finally

diapede-sis, further described in paragraph 1.3 (Figure 2) 5,6_{. The last stage of TEM,}

diapedesis, can occur either via the paracellular route, i.e. through the ECs

junctions 7,8_{, or the transcellular route, i.e. through the EC body} 9–11_{. TEM is a}

close collaboration between leukocytes and the ECs in which rapid remode-ling of the cytoskeleton of the ECs is essential in order to allow leukocytes to

pass the tight EC barrier that normally maintains vascular homeostasis 12–15_.

Actin, GTPases, GEFs and GAPs

The actin cytoskeleton is composed of G-actin monomers that polymerize into larger filaments of which two chains intertwine to form the so-called fila-mentous (F)-actin fibers. There are myosin motor proteins that move along the F-actin fibers and generate intracellular contractile forces. The actin

Chapter 1

12

toskeleton is a highly dynamic structure that controls cell movement, shape and adhesion, intracellular transport and cytokinesis. Changing cell shape is essential for ECs to let leukocytes pass via TEM.

During leukocyte extravasation, the ECs receive several input signals that initiate dynamic rearrangements of the actin cytoskeleton in order to let leukocytes pass, and prevent vascular permeability. In order to do so, these input signals need to be converted into an intracellular response that induces local actin polymerization, degradation, branching and contraction resulting in spatial and temporal cytoskeletal remodeling. A family of proteins that ful-fills the task of inducing actin remodeling in time and place and in response to extra- and intracellular cues are the Rho GTPases including RhoA, Rac1, and Cdc42.

Rho GTPases cycle between an active GTP (guanosine triphospha-te)-bound state and an inactive GDP (guanosine diphosphatriphospha-te)-bound state. This cycling is regulated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). ECs express over 22 Rho GTPases and

more than 70 GEFs and a similar number of GAPs 16_{. GEFs and GAPs are}

the upstream and downstream regulators of Rho-GTPases controlling their spatiotemporal activation and signaling. Combining different GEFs, GAPs and GTPases enables cells to fine-tune complex cellular processes such as maintenance of stable endothelial cell-cell junctions at one side of the endothelial cell, whereas at the other side a leukocyte is penetrating. Cur-rent studies use functional Förster resonance energy transfer (FRET)-based biosensors to study local GEF and GAP activity within the cell to chart spati-otemporal (in)activation of GTPases. A more detailed description of activati-on of the actin cytoskeletactivati-on polymerizatiactivati-on machinery by GTPases and the function of endothelial GEF and GAP proteins is given in Chapter 2.

Step by step regulation of leukocyte extravasation by GTPases

Based on the multistep leukocyte TEM model, we will introduce the function of endothelial actin regulation by GTPases during TEM in 3 parts: Rolling, Firm adhesion and crawling, and Diapedesis (Figure 2A, B, C and D respec-tively).

Rolling

In response to inflammatory stimuli, ECs upregulate adhesion molecules to capture activated leukocytes, and direct them to the site of inflammation. The first step in this process is leukocyte rolling, accomplished through weak binding of sialyl Lewis-x-like structures on the leukocyte- or endothelial sur-face to their selectin ligand on the opposite sursur-face. The three main select-ins are platelet selectin (P-selectin; CD62P), endothelial selectin (E-selectin; CD62E) and leukocyte selectin (L-selectin; CD62L). An example of a selectin ligand is the mucin-like P-selectin glycoprotein ligand 1 (PSGL-1; CD162),

General introduction

13 which is constitutively expressed on a subset of leukocytes and on the

surfa-ce of ECs 17–19_{. It has the potential to bind all members of the selectin family,}

including E- and L-selectin, but it binds with highest affinity to P-selectin 20_.

P-selectin is stored in Weibel-Palade bodies of ECs and is rapidly released

at the cell surface in response to stimuli such as histamine or thrombin 21_.

Ac-tivation of the GTPase Rac1 has been correlated with Weibel-Palade body release, through a mechanism that involves reactive oxygen species (ROS) production. This way, Rac1 regulates P-selectin exposure on the EC surface

22,23_.

Expression of E-selectin on ECs is induced by tumor necrosis

fac-tor-α (TNFα) and interleukin-1 (IL-1) 24_{. The signaling pathway of E-selectin}

upregulation involves nuclear factor κB (NFκB) and c-jun N-terminal kinase

(JNK) /p38 mitogen-activated protein kinase (p38MAPK) 25,26_{. The GTPase}

RhoA is capable of activating the NFκB signaling pathway 27–29 _and

expressi-on of a dominant-negative mutant RhoA has been shown to inhibit E-selectin

expression on the endothelial surface 30,31 _{as a result of suppression of the}

NFκB pathway.

Both the upregulation of P-selectin by Weibel-Palade body release and E-selectin by upregulation of transcription via NFκB signaling involve activation of the GTPases Rac1 and RhoA respectively upon similar stimuli. That is where regulation by Rho-GEF comes in play.

For example Trio, which is activated upon histamine or thrombin sti-mulation, contains two distinct GEF domains; the GEF1 domain activates

Rac1/RhoG; and the GEF2 domain activates RhoA 32–34_{.Confirming the role}

of RhoA in E-selectin expression, silencing of Trio resulted in decreased ex-pression of E-selectin in TNFα-stimulated ECs due to decreased RhoA acti-vity 35_.

Besides upregulation of selectins on ECs to induce leukocyte rolling, TNFα also induces expression of intercellular adhesion molecule-1 (ICAM-1) via activation of the NFκB pathway. Moreover, TNFα induces formation of ICAM-1-rich filopodia on the EC surface, which is essential to facilitate leukocytes to the next step of firm adhesion and crawling. Localization of ICAM-1 into filopodia is regulated by filopodia-specific Myosin-X and

requi-res Cdc42 activity 36_{. Thus inflammatory stimuli not only regulate expression}

of selectins for the rolling stage, also formation of ICAM-1-rich filopodia is induces to enhance progression towards the next step of firm adhesion and crawling.

Firm adhesion & crawling

By bridging the transition between the initial contact and the actual diapesis of leukocytes, the firm adhesion and crawling phase is critical for the de-cision if and where a leukocyte will breach the endothelial barrier. In the last two decades, it has become increasingly clear that there is intensive

Chapter 1

14

stalk between endothelial cell adhesion receptors and the cortical F-actin cy-toskeleton of the endothelium: (i) actin-binding proteins (ABPs) connect ad-hesion receptors to the actin cytoskeleton, and (ii) the signaling downstream of adhesion receptors controls the dynamics of the F-actin cytoskeleton.

Chemokine-induced activation of leukocyte integrins LFA-1 (αLβ2) and VLA-4 (αVβ1) allow these adhesion receptors to interact with their en-dothelial counter ligands ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1), respectively, resulting in firm adhesion of leukocytes to the en-dothelium. ICAM-1 and VCAM-1 are both members of the immunoglobulin family of adhesion receptors and become strongly upregulated by the ECs under inflammatory conditions. Binding of leukocyte integrins to ICAM-1 and VCAM-1 induces higher-order cluster formation in order to support firm ad-hesion of leukocytes 37,38_.

Clustering of these adhesion receptors induces interaction with a number of ABPs of which at least eight (i.e., FilaminA/B/C, α-actinin1/4, cor-tactin, ezrin, moesin) have been reported to bind either directly or indirectly

to the intracellular domain of ICAM-1 39–43_{. Since the intracellular domain of}

ICAM-1 is only 28 amino acids long, spatiotemporal regulation of ABPs inter-acting with 1 is necessary. Distinct molecular complexes with ICAM-1 are formed by Filamin B, α-actinin4 and cortactin, that result in different output signals regarding actin dynamics specific for the ABPs present in the complex 44_.

Each ABP has been shown to regulate actin dynamics in a different

way. Through dimerization, Filamins can crosslink F-actin fibers 45_{. α-actinins}

are involved in crosslinking antiparallel F-actin bundles 46_{, whereas cortactin}

can activate the Arp2/3 complex to initiate local F-actin polymerization 47_.

Surprisingly, these three ABPs are all required for efficient leukocyte adhesi-on 42–44_{. Besides linking adhesion receptors to the F-actin cytoskeleton, these}

ABPs also have important functions as adaptor proteins to facilitate Rho GTPase signaling.

In addition to ABP recruitment, clustering of both ICAM-1 and VCAM-1 seems to be the main mechanism for inducing Rho GTPase signaling. Overall ICAM-1 and VCAM-1 clustering induced by the addition of antibodies directed against these adhesion receptors demonstrated that ICAM-1 is a

potent activator of RhoA in cultured ECs 48,49_{, whereas VCAM-1 signaling}

leads to activation of Rac1 50,51_{. However, later studies were able to cluster}

ICAM-1 more locally by using beads coated with anti-ICAM-1 antibodies. These studies found that ICAM-1 clustering also leads to the activation of RhoG and Rac1, which is mediated by the Rho-GEFs SGEF and Trio,

res-pectively 43,52,53_{. Both Trio and Rac1 were reported as binding partners of}

Filamin 54_{, and Trio-mediated RhoG and Rac1 activation appeared to be}

de-pendent on Filamin 53_{. This demonstrates that Filamin indeed functions as a}

General introduction

15

also cortactin was shown to be required for RhoG activation 43_{. It is therefore}

tempting to speculate that Filamin may scaffold the Trio and RhoG/Rac1 sig-naling cascade, whereas cortactin may be involved in SGEF-mediated RhoG activation.

Although these Rho GTPase signaling pathways are activated upon leukocyte adhesion-induced clustering of ICAM-1 and VCAM-1, their con-tribution to leukocyte firm adhesion is questionable. Knockdown of Rac1 or RhoG expression or use of dominant-negative Rac1 or RhoG did not affect

leukocyte adhesion 55,56_{. For most leukocytes the firm adhesion stage is not}

much more than a quick transition from rolling to crawling on the ECs and therefore the effects of Rho GTPase activation upon adhesion receptor clus-tering is more likely to coincide with and regulate the consequent steps of

crawling and diapedesis 57_.

After establishing firm adhesion, crawling to a site permissive for transmigration is the subsequent step. Pulling forces of leukocytes on en-dothelial ICAM-1 molecules activate a mechanosensitive response, that in-volves RhoA activation via its Rho-GEF LARG, in order to increase EC

stif-fness 58_{. Depletion of LARG resulted in reduced leukocyte crawling speed,}

demonstrating that this signaling pathway contributes to stiffness-dependent leukocyte crawling. More details on the effects of endothelial and/or matrix stiffness on leukocyte crawling and TEM is given later on.

Diapedesis

The final and perhaps most complicated step of the multistep process of leukocyte TEM is the actual diapedesis, where leukocytes breach through the EC barrier in order to continue their way into the underlying tissues. In-tensifying the interactions between the two involved cell types (i.e. the leu-kocyte and the endothelial cell) during this stage is essential to ensure tight regulation of critical processes such as the opening and closure of the endo-thelial barrier to limit vascular leakage. The role of actin and its regulation of Rho GTPases during the transition from adhesion to initiating diapedesis, the opening of endothelial cell-cell junctions, the preservation of vascular per-meability during leukocyte diapedesis, and finally resealing of the EC barrier afterwards are discussed below.

Transition from leukocyte adhesion to the actual diapedesis is regu-lated by a process that involves endothelial membrane protrusions. These protrusions and their formation go by many different names that all describe the same phenomenon: the surrounding of the migrating leukocyte by EC membrane protrusions. The first study that reported these structures

descri-bed them as docking structures 59,60_{, shortly followed by the so-called}

trans-migratory cup studies 10,61_.

Other terms known for these structures are apical cups 55_{, dome}

structures 62–64_{, actin dynamic structures} 65 _{or ICAM-1-rich contact areas} 43,66_.

Chapter 1

16

Whether these structures function as adhesion platforms or are exclusively for transmigration support to limit vascular leakage, in the end they facilitate the transition of leukocytes from adhesion to the last stage of extravasation: diapedesis.

When taking a closer look at the actin structures present in these membrane protrusions, there are actually two different actin structures pre-sent: vertical actin branches towards the apical surface and a F-actin-rich ring surrounding transmigrating leukocytes, localized more at the basal pla-ne of the leukocyte-EC interaction. The vertical membrapla-ne protrusions are formed by polymerized actin filaments that require activation by RhoG and

Rac1 43,53,55 _{and contain adhesion receptors like ICAM-1 and VCAM-1 and}

present pro-inflammatory chemokines such as IL-8 67–69_.

The F-actin ring that surrounds passing leukocytes, is present on the

Rolling Leukocyte Endothelial cell Extracellular matrix P/E-selectin Cdc42 Rac1/RhoA TNFa ICAM1 TNFa-receptor

Firm adhesion and crawling

Trio

RhoGRac1

Actin binding proteins Branched Actin ICAM1 clustering Actin polymerization Cup formation Figure 2 (A)

Upon inflammatory stimuli like TNFα, ECs transmit the intracellular signal re-sulting in activation of Rac1 an RhoA which will release P-selectin from the Weibel Palade bodies, and cause upregu-lation of E-selectin. This makes rolling of leukocytes possible in order to slow them down and activate the integrins on the leukocytes. A second effect occurs in the ECs upon TNFα stimulation, for-mation of ICAM-1-rich filopodia on the apical surface, which is essential for the next step.

Figure 2 (B)

Interaction of the leukocyte integrins with endothelial ICAM-1 and VCAM-1 results in clustering of the adhesion molecules and recruitment of actin bin-ding proteins to the intracellular tail of ICAM-1 or VCAM-1. This anchors the adhesion molecules to the actin cytos-keleton an results in activation of RhoG and Rac1 via the GEF Trio. RhoG and Rac1 rearrange actin dynamics, resulting in formation of endothelial apical mem-brane protrusions.

General introduction

17

Diapedesis _{ICAM1 tension}

LARG/Ect2 RhoA Actomyosin contraction Bundled Actin Closure Rac1 Actin polymerization Actin branching Figure 2 (C)

Pulling forces on ICAM-1 exerted by the moving leukocytes induce activation of RhoA via the GEFs LARG and Ect2. Myosin contractility downstream of RhoA signaling ensures the present actin ring that surrounds breaching leukocytes to seal off the transmigration pore during passage of leukocytes.

Figure 2 (D)

After passage of leukocytes, Rac1 dri-ven dri-ventral lamellipodia ensure closu-re of the transmigration poclosu-re and Rac1 activity is necessary for restoration of VE-cadherin-based endothelial cell-cell junctions.

more basal side inside the ECs. These rings, rich in F-actin and RhoA-dri-ven, will be described and discussed in Chapter 3. Involvement of the other GTPases RhoB and RhoC in regulating leukocyte diapedesis and vascular permeability will be discussed in Chapter 4.

After passage of leukocytes across the EC layer, the formed gaps need to be closed immediately in order to limit vascular leakage (Figure 2D). Dynamic F-actin bursts in lamellipodia-like structures on the ventral side of ECs are observed to close mechanical-induced micro-wounds in an

asym-metrical manner 12_{. These ventral lamellipodia show enrichment of Rac1}

ef-fectors like cortactin, IQGAP and p47phox, indicating that Rac1-driven

ven-tral lamellipodia rapidly restore the EC barrier upon injury 12_{. The formation of}

ventral lamellipodia that close the gaps is induced when leukocytes transmi-grate either via the para- or transcellular route. However, a major difference between the two routes is the need for junction restoration in case of pa-racellular transmigration. Dynamics of the major endothelial cell-cell junction molecule, VE-cadherin, is regulated by the Rac1-regulated actin nucleator

ARP2/3 65,70_{. Thereby, a second role for endothelial Rac1 during leukocyte}

TEM is being described, emphasizing the need for specific spatiotemporal

Chapter 1

18

control by GEFs and GAPs.

Effects of ageing on cardiovascular diseases

More than 80% of all cardiovascular deaths in developed countries occurs

in the population aged 65 _{or older, making aging a major risk factor for}

car-diovascular disease. It is well established that age-induced stiffening of the extracellular matrix (ECM) in the intima alters the condition of blood vessels. These alterations of increased ECM stiffness affect EC health and result in inflammation, hypertension and ultimately in vascular disease development and progression as occurs for atherosclerosis and pulmonary arterial hyper-tension (PAH) 71–75_.

Age related vascular stiffening

Increased vascular ECM stiffness is induced upon aging and/or inflammati-on, as was shown by rigidity measurements, using murine aortas of different

ages 76_{, and in murine and human atherosclerotic plaques} 77,78_{. Vascular}

cal-cification is often observed in human atherosclerotic plaques and is known to induce EC dysfunction, which is one of the main characteristics of arterial

aging 79_{. In mice, age-induced increases in ECM stiffness disrupts}

VE-cad-herin based cell-cell junctions and enhances vascular permeability 76,80_{. Until}

now, it remains very difficult to modulate ECM stiffness, as processes such as calcification seem to be irreversible. A number of studies attempted to

inhibit or reverse ECM stiffening, but were all unsuccessful 81–84_{. Hence,}

fo-cus of current research has shifted towards the translational effects of ECM stiffness on ECs to explore the possibilities of reversing the response of ECs to increased ECM stiffness, instead of directly targeting ECM stiffening.

Effect of ECM stiffness on leukocyte TEM

The relevance of ECM stiffness on EC function becomes clear when per-forming leukocyte TEM experiments. The specific location that leukocytes choose to exit the circulation is presumably not a matter of coincidence. Sites on the endothelium are favored by leukocytes to cross the EC barrier; so-cal-led transmigration “hotspots”. Which components determine these hotspots remains unclear, however, it seems that leukocytes use the crawling stage to find these hotspots.

So far, several key principals of taxis have been studied that each affect the site of leukocyte diapedesis by means of attraction towards an optimal concentration of chemokines (chemotaxis), density of adhesion

mo-lecules (haptotaxis) or cellular stiffness (durotaxis) 15_{. The processes of}

che-motaxis and haptotaxis are described in more detail in Chapter 2.

Durotaxis is the type of cell migration in which a substrate stiffness gradient guides the cells in a certain direction. For most cell types this is

General introduction

19 from the ECM and in case of leukocyte TEM from the subcellular stiffness variations of the ECs. Migration of neutrophils on fibronectin-coated stiffness substrate gradients ranging from soft (4kPa) until stiff (13kPa) show fastest

crawling speeds on 7 kPa 87_{. ECs themselves were also shown to have an}

increasing central-to-peripheral stiffness gradient, which was largely

depen-dent on the ICAM-1-interacting ABPs α-actinin 4 _{and cortactin} 44_{. Using}

po-dosome-like protrusions, leukocytes were shown to probe the ECs to sense

local stiffness and thereby find their path for TEM 9,88_.

Neutrophils have been shown to increase their spreading and migra-tion persistence on stiffer substrates, resulting in increased migramigra-tion

distan-ce on the apical side of ECs 87,89_{. As the lateral migration distance of crawling}

neutrophils increases, their chances of finding the transmigration hotspot increases as well, suggesting that durotaxis is an important aspect of

leuko-cyte TEM 90_{. It is well established by ex vivo studies that areas of increased}

ECM stiffening such as in atherosclerotic lesions or in the lung

microvascu-lature from PAH patients, there is increased leukocyte extravasation 91_{. In}

conclusion, it is now widely accepted that an increase in substrates stiffness

is correlated with increased leukocyte extravasation 76,92–94_.

Scope of the Thesis

In this thesis, we demonstrate different aspects of the active role of ECs in the process of leukocyte TEM. In particular we focused on the role of the ac-tin cytoskeleton, its regulation by the Rho family of small GTPases, and their spatiotemporal activation and deactivation by GEFs and GAPs respectively. Although many efforts have been made, we still know relatively little on the true contribution of Rho GTPases in the process of leukocyte TEM. The work presented in this thesis contributes to the elucidation of some of the most well-studied family members such as RhoA and Rac1, but also shows some more insight in the less investigated family members like RhoB, RhoC and Cdc42.

In Chapter 2, we provide an overview of EC proteins and signaling

pathways that are involved in regulating leukocyte TEM and limiting vascular leakage during this process.

In Chapter 3, we describe a specific role for endothelial RhoA

ac-tivity during leukocyte TEM. Activation of this GTPase is established via ICAM-1-mediated recruitment of the GEFs LARG and Ect2, which results in Myosin II-driven contractility of a F-actin-rich ring that surrounds trans-migrating leukocytes. Operating like an elastic strap, this F-actin ring limits leakage of plasma proteins, while leukocytes are able to pass and migrate to underlying tissues.

In Chapter 4, we report that in contrast to RhoA, two other closely

related GTPases RhoB and RhoC are not involved in regulating leukocyte diapedesis or for maintenance of vascular integrity during leukocyte

Chapter 1

20 desis.

In Chapter 5, we examine the regulation of Rho GTPases in basal

endothelial permeability, leukocyte-induced vascular leakage or leukocy-te TEM efficiency by means of an shRNA-based screen for Rho-GEFs and GAPs. A novel role for the small Rho GTPase Cdc42 in regulating TNFα-in-duced ICAM-1-rich filopodia formation is regulated by the

Rho-GEF FGD5. Moreover, an additional function for Cdc42 is shown to be regulated by the Rho-GEF Tuba and concerns the closure of EC gaps after passage of leukocytes.

In Chapter 6, we show a Rho-GAP-independent function for DLC-1

in translating substrate stiffness towards leukocyte adhesion and TEM ef-ficiency. Increased substrate stiffness due to disease, like atherosclerosis, results in upregulation of endothelial DLC-1. This Rho-GAP facilitates, inde-pendent of its GAP function, the stabilization of the ICAM-1 adhesome. The ICAM-1 adhesome is a protein complex induced upon ICAM-1 clustering by leukocyte binding and involves actin adaptor proteins to locally remodel the EC actin cytoskeleton and thereby to provide a proper surface for leukocyte spreading.

In Chapter 7, we address the effects of Rac1 activation by the GEF

Tiam1 on leukocyte TEM. Tiam1 expression in EC is stiffness- and inflam-mation-dependent, and activation of endothelial Rac1 by constitutive active Tiam1 regulates leukocyte transmigration sites. Rac1 activation results in formation of endothelial dorsal membrane ruffles that guide leukocytes to-wards optimal sites for transmigration.

In Chapter 8, we discuss the findings described in this thesis. We

address our work on leukocyte adhesion (chapter 6), transmigration (chapter 5), leukocyte-induced permeability (chapter 3 & 4) and the effects of aging and substrate stiffness (chapter 6 & 7) from an EC point of view and put it in a broader perspective.

General introduction

21

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me-General introduction

25 chanical forces and signaling pathways

du-ring transendothelial cell migration. FEBS J.

1_{Department of Plasma Proteins, Molecular Cell Biology Lab, Sanquin Research and Landsteiner} Labo-ratory, Academic Medical Center, University of Amsterdam, Plesmanlaan 125, Amsterdam 1066 CX, The

Netherlands

Small GTPases (2017); 1:1-15

Lilian Schimmel

, Niels Heemskerk

&

Jaap D. van Buul

Leukocyte transendothelial

migration: A local affair

28

Chapter 2

Summary

Inflammation is part of the complex biological response of body tissues to harmful stimuli, such as pathogens. It serves as a protective response that involves leukocytes, blood vessels and molecular mediators with the pur-pose to eliminate the initial cause of cell injury and to initiate tissue repair. Inflammation is tightly regulated by the body and is associated with transient crossing of leukocytes through the blood vessel wall, a process called tran-sendothelial migration (TEM) or diapedesis. TEM is a close collaboration between leukocytes on one hand and the endothelium on the other. Limi-ting vascular leakage during TEM but also when the leukocyte has crossed the endothelium is essential for maintaining vascular homeostasis. Although many details have been uncovered during the recent years, the molecular mechanisms from the vascular part that drive TEM still shows significant gaps in our understanding. This review will focus on the local signals that are induced in the endothelium that regulate leukocyte TEM and simultaneous preservation of endothelial barrier function.

29

Review: Leukocyte transendothelial migration

Introduction

The vascular system is a complex network formed by numerous connected blood vessels that are embedded in tissue throughout the human body. Re-moving all tissues leaving only the vascular system intact fully outlines the shape of the human body showing the high density of blood vessels in our tissues and organs. Proper functions of this high density network are essen-tial for human health, since it provides our body with nutrients, oxygen and hormones and regulates body homeostasis such as temperature and pH. In addition the vascular system governs guidance to traveling immune cells and thereby supports protective immune functions that keep our body free

of pathogens, cancer and foreign material 1,2_{. In case of inflammation or}

im-mune surveillance the cells lining the luminal site of blood vessels, known as endothelial cells (ECs), attract and direct traveling immune cells to suitable exit sites in the vasculature allowing cells to enter underlying tissue. ECs therefore fulfill an important supportive role in guidance and directional mi-gration of trafficking immune cells. During inflammation ECs expose a variety of adhesion molecules at their surface that slow down and arrest traveling immune cells in the blood circulation. These adhesive molecules are thought to provide guidance cues to immune cells where to breech the blood vessel wall through a multi-step process known as transendothelial migration (TEM)

or diapedesis 3_{. Although many adhesion molecules have been identified,}

the exact composition of adhesion molecules that determine a suitable exit site for immune cell diapedesis remains elusive. It is well appreciated that blood vessels in inflamed tissues are more permissive for macro molecules. This endothelial leakiness supports several inflammatory functions such as activation of the complement system and recruitment of innate immune cells. Paradoxically, recruitment of innate immune cells occurs through transient

openings in the endothelium without plasma leakage 4–6_{. This indicates that}

vascular permeability for small macromolecules and immune cells are sepa-rately regulated. Which mechanisms protect the endothelial barrier during leukocyte diapedesis is currently poorly understood.

In several diseases, such as thrombocytopenia, ischemia and rheu-matoid arthritis accumulation of immune cells evoke serious tissue damage. In case of thrombocytopenia it is known that the physical movement of

immu-ne cells through the ECs barrier elicits organ hemorrhages 7_{. This bleeding}

disorder is partly caused due to the incapability of ECs to maintain a tight barrier during the physical movement of immune cells through the EC layer. In the past years lots of effort has made into the development of blocking antibodies targeting leukocyte integrins or integrin ligands that are exposed at the endothelial surface. Blocking immune cell exit sites may prevent TEM and consequently reduce patients symptoms. However, 2 clinical trials that

30

Chapter 2

tried to interfere with adhesion molecules evoked serious side effects and aggravated the patients conditions, since the blocking antibodies used in the trial activated the immune cells in contrast to their predicted blocking effect

8_{. In order to improve treatments for diseases that involve immune cell traffic}

it is a necessity that we increase our understanding about what processes occur during leukocyte TEM, both at the cellular and molecular level. In this review, we focus on how immune cells travels through the endothelial barrier and discuss recent insights on how ECs protect their barrier function during immune cell trafficking.

31

Transendothelial migration ‘hotspots’

The regulation of immune cell trafficking is complex and goes far beyond our current understanding. Although much is yet to discover, intensive rese-arch over the past decade has revealed several fundamental principles that regulate cell migration in a variety of immune cell related responses such as hematopoiesis, immune surveillance and innate and adaptive immunity. The current paradigm of TEM is a refined version of the multi-step model

that was first proposed by Butcher and Springer 9,10_{. The current order in the}

multistep paradigm are; leukocyte rolling, arrest, crawling, firm adhesion and transmigration. The latter occurs either through the endothelial junctions

(pa-racellular route) 11,12 _{or through the endothelial cell body (transcellular route)}

13–15_{. Interestingly, leukocytes diapedesis gives the impression to occur at}

predefined places in the endothelium lining. Some locations even favor the migration of multiple immune cells that breech the endothelial lining in rapid succession. In fact, when looking at a transmigrating leukocyte, just prior to exiting, the leukocyte changes its crawling morphology to a more round appearance. This raises some important questions, such as what factors determine these so called ‘hotspots for transmigration’, why do 2 routes exist and what defines the usage of one over the other. Judging on the recordings of transmigrating leukocytes, it appears that the leukocytes search the en-dothelial monolayer to find an exit point, indicating that they use the crawling step as a sort of searching period. However, strong evidence on this latter suggestion is missing and requires future investigations. So far, several key principles have been established, although it needs to be kept in mind that these principles are based on in vitro studies and therefore can only ser-ve as a model that awaits confirmation in future in vivo studies. First of all, immune cells are attracted toward an optimal concentration of chemokines (chemotaxis), density of adhesion molecules (haptotaxis) or cellular stiffness (durotaxis). Secondly, migration into a tissue or organ is believed to follow the path of least resistance (tenertaxis). In addition, shear forces, vessel type and composition of the glycocalyx play an important regulatory role in dictating suitable exit sites. Each principle will be briefly delineated starting with chemotaxis.

Chemotaxis

Chemokines are of key importance for leukocyte TEM not only because of their involvement in chemotaxis but also because of their role in integrin ac-tivation inducing leukocyte arrest (Figure 1A). Chemokines are immobilized by heparan sulfate (HS) proteoglycans that are part of a 50–100 nm nega-tively charged network on the apical surface of EC called the glycocalyx.

Immobilized chemokines elicit integrin-mediated adhesion 16_{. Recently, it has}

Review: Leukocyte transendothelial migration

32

Chapter 2

been shown that perivascular macrophages located between the tissue and blood vessels, secrete chemokines that cause local “hotspots” for neutrophil

diapedesis in vivo 17_{. These chemokines secreted in the extravascular space}

are bound to glycosaminoglycans (GAGs) and are subsequently transcyto-sed to the luminal side of the vasculature. There are some indications that oligomeric chemokine-forms activate leukocyte-integrins that direct leuko-cyte arrest and firm adhesion whereas monomeric-forms activate integrin

subsets on the leukocyte that govern cell movement 18,19_.

Haptotaxis

Similar ideas have been suggested for integrin ligands presented at the api-cal surface of ECs where the amount of leukocyte-integrin ligands regulates leukocyte behavior. A good example of haptotaxis is the amount of ICAM-1 molecules present at the endothelial surface (Figure 1B). Surface density and distribution of endothelial ICAM-1 induced a transition from paracellular to transcellular migration, while intermediate levels favored the paracellular

route 20,21_{. Related to the amount of surface ligands,}

neutrophil-ECinteracti-ons during TEM does increase integrin expression at the surface of

neutrop-hils thereby affecting their activity and behavior after transmigration 22_.

Durotaxis

Migrating cells sense environmental cues that give direction to their move-ment. Migrating cells are attracted to an optimal surface stiffness also called stiffness sensing or durotaxis (Figure 1C). Leukocytes sense and respond to their physical surroundings, for example in vitro neutrophils migrate slower on soft (4 kPa) and very rigid (13 kPa) fibronectin coated surfaces whereas optimal crawling speeds were reached on 7 kPa. Interestingly fibronectin density also affected the outcome of migration speed. Using FN concentrati-ons of 100 µg/ml the optimal stiffness for migration is 4 kPa while on 10 µg/

ml the optimal rigidity for maximal migration is increased to 7 kPa 23_{. This}

suggests that leukocyte TEM in vivo depends on the combination between matrix rigidity (i.e. durotaxis) and the amount of available surface ligands (i.e., haptotaxis) for leukocytes to interact with.

Tenertaxis

Another phenomenon that is often observed in vitro when the endothelial bar-rier is very tight, is the predominant use of the transcellular route, whereas weak endothelial junctional integrity shows high association with paracellular

diapedesis (Figure 1D) 24_{. To find these spots of low resistance, lymphocytes}

dynamically probe the underlying endothelium by extending invadosome-like protrusions into its surface that deform the plasma membrane, depolymerize F-actin filaments at the membrane cortex and ultimately breach the barrier

com-33

mon principle namely ‘the path of least resistance’ 24_{. However, determining}

where the path of least resistance is present in vivo is very difficult, if not impossible. The observations of tenertaxis for TEM are so far only performed in in vitro studies. Another point is the seemingly contradictory effects of du-rotaxis and tenertaxis. According to tenertaxis, leukocytes would prefer a site of low endothelial cytoskeletal density, while according to durotaxis

leukocy-tes need an optimum cytoskeletal stiffness to cross the vessel wall 24_{. This}

opposing arguments for determining the transendothelial migration ‘hotspot’ indicate that the mechanism that determines the TEM hotspot is likely an in-terdependent combination of the TEM factors chemo-, hapto-, duro- and te-nertaxis, where, dependent on specific (patho)physiological conditions, one factor may play a more dominant role over the other in determining the site for leukocytes to cross.

Shear forces

The impact of shear forces on leukocyte behavior has been established by several research groups. Transmigration kinetics of neutrophils was

signifi-cantly faster under shear stress than under static conditions (Figure 1E) 26_.

Leukocyte extravasation primarily takes place in the postcapillary venules

of the inflamed tissue where the flow velocity is between 1–10 dyn/cm 2,27_.

Cinamon and co-workers showed that specifically for lymphocytes TEM was

promoted by a continuous physiological flow between 0.75 and 5 dyn/cm 2,28_.

From these data it was concluded that flow-induced mechanical signals are coupled to Gi protein signaling at the luminal endothelial cell surface,

resul-ting in further enhancing lymphocyte TEM 29_{. Additional work from the same}

group convincingly showed that shear stress promotes extensive filopodia

formation by T-lymphocytes 19_{. Filopodia are small membrane “finger-like”}

protrusions that leukocytes use to probe the luminal endothelial surface be-fore and during TEM. This process of lymphocyte probing the endothelial surface was underscored by a report by Carman and colleagues, who

refer-red to these structures as invading podosomes 30_.

Although the majority of leukocyte extravasation occurs under low shear conditions in postcapillary venules, during some pathological condi-tions such as atherosclerosis, monocytes adhere and transmigrate through the endothelial lining of the artery wall where shear stress is much higher. It was been shown that leukocytes tethered to and rolled on platelet-decorated ultra-large Von Willebrand factor (ULVWF) string-like structures presented

on the luminal side of the endothelium 31_{. Leukocytes scanned for activated}

platelets to interact via P-selectin glycoprotein ligand-1 (PSGL-1) resulting in clustering and activation of the β2 integrin Mac-1 that mediates

neutrop-hil TEM 32,33_{. This simultaneous interaction with activated platelets and the}

endothelium results in rapid neutrophil exit and the onset of inflammation 34_.

Using platelets as intermediate substrates, monocytes are able to

transmi-Review: Leukocyte transendothelial migration

34

Chapter 2

grate under high shear stress varying between 20 and 40 dyn/cm 2,31_{. Thus,}

also matrices generated on the luminal surface of the endothelium can drive leukocyte TEM under high shear conditions.

Vascular beds

Leukocyte diapedesis through the blood brain barrier, into the peritoneum or lungs has been shown to be differentially regulated (Figure 1F). For instan-ce, neutrophil diapedesis in ICAM-1/P-selectin knock-out mice is normal in

the lungs but totally abrogated in the peritoneum 35_{. Recently, it has been}

shown that locking the endothelial junctions prevented leukocyte diapedesis, but not in all tissues. Diapedesis into lung, skin and cremaster was severely reduced, establishing the paracellular route as the dominant route in the-se tissues. However, the migration of naïve lymphocytes into lymph nodes and transmigration of neutrophils into the peritoneum was not affected by

junctional locking 36_{. Moreover, during inflammation in the respiratory tract of}

rats, plasma proteins leakage is predominantly observed in the post-capillary venules whereas capillaries and arterioles did not leak. Under these inflam-matory conditions most leukocytes, in particular neutrophils, transmigrate in

the collecting venules downstream of the leaky post-capillary venules 4_{. This}

landmark paper reveals that plasma protein leakage and leukocyte recruit-ment are 2 separable events that can occur side by side, but this leakage is not necessarily caused by the transmigration of immune cells through the ECs.

Chemotaxis Haptotaxis Durotaxis

Tenertaxis Shear forces Vessel type

Paracellular Transcellular Lung Skin Cremaster Lymph node Peritoneum BBB TEM Substrate stiffness Adhesion molecules Figure 1 Cellular stiffness Para Trans Path of least resistance

Monomeric chemokines GAG + oligomeric

chemokines

a b c