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Filling the gapsThe endothelium in regulating vascular leakage and leukocyte extravasationSchimmel, L.

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Citation for published version (APA):Schimmel, L. (2018). Filling the gaps: The endothelium in regulating vascular leakage and leukocyteextravasation.

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Download date: 18 Aug 2019

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1Depart 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 Schimmel1*, Sofia Morsing1*, Jos van Rijssel1* & Jaap D. van Buul1*

General introduction

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General introductionWilliam 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 1A 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 was too big to be explained by a single-pass open system. Modified from 1

General introduction

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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 migrationNowadays, 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 GAPsThe 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 cy-

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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 diphosphate)-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 cytoskeleton polymerization 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 GTPasesBased 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).

RollingIn 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 surface. 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),

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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 & crawlingBy bridging the transition between the initial contact and the actual diapede-sis 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 cros-

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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 ICAM-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 scaffolding molecule downstream of ICAM-1 signaling. In addition to Filamin,

General introduction

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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.

DiapedesisThe 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.

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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 membrane 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

BranchedActin

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.

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Diapedesis ICAM1 tension

LARG/Ect2

RhoA

Actomyosin contraction

BundledActin

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 ventral lamellipodia ensure closu-re of the transmigration pore 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

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control by GEFs and GAPs.

Effects of ageing on cardiovascular diseasesMore 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 stiffeningIncreased 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 TEMThe 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 towards high stiffness areas 85,86. The differences in substrate stiffness arise

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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 migration 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 ThesisIn 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 diape-

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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.

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