VISTA Expression on Cancer-Associated Endothelium Selectively Prevents T-cell Extravasation.

Cancers evade T-cell immunity by several mechanisms such as secretion of anti-inflammatory cytokines, down regulation of antigen presentation machinery, upregulation of immune checkpoint molecules, and exclusion of T cells from tumor tissues. The distribution and function of immune checkpoint molecules on tumor cells and tumor-infiltrating leukocytes is well established, but less is known about their impact on intratumoral endothelial cells. Here, we demonstrated that V-domain Ig suppressor of T-cell activation (VISTA), a PD-L1 homolog, was highly expressed on endothelial cells in synovial sarcoma, subsets of different carcinomas, and immune-privileged tissues. We created an ex vivo model of the human vasculature and demonstrated that expression of VISTA on endothelial cells selectively prevented T-cell transmigration over endothelial layers under physiologic flow conditions, whereas it does not affect migration of other immune cell types. Furthermore, endothelial VISTA correlated with reduced infiltration of T cells and poor prognosis in metastatic synovial sarcoma. In endothelial cells, we detected VISTA on the plasma membrane and in recycling endosomes, and its expression was upregulated by cancer cell-secreted factors in a VEGF-A-dependent manner. Our study reveals that endothelial VISTA is upregulated by cancer-secreted factors and that it regulates T-cell accessibility to cancer and healthy tissues. This newly identified mechanism should be considered when using immunotherapeutic approaches aimed at unleashing T cell-mediated cancer immunity.

The endothelial diapedesis synapse regulates transcellular migration of human T lymphocytes in a CX3CL1- and SNAP23-dependent manner.

Understanding how cytotoxic T lymphocytes (CTLs) efficiently leave the circulation to target cancer cells or contribute to inflammation is of high medical interest. Here, we demonstrate that human central memory CTLs cross the endothelium in a predominantly paracellular fashion, whereas effector and effector memory CTLs cross the endothelium preferably in a transcellular fashion. We find that effector CTLs show a round morphology upon adhesion and induce a synapse-like interaction with the endothelium where ICAM-1 is distributed at the periphery. Moreover, the interaction of ICAM-1:ß2integrin and endothelial-derived CX3CL1:CX3CR1 enables transcellular migration. Mechanistically, we find that ICAM-1 clustering recruits the SNARE-family protein SNAP23, as well as syntaxin-3 and -4, for the local release of endothelial-derived chemokines like CXCL1/8/10. In line, silencing of endothelial SNAP23 drives CTLs across the endothelium in a paracellular fashion. In conclusion, our data suggest that CTLs trigger local chemokine release from the endothelium through ICAM-1-driven signals driving transcellular migration.

Rho GTPase signalling networks in cancer cell transendothelial migration.

Rho GTPases are small signalling G-proteins that are central regulators of cytoskeleton dynamics, and thereby regulate many cellular processes, including the shape, adhesion and migration of cells. As such, Rho GTPases are also essential for the invasive behaviour of cancer cells, and thus involved in several steps of the metastatic cascade, including the extravasation of cancer cells. Extravasation, the process by which cancer cells leave the circulation by transmigrating through the endothelium that lines capillary walls, is an essential step for metastasis towards distant organs. During extravasation, Rho GTPase signalling networks not only regulate the transmigration of cancer cells but also regulate the interactions between cancer and endothelial cells and are involved in the disruption of the endothelial barrier function, ultimately allowing cancer cells to extravasate into the underlying tissue and potentially form metastases. Thus, targeting Rho GTPase signalling networks in cancer may be an effective approach to inhibit extravasation and metastasis. In this review, the complex process of cancer cell extravasation will be discussed in detail. Additionally, the roles and regulation of Rho GTPase signalling networks during cancer cell extravasation will be discussed, both from a cancer cell and endothelial cell point of view.

Hematopoietic stem and progenitor cells use podosomes to transcellularly cross the bone marrow endothelium.

Bone marrow endothelium plays an important role in the homing of hematopoietic stem and progenitor cells upon transplantation, but surprisingly little is known on how the bone marrow endothelial cells regulate local permeability and hematopoietic stem and progenitor cells transmigration. We show that temporal loss of vascular endothelial-cadherin function promotes vascular permeability in BM, even upon low-dose irradiation. Loss of vascular endothelial-cadherin function also enhances homing of transplanted hematopoietic stem and progenitor cells to the bone marrow of irradiated mice although engraftment is not increased. Intriguingly, stabilizing junctional vascular endothelial-cadherin in vivo reduced bone marrow permeability, but did not prevent hematopoietic stem and progenitor cells migration into the bone marrow, suggesting that hematopoietic stem and progenitor cells use the transcellular migration route to enter the bone marrow. Indeed, using an in vitro migration assay, we show that human hematopoietic stem and progenitor cells predominantly cross bone marrow endothelium in a transcellular manner in homeostasis by inducing podosome-like structures. Taken together, vascular endothelial-cadherin is crucial for BM vascular homeostasis but dispensable for the homing of hematopoietic stem and progenitor cells. These findings are important in the development of potential therapeutic targets to improve hematopoietic stem and progenitor cell homing strategies.

The GEF Trio controls endothelial cell size and arterial remodeling downstream of Vegf signaling in both zebrafish and cell models.

Arterial networks enlarge in response to increase in tissue metabolism to facilitate flow and nutrient delivery. Typically, the transition of a growing artery with a small diameter into a large caliber artery with a sizeable diameter occurs upon the blood flow driven change in number and shape of endothelial cells lining the arterial lumen. Here, using zebrafish embryos and endothelial cell models, we describe an alternative, flow independent model, involving enlargement of arterial endothelial cells, which results in the formation of large diameter arteries. Endothelial enlargement requires the GEF1 domain of the guanine nucleotide exchange factor Trio and activation of Rho-GTPases Rac1 and RhoG in the cell periphery, inducing F-actin cytoskeleton remodeling, myosin based tension at junction regions and focal adhesions. Activation of Trio in developing arteries in vivo involves precise titration of the Vegf signaling strength in the arterial wall, which is controlled by the soluble Vegf receptor Flt1.

DLC1 is a direct target of activated YAP/TAZ that drives collective migration and sprouting angiogenesis.

Endothelial YAP/TAZ (YAP is also known as YAP1, and TAZ as WWTR1) signaling is crucial for sprouting angiogenesis and vascular homeostasis. However, the underlying molecular mechanisms that explain how YAP/TAZ control the vasculature remain unclear. This study reveals that the focal adhesion protein deleted-in-liver-cancer 1 (DLC1) is a direct transcriptional target of the activated YAP/TAZ-TEAD complex. We find that substrate stiffening and VEGF stimuli promote expression of DLC1 in endothelial cells. In turn, DLC1 expression levels are YAP and TAZ dependent, and constitutive activation of YAP is sufficient to drive DLC1 expression. DLC1 is needed to limit F-actin fiber formation, integrin-based focal adhesion lifetime and integrin-mediated traction forces. Depletion of endothelial DLC1 strongly perturbs cell polarization in directed collective migration and inhibits the formation of angiogenic sprouts. Importantly, ectopic expression of DLC1 is sufficient to restore migration and angiogenic sprouting in YAP-depleted cells. Together, these findings point towards a crucial and prominent role for DLC1 in YAP/TAZ-driven endothelial adhesion remodeling and collective migration during angiogenesis.This article has an associated First Person interview with the first author of the paper.

Nuclear shape, protrusive behaviour and in vivo retention of human bone marrow mesenchymal stromal cells is controlled by Lamin-A/C expression.

Culture expanded mesenchymal stromal cells (MSCs) are being extensively studied for therapeutic applications, including treatment of graft-versus-host disease, osteogenesis imperfecta and for enhancing engraftment of hematopoietic stem cells after transplantation. Thus far, clinical trials have shown that the therapeutic efficiency of MSCs is variable, which may in part be due to inefficient cell migration. Here we demonstrate that human MSCs display remarkable low migratory behaviour compared to other mesodermal-derived primary human cell types. We reveal that specifically in MSCs the nucleus is irregularly shaped and nuclear lamina are prone to wrinkling. In addition, we show that expression of Lamin A/C is relatively high in MSCs. We further demonstrate that in vitro MSC migration through confined pores is limited by their nuclei, a property that might correlate to the therapeutic inefficiency of administered MSC in vivo. Silencing expression of Lamin A/C in MSCs improves nuclear envelope morphology, promotes the protrusive activity of MSCs through confined pores and enhances their retention in the lung after intravenous administration in vivo. Our findings suggest that the intrinsic nuclear lamina properties of MSCs underlie their limited capacity to migrate, and that strategies that target the nuclear lamina might alter MSC-based cellular therapies.

Human B Cells Engage the NCK/PI3K/RAC1 Axis to Internalize Large Particles via the IgM-BCR.

Growing evidence indicate that large antigen-containing particles induce potent T cell-dependent high-affinity antibody responses. These responses require large particle internalization after recognition by the B cell receptor (BCR) on B cells. However, the molecular mechanisms governing BCR-mediated internalization remain unclear. Here we use a high-throughput quantitative image analysis approach to discriminate between B cell particle binding and internalization. We systematically show, using small molecule inhibitors, that human B cells require a SYK-dependent IgM-BCR signaling transduction via PI3K to efficiently internalize large anti-IgM-coated particles. IgM-BCR-mediated activation of PI3K involves both the adaptor protein NCK and the co-receptor CD19. Interestingly, we here reveal a strong NCK-dependence without profound requirement of the co-receptor CD19 in B cell responses to large particles. Furthermore, we demonstrate that the IgM-BCR/NCK signaling event facilitates RAC1 activation to promote actin cytoskeleton remodeling necessary for particle engulfment. Thus, we establish NCK/PI3K/RAC1 as an attractive IgM-BCR signaling axis for biological intervention to prevent undesired antibody responses to large particles.

CXCR4, but not CXCR3, drives CD8+ T-cell entry into and migration through the murine bone marrow.

The BM serves as a blood-forming organ, but also supports the maintenance and immune surveillance function of many T cells. Yet, in contrast to other organs, little is known about the molecular mechanisms that drive T-cell migration to and localization inside the BM. As BM accumulates many CXCR3-expressing memory CD8+ T cells, we tested the involvement of this chemokine receptor, but found that CXCR3 is not required for BM entry. In contrast, we could demonstrate that CXCR4, which is highly expressed on both naive and memory CD8+ T cells in BM, is critically important for homing of all CD8+ T-cell subsets to the BM in mice. Upon entry into the BM parenchyma, both naïve and memory CD8+ T cells locate close to sinusoidal vessels. Intravital imaging experiments revealed that CD8 T cells are surprisingly immobile and we found that they interact with ICAM-1+VCAM-1+BP-1+ perivascular stromal cells. These cells are the major source of CXCL12, but also express key survival factors and maintenance cytokines IL-7 and IL-15. We therefore conclude that CXCR4 is not only crucial for entry of CD8+ T cells into the BM, but also controls their subsequent localization toward BM niches that support their survival.

Stiffness-Induced Endothelial DLC-1 Expression Forces Leukocyte Spreading through Stabilization of the ICAM-1 Adhesome.

Leukocytes follow the well-defined steps of rolling, spreading, and crawling prior to diapedesis through endothelial cells (ECs). We found increased expression of DLC-1 in stiffness-associated diseases like atherosclerosis and pulmonary arterial hypertension. Depletion of DLC-1 in ECs cultured on stiff substrates drastically reduced cell stiffness and mimicked leukocyte transmigration kinetics observed for ECs cultured on soft substrates. Mechanistic studies revealed that DLC-1-depleted ECs or ECs cultured on soft substrates failed to recruit the actin-adaptor proteins filamin B, α-actinin-4, and cortactin to clustered ICAM-1, thereby preventing the ICAM-1 adhesome formation and impairing leukocyte spreading. This was rescued by overexpressing DLC-1, resulting in ICAM-1 adhesome stabilization and leukocyte spreading. Our results reveal an essential role for substrate stiffness-regulated endothelial DLC-1, independent of its GAP domain, in locally stabilizing the ICAM-1 adhesome to promote leukocyte spreading, essential for efficient leukocyte transendothelial migration.

Interferon-Gamma Impairs Maintenance and Alters Hematopoietic Support of Bone Marrow Mesenchymal Stromal Cells.

Bone marrow (BM) mesenchymal stromal cells (MSCs) provide microenvironmental support to hematopoietic stem and progenitor cells (HSPCs). Culture-expanded MSCs are interesting candidates for cellular therapies due to their immunosuppressive and regenerative potential which can be further enhanced by pretreatment with interferon-gamma (IFN-γ). However, it remains unknown whether IFN-γ can also influence hematopoietic support by BM-MSCs. In this study, we elucidate the impact of IFN-γ on the hematopoietic support of BM-MSCs. We found that IFN-γ increases expression of interleukin (IL)-6 and stem cell factor by human BM-MSCs. IFN-γ-treated BM-MSCs drive HSPCs toward myeloid commitment in vitro, but impair subsequent differentiation of HSPC. Moreover, IFN-γ-ARE-Del mice with increased IFN-γ production specifically lose their BM-MSCs, which correlates with a loss of hematopoietic stem cells' quiescence. Although IFN-γ treatment enhances the immunomodulatory function of MSCs in a clinical setting, we conclude that IFN-γ negatively affects maintenance of BM-MSCs and their hematopoietic support in vitro and in vivo.

Inflammation-Sensitive Myosin-X Functionally Supports Leukocyte Extravasation by Cdc42-Mediated ICAM-1-Rich Endothelial Filopodia Formation.

Leukocyte transendothelial migration is key to inflammation. Leukocytes first start rolling over the inflamed endothelium, followed by firmly adhering to it. Under inflammatory conditions, endothelial cells express small finger-like protrusions that stick out into the lumen. The function and regulation of these structures are unclear. We present evidence that these ICAM-1- and F-actin-rich endothelial finger-like protrusions are filopodia and function as adhesive structures for leukocytes to transit from rolling to crawling but are dispensable for diapedesis. Mechanistically, these structures require the motor function of myosin-X, activity of the small GTPase Cdc42, and p21-activated kinase 4. Moreover, myosin-X expression is under control of TNF-α-mediated c-Jun N-terminal kinase activity and is upregulated in human atherosclerotic regions. To our knowledge, this is the first study to identify that regulation of endothelial filopodia is crucial for leukocyte extravasation, in particular for the initiation of leukocyte adhesion under flow conditions.

Tissue Transglutaminase contributes to experimental multiple sclerosis pathogenesis and clinical outcome by promoting macrophage migration.

Multiple sclerosis is a serious neurological disorder, resulting in e.g., sensory, motor and cognitive deficits. A critical pathological aspect of multiple sclerosis (MS) is the influx of immunomodulatory cells into the central nervous system (CNS). Identification of key players that regulate cellular trafficking into the CNS may lead to the development of more selective treatment to halt this process. The multifunctional enzyme tissue Transglutaminase (TG2) can participate in various inflammation-related processes, and is known to be expressed in the CNS. In the present study, we question whether TG2 activity contributes to the pathogenesis of experimental MS, and could be a novel therapeutic target. In human post-mortem material, we showed the appearance of TG2 immunoreactivity in leukocytes in MS lesions, and particular in macrophages in rat chronic-relapsing experimental autoimmune encephalomyelitis (cr-EAE), an experimental MS model. Clinical deficits as observed in mouse EAE were reduced in TG2 knock-out mice compared to littermate wild-type mice, supporting a role of TG2 in EAE pathogenesis. To establish if the enzyme TG2 represents an attractive therapeutic target, cr-EAE rats were treated with TG2 activity inhibitors during ongoing disease. Reduction of TG2 activity in cr-EAE animals dramatically attenuated clinical deficits and demyelination. The mechanism underlying these beneficial effects pointed toward a reduction in macrophage migration into the CNS due to attenuated cytoskeletal flexibility and RhoA GTPase activity. Moreover, iNOS and TNFα levels were selectively reduced in the CNS of cr-EAE rats treated with a TG2 activity inhibitor, whereas other relevant inflammatory mediators were not affected in CNS or spleen by reducing TG2 activity. We conclude that modulating TG2 activity opens new avenues for therapeutic intervention in MS which does not affect peripheral levels of inflammatory mediators.

P-glycoprotein regulates trafficking of CD8(+) T cells to the brain parenchyma.

The trafficking of cytotoxic CD8(+) T lymphocytes across the lining of the cerebral vasculature is key to the onset of the chronic neuro-inflammatory disorder multiple sclerosis. However, the mechanisms controlling their final transmigration across the brain endothelium remain unknown. Here, we describe that CD8(+) T lymphocyte trafficking into the brain is dependent on the activity of the brain endothelial adenosine triphosphate-binding cassette transporter P-glycoprotein. Silencing P-glycoprotein activity selectively reduced the trafficking of CD8(+) T cells across the brain endothelium in vitro as well as in vivo. In response to formation of the T cell-endothelial synapse, P-glycoprotein was found to regulate secretion of endothelial (C-C motif) ligand 2 (CCL2), a chemokine that mediates CD8(+) T cell migration in vitro. Notably, CCL2 levels were significantly enhanced in microvessels isolated from human multiple sclerosis lesions in comparison with non-neurological controls. Endothelial cell-specific elimination of CCL2 in mice subjected to experimental autoimmune encephalomyelitis also significantly diminished the accumulation of CD8(+) T cells compared to wild-type animals. Collectively, these results highlight a novel (patho)physiological role for P-glycoprotein in CD8(+) T cell trafficking into the central nervous system during neuro-inflammation and illustrate CCL2 secretion as a potential link in this mechanism.

Two CGD Families with a Hypomorphic Mutation in the Activation Domain of p67phox.

STUDY BACKGROUND: Chronic granulomatous Disease (CGD) is a rare immunodeficiency caused by a defect in the leukocyte NADPH oxidase. This enzyme generates superoxide, which is needed for the killing of bacteria and fungi by phagocytic leukocytes. Most CGD patients have mutations in CYBB, the X-linked gene that encodes gp91phox, the catalytic subunit of the leukocyte NADPH oxidase. We report here three autosomal recessive CGD patients from two families with a homozygous mutation in NCF2, the gene that encodes p67phox, the activator subunit of the NADPH oxidase. METHODS: Leukocyte NADPH oxidase activity, expression of oxidase components and gene sequences were measured with standard methods. The mutation found in the patients' NCF2 gene was expressed as Ala202Val-p67phox in K562 cells to measure its effect on NADPH oxidase activity. Translocation of the mutated p67phox from the cytosol of the patients' neutrophils to the plasma membrane was measured by confocal microscopy and by Western blotting after membrane purification. RESULTS: The exceptional feature of the A67 CGD patients reported here is that the p.Ala202Val mutation in the activation domain of p67phox was clearly hypomorphic: substantial expression of p67phox protein was noted and the NADPH oxidase activity in the neutrophils of the patients was 20-70% of normal, dependent on the stimulus used to activate the cells. The extent of Ala202Val-p67phox translocation to the plasma membrane during cell activation was also stimulus dependent. Ala202Val-p67phox in K562 cells mediated only about 3% of normal oxidase activity compared to cells transfected with the wild-type p67phox. CONCLUSION: The mutation found in NCF2 is the cause of the decreased NADPH oxidase activity and the (mild) clinical problems of the patients. We propose that the p.Ala202Val mutation has changed the conformation of the activation domain of p67phox, resulting in reduced activation of gp91phox.

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

OBJECTIVE: In aortic aneurysms the arterial vessel wall is dilated because of destruction of its integrity, which may lead to lethal vessel rupture. Chronic infiltration of inflammatory cells into the vessel wall is fundamental to aneurysm pathology. We aim to limit aneurysm growth by inhibition of inflammation and reducing endothelial cell (EC) activation with immunosuppressive drug azathioprine (Aza). APPROACH AND RESULTS: Aza and its metabolite 6-mercaptopurine have anti-inflammatory effects on leukocytes. We here demonstrate that treatment of ECs with 6-mercaptopurine inhibits cell activation as illustrated by reduced expression of interleukin-12, CCL5, CCL2, and vascular cell adhesion molecule-1 and inhibition of monocyte-EC adhesion. The underlying mechanism of 6-mercaptopurine involves suppression of GTPase Rac1 activation, resulting in reduced phosphorylation of c-Jun-terminal-N-kinase and c-Jun. Subsequently, the effect of Aza was investigated in aneurysm formation in the angiotensin II aneurysm mouse model in apolipoprotein E-deficient mice. We demonstrated that Aza decreases de novo aortic aneurysm formation from an average aneurysm severity score of 2.1 (control group) to 0.6 (Aza group), and that Aza effectively delays aorta pathology in a progression experiment, resulting in a reduced severity score from 2.8 to 1.7 in Aza-treated mice. In line with the in vitro observations, Aza-treated mice showed less c-Jun-terminal-N-kinase activation in ECs and reduced leukocyte influx in the aortic wall. CONCLUSIONS: The immunosuppressive drug Aza has an anti-inflammatory effect and in ECs inhibits Rac1 and c-Jun-terminal-N-kinase activation, which may explain the protective effect of Aza in aneurysm development and, most importantly for clinical implications, aneurysm severity.

Endothelial cell preservation at hypothermic to normothermic conditions using clinical and experimental organ preservation solutions.

INTRODUCTION: Endothelial barrier function is pivotal for the outcome of organ transplantation. Since hypothermic preservation (gold standard) is associated with cold-induced endothelial damage, endothelial barrier function may benefit from organ preservation at warmer temperatures. We therefore assessed endothelial barrier integrity and viability as function of preservation temperature and perfusion solution, and hypothesized that endothelial cell preservation at subnormothermic conditions using metabolism-supporting solutions constitute optimal preservation conditions. METHODS: Human umbilical vein endothelial cells (HUVEC) were preserved at 4-37°C for up to 20 h using Ringer's lactate, histidine-tryptophan-ketoglutarate solution, University of Wisconsin (UW) solution, Polysol, or endothelial cell growth medium (ECGM). Following preservation, the monolayer integrity, metabolic capacity, and ATP content were determined as positive parameters of endothelial cell viability. As negative parameters, apoptosis, necrosis, and cell activation were assayed. A viability index was devised on the basis of these parameters. RESULTS: HUVEC viability and barrier integrity was compromised at 4°C regardless of the preservation solution. At temperatures above 20°C, the cells' metabolic demands outweighed the preservation solutions' supporting capacity. Only UW maintained HUVEC viability up to 20°C. Despite high intracellular ATP content, none of the solutions were capable of sufficiently preserving HUVEC above 20°C except for ECGM. CONCLUSION: Optimal HUVEC preservation is achieved with UW up to 20°C. Only ECGM maintains HUVEC viability at temperatures above 20°C.

Shear stress-dependent downregulation of the adhesion-G protein-coupled receptor CD97 on circulating leukocytes upon contact with its ligand CD55.

Adhesion G protein-coupled receptors (aGPCRs) are two-subunit molecules, consisting of an adhesive extracellular α subunit that couples noncovalently to a seven-transmembrane ß subunit. The cooperation between the two subunits and the effect of endogenous ligands on the functioning of aGPCRs is poorly understood. In this study, we investigated the interaction between the pan-leukocyte aGPCR CD97 and its ligand CD55. We found that leukocytes from CD55-deficient mice express significantly increased levels of cell surface CD97 that normalized after transfer into wild-type mice because of contact with CD55 on both leukocytes and stromal cells. Downregulation of both CD97 subunits occurred within minutes after first contact with CD55 in vivo, which correlated with an increase in plasma levels of soluble CD97. In vitro, downregulation of CD97 on CD55-deficient leukocytes cocultured with wild-type blood cells was strictly dependent on shear stress. In vivo, CD55-mediated downregulation of CD97 required an intact circulation and was not observed on cells that lack contact with the blood stream, such as microglia. Notably, de novo ligation of CD97 did not activate signaling molecules constitutively engaged by CD97 in cancer cells, such as ERK and protein kinase B/Akt. We conclude that CD55 downregulates CD97 surface expression on circulating leukocytes by a process that requires physical forces, but based on current evidence does not induce receptor signaling. This regulation can restrict CD97-CD55-mediated cell adhesion to tissue sites.

The many faces of the guanine-nucleotide exchange factor trio.

Small Rho-GTPases are enzymes that are bound to GDP or GTP, which determines their inactive or active state, respectively. The exchange of GDP for GTP is catalyzed by so-called Rho-guanine nucleotide exchange factors (GEFs). Rho-GEFs are characterized by a Dbl-homology (DH) and adjacent Pleckstrin-homology (PH) domain that serves as enzymatic unit for the GDP/GTP exchange. Rho-GEFs show different GTPase specificities, meaning that a particular GEF can activate either multiple GTPases or only one specific GTPase. We recently reported that the Rho-GEF Trio, known to be able to exchange GTP on Rac1, RhoG and RhoA, regulates lamellipodia formation to mediate cell spreading and migration in a Rac1-dependent manner. In this commentary, we review the current knowledge of Trio in several aspects of cell biology.

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

OBJECTIVE: Atherosclerosis preferentially develops at sites of disturbed blood flow. We tested the hypothesis that transglutaminase activity plays a role in plaque development at these locations. METHODS AND RESULTS: Exposure of endothelial cells to steady flow (7 dynes/cm(2)) was associated with relatively low transglutaminase activity, whereas under low oscillatory flow (1.3 ± 2.6 dynes/cm(2)) endothelial cells showed a >4-fold higher level of transglutaminase activity. Under oscillatory flow, transglutaminase activity increased the expression of the chemokine MCP-1 (CCL2). In vivo, oscillatory flow was induced by placement of a tapered perivascular cast around the carotid artery of type 2 transglutaminase (TGM2) knockout mice and WT counterparts. After 2 days, significantly less monocytes adhered to the endothelium in TGM2 knockout mice as compared to WT. In a more chronic setting, ApoE knockout mice that were equipped with the flow-modifying cast developed lesions proximal to the cast (low shear stress), and distal to the cast (oscillatory shear stress). Inhibition of transglutaminase induced a marked reduction in macrophage and fat content in distal lesions only. In addition, lesion size was increased in this area, which was attributed to an increase in smooth muscle content. CONCLUSION: Oscillatory shear stress increases endothelial transglutaminase activity. In turn, transglutaminase activity affects the expression of MCP-1 in vitro and monocyte recruitment in vivo. In a mouse model of atherosclerosis, transglutaminase activity has a major effect on plaque composition under oscillatory shear stress.