Publications by authors named "Mark Hoogenboezem"

30 Publications

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Labeling and tracking of immune cells in ex vivo human skin.

Nat Protoc 2021 02 21;16(2):791-811. Epub 2020 Dec 21.

Division of Molecular Oncology and Immunology, Oncode Institute, The Netherlands Cancer Institute, Amsterdam, the Netherlands.

Human skin harbors various immune cells that are crucial for the control of injury and infection. However, the current understanding of immune cell function within viable human skin tissue is limited. We developed an ex vivo imaging approach in which fresh skin biopsies are mounted and then labeled with nanobodies or antibodies against cell surface markers on tissue-resident memory CD8 T cells, other immune cells of interest, or extracellular tissue components. Subsequent longitudinal imaging allows one to describe the dynamic behavior of human skin-resident cells in situ. In addition, this strategy can be used to study immune cell function in murine skin. The ability to follow the spatiotemporal behavior of CD8 T cells and other immune cells in skin, including their response to immune stimuli, provides a platform to investigate physiological immune cell behavior and immune cell behavior in skin diseases. The mounting, staining and imaging of skin samples requires ~1.5 d, and subsequent tracking analysis requires a minimum of 1 d. The optional production of fluorescently labeled nanobodies takes ~5 d.
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http://dx.doi.org/10.1038/s41596-020-00435-8DOI Listing
February 2021

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

Haematologica 2020 12 1;105(12):2746-2756. Epub 2020 Dec 1.

Sanquin Research and Landsteiner Laboratory, Academic Medical Center, Amsterdam, the Netherlands.

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.
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http://dx.doi.org/10.3324/haematol.2018.196329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7716366PMC
December 2020

SIRPα on Mouse B1 Cells Restricts Lymphoid Tissue Migration and Natural Antibody Production.

Front Immunol 2020 9;11:570963. Epub 2020 Oct 9.

Sanquin Research and Landsteiner Laboratory, Department of Blood Cell Research, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands.

The inhibitory immunoreceptor SIRPα is expressed on myeloid and neuronal cells and interacts with the broadly expressed CD47. CD47-SIRPα interactions form an innate immune checkpoint and its targeting has shown promising results in cancer patients. Here, we report expression of SIRPα on B1 lymphocytes, a subpopulation of murine B cells responsible for the production of natural antibodies. Mice defective in SIRPα signaling (SIRPα mice) displayed an enhanced CD11b/CD18 integrin-dependent B1 cell migration from the peritoneal cavity to the spleen, local B1 cell accumulation, and enhanced circulating natural antibody levels, which was further amplified upon immunization with T-independent type 2 antigen. As natural antibodies are atheroprotective, we investigated the involvement of SIRPα signaling in atherosclerosis development. Bone marrow (SIRPα>LDLR) chimaeric mice developed reduced atherosclerosis accompanied by increased natural antibody production. Collectively, our data identify SIRPα as a unique B1 cell inhibitory receptor acting to control B1 cell migration, and imply SIRPα as a potential therapeutic target in atherosclerosis.
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http://dx.doi.org/10.3389/fimmu.2020.570963DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7581795PMC
October 2020

Flow-induced Reorganization of Laminin-integrin Networks Within the Endothelial Basement Membrane Uncovered by Proteomics.

Mol Cell Proteomics 2020 07 24;19(7):1179-1192. Epub 2020 Apr 24.

Department of Molecular and Cellular Hemostasis, Sanquin Research, Amsterdam, The Netherlands

The vessel wall is continuously exposed to hemodynamic forces generated by blood flow. Endothelial mechanosensors perceive and translate mechanical signals via cellular signaling pathways into biological processes that control endothelial development, phenotype and function. To assess the hemodynamic effects on the endothelium on a system-wide level, we applied a quantitative mass spectrometry approach combined with cell surface chemical footprinting. SILAC-labeled endothelial cells were subjected to flow-induced shear stress for 0, 24 or 48 h, followed by chemical labeling of surface proteins using a non-membrane permeable biotin label, and analysis of the whole proteome and the cell surface proteome by LC-MS/MS analysis. These studies revealed that of the >5000 quantified proteins 104 were altered, which were highly enriched for extracellular matrix proteins and proteins involved in cell-matrix adhesion. Cell surface proteomics indicated that LAMA4 was proteolytically processed upon flow-exposure, which corresponded to the decreased LAMA4 mass observed on immunoblot. Immunofluorescence microscopy studies highlighted that the endothelial basement membrane was drastically remodeled upon flow exposure. We observed a network-like pattern of LAMA4 and LAMA5, which corresponded to the localization of laminin-adhesion molecules ITGA6 and ITGB4. Furthermore, the adaptation to flow-exposure did not affect the inflammatory response to tumor necrosis factor α, indicating that inflammation and flow trigger fundamentally distinct endothelial signaling pathways with limited reciprocity and synergy. Taken together, this study uncovers the blood flow-induced remodeling of the basement membrane and stresses the importance of the subendothelial basement membrane in vascular homeostasis.
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http://dx.doi.org/10.1074/mcp.RA120.001964DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7338090PMC
July 2020

Author Correction: Tissue patrol by resident memory CD8 T cells in human skin.

Nat Immunol 2020 06;21(6):696

Division of Molecular Oncology and Immunology, Oncode Institute, The Netherlands Cancer Institute, Amsterdam, the Netherlands.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41590-020-0665-xDOI Listing
June 2020

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

Sci Rep 2019 10 7;9(1):14401. Epub 2019 Oct 7.

Amsterdam UMC, University of Amsterdam, Department of Medical Biochemistry, Amsterdam Cardiovascular Sciences, Amsterdam, The Netherlands.

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.
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http://dx.doi.org/10.1038/s41598-019-50955-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6779744PMC
October 2019

LIM-only protein FHL2 attenuates vascular tissue factor activity, inhibits thrombus formation in mice and genetic variation associates with human venous thrombosis.

Haematologica 2020 Jun 29;105(6):1677-1685. Epub 2019 Aug 29.

Department of Medical Biochemistry, Amsterdam Cardiovascular Sciences, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands

Bleeding disorders and thrombotic complications are major causes of morbidity and mortality with many cases being unexplained. Thrombus formation involves aberrant expression and activation of tissue factor (TF) in vascular endothelial and smooth muscle cells. Here, we sought to identify factors that modulate gene expression and activity in these vascular cells. The LIM-only protein FHL2 is a scaffolding protein that modulates signal transduction pathways with crucial functions in endothelial and smooth muscle cells. However, the role of FHL2 in TF regulation and thrombosis remains unexplored. Using a murine model of venous thrombosis in mesenteric vessels, we demonstrated that FHL2 deficiency results in exacerbated thrombus formation. Gain- and loss-of-function experiments revealed that represses TF expression in endothelial and smooth muscle cells through inhibition of the transcription factors nuclear factor κB and activating protein-1. Furthermore, we observed that FHL2 interacts with the cytoplasmic tail of TF. In line with our observations, decreases TF activity in endothelial and smooth muscle cells whereas knockdown or deficiency results in enhanced TF activity. Finally, the single nucleotide polymorphism rs4851770 was associated with the risk of venous thrombosis in a large population of venous thrombosis cases and control subjects from 12 studies (INVENT consortium). Altogether, our results highlight functional involvement of FHL2 in TF-mediated coagulation and identify as a novel gene associated with venous thrombosis in humans.
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http://dx.doi.org/10.3324/haematol.2018.203026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7271603PMC
June 2020

Tissue patrol by resident memory CD8 T cells in human skin.

Nat Immunol 2019 06 20;20(6):756-764. Epub 2019 May 20.

Division of Molecular Oncology and Immunology, Oncode Institute, The Netherlands Cancer Institute, Amsterdam, the Netherlands.

Emerging data show that tissue-resident memory T (T) cells play an important protective role at murine and human barrier sites. T cells in the epidermis of mouse skin patrol their surroundings and rapidly respond when antigens are encountered. However, whether a similar migratory behavior is performed by human T cells is unclear, as technology to longitudinally follow them in situ has been lacking. To address this issue, we developed an ex vivo culture system to label and track T cells in fresh skin samples. We validated this system by comparing in vivo and ex vivo properties of murine T cells. Using nanobody labeling, we subsequently demonstrated in human ex vivo skin that CD8 T cells migrated through the papillary dermis and the epidermis, below sessile Langerhans cells. Collectively, this work allows the dynamic study of resident immune cells in human skin and provides evidence of tissue patrol by human CD8 T cells.
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http://dx.doi.org/10.1038/s41590-019-0404-3DOI Listing
June 2019

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

Eur J Immunol 2019 04 15;49(4):576-589. Epub 2019 Feb 15.

Department of Hematopoiesis, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

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.
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http://dx.doi.org/10.1002/eji.201747438DOI Listing
April 2019

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

Cell Rep 2018 09;24(12):3115-3124

Molecular Cell Biology Laboratory, Department of Plasma Proteins, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, 1066 CX Amsterdam, the Netherlands. Electronic address:

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.
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http://dx.doi.org/10.1016/j.celrep.2018.08.045DOI Listing
September 2018

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

J Immunol 2018 03 31;200(5):1790-1801. Epub 2018 Jan 31.

Molecular Cell Biology Laboratory, Department of Plasma Proteins, Sanquin Research and Landsteiner Laboratory, Academic Medical Center Amsterdam, University of Amsterdam, 1066 CX Amsterdam, the Netherlands;

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.
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http://dx.doi.org/10.4049/jimmunol.1700702DOI Listing
March 2018

GATA1-Deficient Dendritic Cells Display Impaired CCL21-Dependent Migration toward Lymph Nodes Due to Reduced Levels of Polysialic Acid.

J Immunol 2016 12 4;197(11):4312-4324. Epub 2016 Nov 4.

Department of Blood Cell Research, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam 1066CX, the Netherlands;

Dendritic cells (DCs) play a pivotal role in the regulation of the immune response. DC development and activation is finely orchestrated through transcriptional programs. GATA1 transcription factor is required for murine DC development, and data suggest that it might be involved in the fine-tuning of the life span and function of activated DCs. We generated DC-specific Gata1 knockout mice (Gata1-KO), which presented a 20% reduction of splenic DCs, partially explained by enhanced apoptosis. RNA sequencing analysis revealed a number of deregulated genes involved in cell survival, migration, and function. DC migration toward peripheral lymph nodes was impaired in Gata1-KO mice. Migration assays performed in vitro showed that this defect was selective for CCL21, but not CCL19. Interestingly, we show that Gata1-KO DCs have reduced polysialic acid levels on their surface, which is a known determinant for the proper migration of DCs toward CCL21.
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http://dx.doi.org/10.4049/jimmunol.1600103DOI Listing
December 2016

Repercussion of Megakaryocyte-Specific Gata1 Loss on Megakaryopoiesis and the Hematopoietic Precursor Compartment.

PLoS One 2016 6;11(5):e0154342. Epub 2016 May 6.

Dept. of Blood Cell Research, Sanquin Research and Landsteiner Laboratory, Academic Medical Centre (AMC), University of Amsterdam (UvA), Amsterdam, the Netherlands.

During hematopoiesis, transcriptional programs are essential for the commitment and differentiation of progenitors into the different blood lineages. GATA1 is a transcription factor expressed in several hematopoietic lineages and essential for proper erythropoiesis and megakaryopoiesis. Megakaryocyte-specific genes, such as GP1BA, are known to be directly regulated by GATA1. Mutations in GATA1 can lead to dyserythropoietic anemia and pseudo gray-platelet syndrome. Selective loss of Gata1 expression in adult mice results in macrothrombocytopenia with platelet dysfunction, characterized by an excess of immature megakaryocytes. To specifically analyze the impact of Gata1 loss in mature committed megakaryocytes, we generated Gata1-Lox|Pf4-Cre mice (Gata1cKOMK). Consistent with previous findings, Gata1cKOMK mice are macrothrombocytopenic with platelet dysfunction. Supporting this notion we demonstrate that Gata1 regulates directly the transcription of Syk, a tyrosine kinase that functions downstream of Clec2 and GPVI receptors in megakaryocytes and platelets. Furthermore, we show that Gata1cKOMK mice display an additional aberrant megakaryocyte differentiation stage. Interestingly, these mice present a misbalance of the multipotent progenitor compartment and the erythroid lineage, which translates into compensatory stress erythropoiesis and splenomegaly. Despite the severe thrombocytopenia, Gata1cKOMK mice display a mild reduction of TPO plasma levels, and Gata1cKOMK megakaryocytes show a mild increase in Pf4 mRNA levels; such a misbalance might be behind the general hematopoietic defects observed, affecting locally normal TPO and Pf4 levels at hematopoietic stem cell niches.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0154342PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4859556PMC
July 2017

Human Neutrophils Use Different Mechanisms To Kill Aspergillus fumigatus Conidia and Hyphae: Evidence from Phagocyte Defects.

J Immunol 2016 Feb 30;196(3):1272-83. Epub 2015 Dec 30.

Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, 1066 CX Amsterdam, the Netherlands; Department of Pediatric Hematology, Immunology, and Infectious Diseases, Emma Children's Hospital, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, the Netherlands;

Neutrophils are known to play a pivotal role in the host defense against Aspergillus infections. This is illustrated by the prevalence of Aspergillus infections in patients with neutropenia or phagocyte functional defects, such as chronic granulomatous disease. However, the mechanisms by which human neutrophils recognize and kill Aspergillus are poorly understood. In this work, we have studied in detail which neutrophil functions, including neutrophil extracellular trap (NET) formation, are involved in the killing of Aspergillus fumigatus conidia and hyphae, using neutrophils from patients with well-defined genetic immunodeficiencies. Recognition of conidia involves integrin CD11b/CD18 (and not dectin-1), which triggers a PI3K-dependent nonoxidative intracellular mechanism of killing. When the conidia escape from early killing and germinate, the extracellular destruction of the Aspergillus hyphae needs opsonization by Abs and involves predominantly recognition via Fcγ receptors, signaling via Syk, PI3K, and protein kinase C to trigger the production of toxic reactive oxygen metabolites by the NADPH oxidase and myeloperoxidase. A. fumigatus induces NET formation; however, NETs did not contribute to A. fumigatus killing. Thus, our findings reveal distinct killing mechanisms of Aspergillus conidia and hyphae by human neutrophils, leading to a comprehensive insight in the innate antifungal response.
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http://dx.doi.org/10.4049/jimmunol.1501811DOI Listing
February 2016

Acid Sphingomyelinase-Derived Ceramide Regulates ICAM-1 Function during T Cell Transmigration across Brain Endothelial Cells.

J Immunol 2016 Jan 23;196(1):72-9. Epub 2015 Nov 23.

Department of Molecular Cell Biology and Immunology, Neuroscience Campus Amsterdam, VU University Medical Center, 1007 MB Amsterdam, the Netherlands;

Multiple sclerosis (MS) is a chronic demyelinating disorder of the CNS characterized by immune cell infiltration across the brain vasculature into the brain, a process not yet fully understood. We previously demonstrated that the sphingolipid metabolism is altered in MS lesions. In particular, acid sphingomyelinase (ASM), a critical enzyme in the production of the bioactive lipid ceramide, is involved in the pathogenesis of MS; however, its role in the brain vasculature remains unknown. Transmigration of T lymphocytes is highly dependent on adhesion molecules in the vasculature such as intercellular adhesion molecule-1 (ICAM-1). In this article, we hypothesize that ASM controls T cell migration by regulating ICAM-1 function. To study the role of endothelial ASM in transmigration, we generated brain endothelial cells lacking ASM activity using a lentiviral shRNA approach. Interestingly, although ICAM-1 expression was increased in cells lacking ASM activity, we measured a significant decrease in T lymphocyte adhesion and consequently transmigration both in static and under flow conditions. As an underlying mechanism, we revealed that upon lack of endothelial ASM activity, the phosphorylation of ezrin was perturbed as well as the interaction between filamin and ICAM-1 upon ICAM-1 clustering. Functionally this resulted in reduced microvilli formation and impaired transendothelial migration of T cells. In conclusion, in this article, we show that ASM coordinates ICAM-1 function in brain endothelial cells by regulating its interaction with filamin and phosphorylation of ezrin. The understanding of these underlying mechanisms of T lymphocyte transmigration is of great value to develop new strategies against MS lesion formation.
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http://dx.doi.org/10.4049/jimmunol.1500702DOI Listing
January 2016

Characterization of hematopoietic GATA transcription factor expression in mouse and human dendritic cells.

Blood Cells Mol Dis 2015 Dec 14;55(4):293-303. Epub 2015 Jul 14.

Department of Blood Cell Research, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. Electronic address:

Dendritic cells (DCs) are key initiators and regulators of the immune response. The development of the DC lineage and their subsets requires an orchestrated regulation of their transcriptional program. Gata1, a transcription factor expressed in several hematopoietic cell lineages, has been recently reported to be required for mouse DC development and function. In humans, GATA1 is involved in the lineage separation between monocyte-derived DCs and Langerhans cells (LC) and loss of GATA1 results in differentiation arrest at the monocyte stage. The hematopoietic GATA factors (i.e. Gata1, Gata2, Gata3) are known to regulate each other's expression and to function consecutively throughout lineage commitment (so-called GATA switch). In humans, mutations in GATA2 are causative of MonoMAC disease, a human immunodeficiency syndrome characterized by loss of DCs, monocytes, B and NK cells. However, additional data on the expression of hematopoietic GATA factors in the DC lineage is missing. In this study, we have characterized the expression of hematopoietic GATA factors in murine and human DCs and their expression dynamics upon TLR stimulation. We found that all hematopoietic GATA factors are expressed in DCs, but identified species-specific differences in the relative expression of each GATA factor, and how their expression fluctuates upon stimulation.
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http://dx.doi.org/10.1016/j.bcmd.2015.07.006DOI Listing
December 2015

A local VE-cadherin and Trio-based signaling complex stabilizes endothelial junctions through Rac1.

J Cell Sci 2015 Aug 26;128(16):3041-54. Epub 2015 Jun 26.

Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam 1066 CX, The Netherlands

Endothelial cell-cell junctions maintain a restrictive barrier that is tightly regulated to allow dynamic responses to permeability-inducing angiogenic factors, as well as to inflammatory agents and adherent leukocytes. The ability of these stimuli to transiently remodel adherens junctions depends on Rho-GTPase-controlled cytoskeletal rearrangements. How the activity of Rho-GTPases is spatio-temporally controlled at endothelial adherens junctions by guanine-nucleotide exchange factors (GEFs) is incompletely understood. Here, we identify a crucial role for the Rho-GEF Trio in stabilizing junctions based around vascular endothelial (VE)-cadherin (also known as CDH5). Trio interacts with VE-cadherin and locally activates Rac1 at adherens junctions during the formation of nascent contacts, as assessed using a novel FRET-based Rac1 biosensor and biochemical assays. The Rac-GEF domain of Trio is responsible for the remodeling of junctional actin from radial into cortical actin bundles, a crucial step for junction stabilization. This promotes the formation of linear adherens junctions and increases endothelial monolayer resistance. Collectively, our data show the importance of spatio-temporal regulation of the actin cytoskeleton through Trio and Rac1 at VE-cadherin-based cell-cell junctions in the maintenance of the endothelial barrier.
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http://dx.doi.org/10.1242/jcs.168674DOI Listing
August 2015

Sp1/Sp3 transcription factors regulate hallmarks of megakaryocyte maturation and platelet formation and function.

Blood 2015 Mar 23;125(12):1957-67. Epub 2014 Dec 23.

Department of Cell Biology, Erasmus MC, Rotterdam, The Netherlands;

Sp1 and Sp3 belong to the specificity proteins (Sp)/Krüppel-like transcription factor family. They are closely related, ubiquitously expressed, and recognize G-rich DNA motifs. They are thought to regulate generic processes such as cell-cycle and growth control, metabolic pathways, and apoptosis. Ablation of Sp1 or Sp3 in mice is lethal, and combined haploinsufficiency results in hematopoietic defects during the fetal stages. Here, we show that in adult mice, conditional pan-hematopoietic (Mx1-Cre) ablation of either Sp1 or Sp3 has minimal impact on hematopoiesis, whereas the simultaneous loss of Sp1 and Sp3 results in severe macrothrombocytopenia. This occurs in a cell-autonomous manner as shown by megakaryocyte-specific (Pf4-Cre) double-knockout mice. We employed flow cytometry, cell culture, and electron microscopy and show that although megakaryocyte numbers are normal in bone marrow and spleen, they display a less compact demarcation membrane system and a striking inability to form proplatelets. Through megakaryocyte transcriptomics and platelet proteomics, we identified several cytoskeleton-related proteins and downstream effector kinases, including Mylk, that were downregulated upon Sp1/Sp3 depletion, providing an explanation for the observed defects in megakaryopoiesis. Supporting this notion, selective Mylk inhibition by ML7 affected proplatelet formation and stabilization and resulted in defective ITAM receptor-mediated platelet aggregation.
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http://dx.doi.org/10.1182/blood-2014-08-593343DOI Listing
March 2015

Real-time imaging of endothelial cell-cell junctions during neutrophil transmigration under physiological flow.

J Vis Exp 2014 Aug 14(90):e51766. Epub 2014 Aug 14.

Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory, AMC at University of Amsterdam;

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.
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http://dx.doi.org/10.3791/51766DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4758758PMC
August 2014

Actin-binding proteins differentially regulate endothelial cell stiffness, ICAM-1 function and neutrophil transmigration.

J Cell Sci 2014 Oct 8;127(Pt 20):4470-82. Epub 2014 Aug 8.

Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory, Academic Medical Center and Swammerdam Institute of Life Sciences, University of Amsterdam, 1066 CX Amsterdam, The Netherlands

Chronic vascular inflammation is driven by interactions between activated leukocytes and the endothelium. Leukocyte β2-integrins bind to endothelial intercellular adhesion molecule 1 (ICAM-1), which allows leukocyte spreading, crawling and transendothelial migration. Leukocytes scan the vascular endothelium for permissive sites to transmigrate, which suggests that there is apical membrane heterogeneity within the endothelium. However, the molecular basis for this heterogeneity is unknown. Leukocyte adhesion induces ICAM-1 clustering, which promotes its association to the actin-binding proteins filamin B, α-actinin-4 and cortactin. We show that these endothelial proteins differentially control adhesion, spreading and transmigration of neutrophils. Loss of filamin B, α-actinin-4 and cortactin revealed adaptor-specific effects on a nuclear-to-peripheral gradient of endothelial cell stiffness. By contrast, increasing endothelial cell stiffness stimulates ICAM-1 function. We identify endothelial α-actinin-4 as a key regulator of endothelial cell stiffness and of ICAM-1-mediated neutrophil transmigration. Finally, we found that the endothelial lining of human and murine atherosclerotic plaques shows elevated levels of α-actinin-4. These results identify endothelial cell stiffness as an important regulator of endothelial surface heterogeneity and of ICAM-1 function, which in turn controls the adhesion and transmigration of neutrophils.
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http://dx.doi.org/10.1242/jcs.154708DOI Listing
October 2014

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

J Immunol 2014 May 26;192(9):4370-8. Epub 2014 Mar 26.

Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands;

Azathioprine and its metabolite 6-mercaptopurine (6-MP) are well established immunosuppressive drugs. Common understanding of their immunosuppressive properties is largely limited to immune cells. However, in this study, the mechanism underlying the protective role of 6-MP in endothelial cell activation is investigated. Because 6-MP and its derivative 6-thioguanosine-5'-triphosphate (6-T-GTP) were shown to block activation of GTPase Rac1 in T lymphocytes, we focused on Rac1-mediated processes in endothelial cells. Indeed, 6-MP and 6-T-GTP decreased Rac1 activation in endothelial cells. As a result, the compounds inhibited TNF-α-induced downstream signaling via JNK and reduced activation of transcription factors c-Jun, activating transcription factor-2 and, in addition, NF κ-light-chain-enhancer of activated B cells (NF-κB), which led to decreased transcription of proinflammatory cytokines. Moreover, 6-MP and 6-T-GTP selectively decreased TNF-α-induced VCAM-1 but not ICAM-1 protein levels. Rac1-mediated generation of cell membrane protrusions, which form docking structures to capture leukocytes, also was reduced by 6-MP/6-T-GTP. Consequently, leukocyte transmigration was inhibited after 6-MP/6-T-GTP treatment. These data underscore the anti-inflammatory effect of 6-MP and 6-T-GTP on endothelial cells by blocking Rac1 activation. Our data provide mechanistic insight that supports development of novel Rac1-specific therapeutic approaches against chronic inflammatory diseases.
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http://dx.doi.org/10.4049/jimmunol.1302527DOI Listing
May 2014

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

Arterioscler Thromb Vasc Biol 2013 Oct 15;33(10):2380-8. Epub 2013 Aug 15.

From the Department of Medical Biochemistry (G.M., S.H., A.v.B., A.F.G., N.B., A.A.J.H., C.J.M.d.V., V.d.W.), Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory (M.H., J.D.v.B.), and Department of Cell Biology and Histology (J.S.), Academic Medical Center, University of Amsterdam, The Netherlands.

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.
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http://dx.doi.org/10.1161/ATVBAHA.113.301394DOI Listing
October 2013

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

Biol Open 2013 Jun 16;2(6):569-79. Epub 2013 Apr 16.

Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam , Amsterdam, 1066CX , The Netherlands.

Inflammation is characterized by endothelium that highly expresses numerous adhesion molecules to trigger leukocyte extravasation. Central to this event is increased gene transcription. Small Rho-GTPases not only control the actin cytoskeleton, but are also implicated in gene regulation. However, in inflammation, it is not clear how this is regulated. Here, we show that the guanine-nucleotide exchange factor Trio expression is increased upon inflammatory stimuli in endothelium. Additionally, increased Trio expression was found in the vessel wall of rheumatoid arthritis patients. Trio silencing impaired VCAM-1 expression. Finally, we excluded that Trio-controlled VCAM-1 expression used the classical NFκB or MAP-kinase pathways, but rather acts on the transcriptional level by increasing phosphorylation and nuclear translocalization of Ets2. These data implicate Trio in regulating inflammation and provide novel targets for therapeutic purposes to treat inflammatory diseases such as rheumatoid arthritis.
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http://dx.doi.org/10.1242/bio.20134382DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3683159PMC
June 2013

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

Mol Biol Cell 2012 Nov 5;23(21):4212-25. Epub 2012 Sep 5.

Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, 1066 CX Amsterdam, The Netherlands.

Impaired endothelial barrier function results in a persistent increase in endothelial permeability and vascular leakage. Repair of a dysfunctional endothelial barrier requires controlled restoration of adherens junctions, comprising vascular endothelial (VE)-cadherin and associated β-, γ-, α-, and p120-catenins. Little is known about the mechanisms by which recovery of VE-cadherin-mediated cell-cell junctions is regulated. Using the inflammatory mediator thrombin, we demonstrate an important role for the Src homology 2-domain containing tyrosine phosphatase (SHP2) in mediating recovery of the VE-cadherin-controlled endothelial barrier. Using SHP2 substrate-trapping mutants and an in vitro phosphatase activity assay, we validate β-catenin as a bona fide SHP2 substrate. SHP2 silencing and SHP2 inhibition both result in delayed recovery of endothelial barrier function after thrombin stimulation. Moreover, on thrombin challenge, we find prolonged elevation in tyrosine phosphorylation levels of VE-cadherin-associated β-catenin in SHP2-depleted cells. No disassembly of the VE-cadherin complex is observed throughout the thrombin response. Using fluorescence recovery after photobleaching, we show that loss of SHP2 reduces the mobility of VE-cadherin at recovered cell-cell junctions. In conclusion, our data show that the SHP2 phosphatase plays an important role in the recovery of disrupted endothelial cell-cell junctions by dephosphorylating VE-cadherin-associated β-catenin and promoting the mobility of VE-cadherin at the plasma membrane.
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http://dx.doi.org/10.1091/mbc.E12-01-0038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3484100PMC
November 2012

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

Atherosclerosis 2012 Oct 7;224(2):355-62. Epub 2012 Aug 7.

Department of Biomedical Engineering and Physics, Academic Medical Center, PO Box 22700, 1100 DE Amsterdam, The Netherlands.

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.
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http://dx.doi.org/10.1016/j.atherosclerosis.2012.07.044DOI Listing
October 2012

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

Mol Biol Cell 2012 Aug 13;23(15):2831-44. Epub 2012 Jun 13.

Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, 1066 Amsterdam, The Netherlands.

Leukocyte transendothelial migration involves the active participation of the endothelium through the formation of apical membrane protrusions that embrace adherent leukocytes, termed docking structures. Using live-cell imaging, we find that prior to transmigration, endothelial docking structures form around 80% of all neutrophils. Previously we showed that endothelial RhoG and SGEF control leukocyte transmigration. In this study, our data reveal that both full-length Trio and the first DH-PH (TrioD1) domain of Trio, which can activate Rac1 and RhoG, interact with ICAM-1 and are recruited to leukocyte adhesion sites. Moreover, upon clustering of ICAM-1, the Rho-guanine nucleotide exchange factor Trio activates Rac1, prior to activating RhoG, in a filamin-dependent manner. We further show that docking structure formation is initiated by ICAM-1 clustering into ring-like structures, which is followed by apical membrane protrusion. Interestingly, we find that Rac1 is required for ICAM-1 clustering, whereas RhoG controls membrane protrusion formation. Finally, silencing endothelial Trio expression or reducing TrioD1 activity without affecting SGEF impairs both docking structure formation and leukocyte transmigration. We conclude that Trio promotes leukocyte transendothelial migration by inducing endothelial docking structure formation in a filamin-dependent manner through the activation of Rac1 and RhoG.
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http://dx.doi.org/10.1091/mbc.E11-11-0907DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3408411PMC
August 2012

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

PLoS One 2012 6;7(1):e29912. Epub 2012 Jan 6.

Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

The guanine-nucleotide exchange factor Trio encodes two DH-PH domains that catalyze nucleotide exchange on Rac1, RhoG and RhoA. The N-terminal DH-PH domain is known to activate Rac1 and RhoG, whereas the C-terminal DH-PH domain can activate RhoA. The current study shows that the N-terminal DH-PH domain, upon expression in HeLa cells, activates Rac1 and RhoG independently from each other. In addition, we show that the flanking SH3 domain binds to the proline-rich region of the C-terminus of Rac1, but not of RhoG. However, this SH3 domain is not required for Rac1 or RhoG GDP-GTP exchange. Rescue experiments in Trio-shRNA-expressing cells showed that the N-terminal DH-PH domain of Trio, but not the C-terminal DH-PH domain, restored fibronectin-mediated cell spreading and migration defects that are observed in Trio-silenced cells. Kymograph analysis revealed that the N-terminal DH-PH domain, independent of its SH3 domain, controls the dynamics of lamellipodia. Using siRNA against Rac1 or RhoG, we found that Trio-D1-induced lamellipodia formation required Rac1 but not RhoG expression. Together, we conclude that the GEF Trio is responsible for lamellipodia formation through its N-terminal DH-PH domain in a Rac1-dependent manner during fibronectin-mediated spreading and migration.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0029912PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3253119PMC
May 2012

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

PLoS One 2010 Jun 28;5(6):e11336. Epub 2010 Jun 28.

Sanquin Research and Landsteiner Laboratory, Department of Molecular Cell Biology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

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

Principal Findings: Detailed scanning electron and fluorescent microscopy showed that the apical surface of endothelial cells constitutively forms small filopodia-like protrusions that are positive for ICAM-1 and freely move within the lateral plane of the membrane. Clustering of ICAM-1, using anti-ICAM-1 antibody-coated beads, efficiently and rapidly recruits ICAM-1. Using fluorescence recovery after photo-bleaching (FRAP), we found that clustering increased the immobile fraction of ICAM-1, compared to non-clustered ICAM-1. This shift required the intracellular portion of ICAM-1. Moreover, biochemical assays showed that ICAM-1 clustering recruited beta-actin and filamin. Cytochalasin B, which interferes with actin polymerization, delayed the clustering of ICAM-1. In addition, we could show that cytochalasin B decreased the immobile fraction of clustered ICAM-1-GFP, but had no effect on non-clustered ICAM-1. Also, the motor protein myosin-II is recruited to ICAM-1 adhesion sites and its inhibition increased the immobile fraction of both non-clustered and clustered ICAM-1. Finally, blocking Rac1 activation, the formation of lipid rafts, myosin-II activity or actin polymerization, but not Src, reduced the adhesive function of ICAM-1, tested under physiological flow conditions.

Conclusions: Together, these findings indicate that ICAM-1 clustering is regulated in an inside-out fashion through the actin cytoskeleton. Overall, these data indicate that signaling events within the endothelium are required for efficient ICAM-1-mediated leukocyte adhesion.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0011336PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2893160PMC
June 2010