Publications by authors named "Stephan Huveneers"

57 Publications

Vinculin controls endothelial cell junction dynamics during vascular lumen formation.

Cell Rep 2022 04;39(2):110658

Biozentrum der Universität Basel, Spitalstrasse 41, 4056 Basel, Switzerland. Electronic address:

Blood vessel morphogenesis is driven by coordinated endothelial cell behaviors. Active remodeling of cell-cell junctions promotes cellular plasticity while preserving vascular integrity. Here, we analyze the dynamics of endothelial adherens junctions during lumen formation in angiogenic sprouts in vivo. Live imaging in zebrafish reveals that lumen expansion is accompanied by the formation of transient finger-shaped junctions. Junctional fingers are positively regulated by blood pressure, whereas flow inhibition prevents their formation. Using fluorescent reporters, we show that junctional fingers contain the mechanotransduction protein vinculin. Furthermore, genetic deletion of vinculin prevents finger formation, a junctional defect that could be rescued by transient endothelial expression of vinculin. Our findings suggest a mechanism whereby lumen expansion leads to an increase in junctional tension, triggering recruitment of vinculin and formation of junctional fingers. We propose that endothelial cells employ force-dependent junctional remodeling to counteract external forces in order to maintain vascular integrity during sprouting angiogenesis.
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http://dx.doi.org/10.1016/j.celrep.2022.110658DOI Listing
April 2022

Bone Marrow Harbors a Unique Population of Dendritic Cells with the Potential to Boost Neutrophil Formation upon Exposure to Fungal Antigen.

Cells 2021 12 24;11(1). Epub 2021 Dec 24.

Department of Hematopoiesis, Sanquin Research, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands.

Apart from controlling hematopoiesis, the bone marrow (BM) also serves as a secondary lymphoid organ, as it can induce naïve T cell priming by resident dendritic cells (DC). When analyzing DCs in murine BM, we uncovered that they are localized around sinusoids, can (cross)-present antigens, become activated upon intravenous LPS-injection, and for the most part belong to the cDC2 subtype which is associated with Th2/Th17 immunity. Gene-expression profiling revealed that BM-resident DCs are enriched for several c-type lectins, including Dectin-1, which can bind beta-glucans expressed on fungi and yeast. Indeed, DCs in BM were much more efficient in phagocytosis of both yeast-derived zymosan-particles and than their splenic counterparts, which was highly dependent on Dectin-1. DCs in human BM could also phagocytose zymosan, which was dependent on β1-integrins. Moreover, zymosan-stimulated BM-resident DCs enhanced the differentiation of hematopoietic stem and progenitor cells towards neutrophils, while also boosting the maintenance of these progenitors. Our findings signify an important role for BM DCs as translators between infection and hematopoiesis, particularly in anti-fungal immunity. The ability of BM-resident DCs to boost neutrophil formation is relevant from a clinical perspective and contributes to our understanding of the increased susceptibility for fungal infections following BM damage.
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http://dx.doi.org/10.3390/cells11010055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8750392PMC
December 2021

Force-induced changes of α-catenin conformation stabilize vascular junctions independently of vinculin.

J Cell Sci 2021 12 22;134(24). Epub 2021 Dec 22.

Department of Vascular Cell Biology, Max Planck Institute for Molecular Biomedicine, D-48149 Münster, Germany.

Cadherin-mediated cell adhesion requires anchoring via the β-catenin-α-catenin complex to the actin cytoskeleton, yet, α-catenin only binds F-actin weakly. A covalent fusion of VE-cadherin to α-catenin enhances actin anchorage in endothelial cells and strongly stabilizes endothelial junctions in vivo, blocking inflammatory responses. Here, we have analyzed the underlying mechanism. We found that VE-cadherin-α-catenin constitutively recruits the actin adaptor vinculin. However, removal of the vinculin-binding region of α-catenin did not impair the ability of VE-cadherin-α-catenin to enhance junction integrity. Searching for an alternative explanation for the junction-stabilizing mechanism, we found that an antibody-defined epitope, normally buried in a short α1-helix of the actin-binding domain (ABD) of α-catenin, is openly displayed in junctional VE-cadherin-α-catenin chimera. We found that this epitope became exposed in normal α-catenin upon triggering thrombin-induced tension across the VE-cadherin complex. These results suggest that the VE-cadherin-α-catenin chimera stabilizes endothelial junctions due to conformational changes in the ABD of α-catenin that support constitutive strong binding to actin.
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http://dx.doi.org/10.1242/jcs.259012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8729784PMC
December 2021

Diverse ultrastructural landscape of atherosclerotic endothelium.

Atherosclerosis 2021 12 19;339:35-45. Epub 2021 Nov 19.

Experimental Vascular Biology, Department of Medical Biochemistry, Amsterdam University Medical Center, Amsterdam, 1105, AZ, the Netherlands; Institute for Cardiovascular Prevention, Ludwig Maximilians University, Munich, 80336, Germany; German Center for Cardiovascular Research (DZHK), Partner Site Munich Heart Alliance, Munich, Germany.

Background And Aims: The endothelium plays a major role in atherosclerosis, yet the endothelial plaque surface is a largely uncharted territory. Here we hypothesize that atherosclerosis-driven remodeling of the endothelium is a dynamic process, involving both damaging and regenerative mechanisms.

Methods: Using scanning electron microscopy (SEM) and immuno-SEM, we studied endothelial junction ultrastructure, endothelial openings and immune cell-endothelium interactions in eight apoe mice and two human carotid plaques.

Results: The surface of early mouse plaques (n = 11) displayed a broad range of morphological alterations, including junctional disruptions and large transcellular endothelial pores with the average diameter between 0.6 and 3 μm. The shoulder region of advanced atherosclerotic lesions (n = 7) had a more aggravated morphology with 8 μm-size paracellular openings at two-fold higher density. In contrast, the central apical surface of advanced plaques, i.e., the plaque body (n = 7), displayed endothelial normalization, as shown by a significantly higher frequency of intact endothelial junctions and a lower incidence of paracellular pores. This normalized endothelial phenotype correlated with low immune cell density (only 5 cells/mm). The human carotid plaque surface (n = 2) displayed both well-organized and disrupted endothelium with similar features as described above. In addition, they were accompanied by extensive thrombotic areas.

Conclusions: Our study unveils the spectrum of endothelial abnormalities associated with the development of atherosclerosis. These were highly abundant in early lesions and in the shoulder region of advanced plaques, while normalized at the advanced plaque's body. Similar endothelial features were observed in human atherosclerotic plaques, underlining the versatility of endothelial transformations in atherosclerosis.
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http://dx.doi.org/10.1016/j.atherosclerosis.2021.11.017DOI Listing
December 2021

Immune Checkpoint Inhibitor Therapy Aggravates T Cell-Driven Plaque Inflammation in Atherosclerosis.

JACC CardioOncol 2020 Nov 6;2(4):599-610. Epub 2020 Oct 6.

Department of Medical Biochemistry, Amsterdam Cardiovascular Sciences, Amsterdam University Medical Centers, University of Amsterdam, Amsterdam, the Netherlands.

Background: Immunotherapy has revolutionized cancer treatment. However, immune checkpoint inhibitors (ICIs) that target PD-1 (programmed cell death protein-1) and/or CTLA-4 (cytotoxic T lymphocyte-associated antigen-4) are commonly associated with acute immune-related adverse events. Accumulating evidence also suggests that ICIs aggravate existing inflammatory diseases.

Objectives: As inflammation drives atherosclerotic cardiovascular disease, we studied the propensity of short-term ICI therapy to aggravate atherosclerosis.

Methods: We used F-FDG (2-deoxy-2-[fluorine-18]fluoro-D-glucose) positron emission tomography-computed tomography to detect macrophage-driven vascular and systemic inflammation in pembrolizumab and nivolumab/ipilimumab-treated melanoma patients. In parallel, atherosclerotic mice were treated with CTLA-4 and PD-1 inhibition to study the proinflammatory consequences of immune checkpoint inhibition.

Results: ICI treatment did not affect F-FDG uptake in the large arteries, spleen, and bone marrow of melanoma patients, nor myeloid cell activation in blood and lymphoid organs in hyperlipidemic mice. In contrast, we found marked changes in the adaptive immune response (i.e., increased CD4 effector T cell and CD8 cytotoxic T cell numbers in lymphoid organs and the arterial wall of our hyperlipidemic mice). Although plaque size was unaffected, plaques had progressed toward a lymphoid-based inflammatory phenotype, characterized by a 2.7-fold increase of CD8 T cells and a 3.9-fold increase in necrotic core size. Increased endothelial activation was observed with a 2.2-fold and 1.6-fold increase in vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, respectively.

Conclusions: This study demonstrates that combination therapy with anti-CTLA-4 and anti-PD-1 antibodies does not affect myeloid-driven vascular and systemic inflammation in melanoma patients and hyperlipidemic mice. However, short-term ICI therapy in mice induces T cell-mediated plaque inflammation and drives plaque progression.
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http://dx.doi.org/10.1016/j.jaccao.2020.08.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8352210PMC
November 2020

Endothelial Focal Adhesions Are Functional Obstacles for Leukocytes During Basolateral Crawling.

Front Immunol 2021 18;12:667213. Epub 2021 May 18.

Molecular Cell Biology Lab, Department of Molecular Hematology, Sanquin Research and Landsteiner Laboratory, Amsterdam, Netherlands.

An inflammatory response requires leukocytes to migrate from the circulation across the vascular lining into the tissue to clear the invading pathogen. Whereas a lot of attention is focused on how leukocytes make their way through the endothelial monolayer, it is less clear how leukocytes migrate underneath the endothelium before they enter the tissue. Upon finalization of the diapedesis step, leukocytes reside in the subendothelial space and encounter endothelial focal adhesions. Using TIRF microscopy, we show that neutrophils navigate around these focal adhesions. Neutrophils recognize focal adhesions as physical obstacles and deform to get around them. Increasing the number of focal adhesions by silencing the small GTPase RhoJ slows down basolateral crawling of neutrophils. However, apical crawling and diapedesis itself are not affected by RhoJ depletion. Increasing the number of focal adhesions drastically by expressing the Rac1 GEF Tiam1 make neutrophils to avoid migrating underneath these Tiam1-expressing endothelial cells. Together, our results show that focal adhesions mark the basolateral migration path of neutrophils.
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http://dx.doi.org/10.3389/fimmu.2021.667213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8167051PMC
October 2021

A junctional PACSIN2/EHD4/MICAL-L1 complex coordinates VE-cadherin trafficking for endothelial migration and angiogenesis.

Nat Commun 2021 05 10;12(1):2610. Epub 2021 May 10.

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

Angiogenic sprouting relies on collective migration and coordinated rearrangements of endothelial leader and follower cells. VE-cadherin-based adherens junctions have emerged as key cell-cell contacts that transmit forces between cells and trigger signals during collective cell migration in angiogenesis. However, the underlying molecular mechanisms that govern these processes and their functional importance for vascular development still remain unknown. We previously showed that the F-BAR protein PACSIN2 is recruited to tensile asymmetric adherens junctions between leader and follower cells. Here we report that PACSIN2 mediates the formation of endothelial sprouts during angiogenesis by coordinating collective migration. We show that PACSIN2 recruits the trafficking regulators EHD4 and MICAL-L1 to the rear end of asymmetric adherens junctions to form a recycling endosome-like tubular structure. The junctional PACSIN2/EHD4/MICAL-L1 complex controls local VE-cadherin trafficking and thereby coordinates polarized endothelial migration and angiogenesis. Our findings reveal a molecular event at force-dependent asymmetric adherens junctions that occurs during the tug-of-war between endothelial leader and follower cells, and allows for junction-based guidance during collective migration in angiogenesis.
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http://dx.doi.org/10.1038/s41467-021-22873-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8110786PMC
May 2021

Endothelial Barrier Function and Leukocyte Transmigration in Atherosclerosis.

Biomedicines 2021 Mar 24;9(4). Epub 2021 Mar 24.

Department of Vascular Surgery, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands.

The vascular endothelium is a highly specialized barrier that controls passage of fluids and migration of cells from the lumen into the vessel wall. Endothelial cells assist leukocytes to extravasate and despite the variety in the specific mechanisms utilized by different leukocytes to cross different vascular beds, there is a general principle of capture, rolling, slow rolling, arrest, crawling, and ultimately diapedesis via a paracellular or transcellular route. In atherosclerosis, the barrier function of the endothelium is impaired leading to uncontrolled leukocyte extravasation and vascular leakage. This is also observed in the neovessels that grow into the atherosclerotic plaque leading to intraplaque hemorrhage and plaque destabilization. This review focuses on the vascular endothelial barrier function and the interaction between endothelial cells and leukocytes during transmigration. We will discuss the role of endothelial dysfunction, transendothelial migration of leukocytes and plaque angiogenesis in atherosclerosis.
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http://dx.doi.org/10.3390/biomedicines9040328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8063931PMC
March 2021

Endothelial YAP/TAZ Signaling in Angiogenesis and Tumor Vasculature.

Front Oncol 2020 4;10:612802. Epub 2021 Feb 4.

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

Solid tumors are dependent on vascularization for their growth. The hypoxic, stiff, and pro-angiogenic tumor microenvironment induces angiogenesis, giving rise to an immature, proliferative, and permeable vasculature. The tumor vessels promote tumor metastasis and complicate delivery of anti-cancer therapies. In many types of tumors, YAP/TAZ activation is correlated with increased levels of angiogenesis. In addition, endothelial YAP/TAZ activation is important for the formation of new blood and lymphatic vessels during development. Oncogenic activation of YAP/TAZ in tumor cell growth and invasion has been studied in great detail, however the role of YAP/TAZ within the tumor endothelium remains insufficiently understood, which complicates therapeutic strategies aimed at targeting YAP/TAZ in cancer. Here, we overview the upstream signals from the tumor microenvironment that control endothelial YAP/TAZ activation and explore the role of their downstream targets in driving tumor angiogenesis. We further discuss the potential for anti-cancer treatments and vascular normalization strategies to improve tumor therapies.
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http://dx.doi.org/10.3389/fonc.2020.612802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7890025PMC
February 2021

Editorial: Endothelial Dynamics in Health and Disease.

Front Physiol 2020 19;11:611117. Epub 2020 Nov 19.

Department of Medical Biochemistry, Amsterdam Cardiovascular Sciences, Amsterdam University Medical Center, Location AMC, University of Amsterdam, Amsterdam, Netherlands.

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http://dx.doi.org/10.3389/fphys.2020.611117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7711136PMC
November 2020

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

Microembolus clearance through angiophagy is an auxiliary mechanism preserving tissue perfusion in the rat brain.

Acta Neuropathol Commun 2020 11 17;8(1):195. Epub 2020 Nov 17.

Amsterdam UMC, Biomedical Engineering and Physics, Amsterdam Cardiovascular Sciences, University of Amsterdam, Meibergdreef 9, Amsterdam, The Netherlands.

Considering its intolerance to ischemia, it is of critical importance for the brain to efficiently process microvascular occlusions and maintain tissue perfusion. In addition to collateral microvascular flow and enzymatic degradation of emboli, the endothelium has the potential to engulf microparticles and thereby recanalize the vessel, through a process called angiophagy. Here, we set out to study the dynamics of angiophagy in relation to cytoskeletal remodeling in vitro and reperfusion in vivo. We show that polystyrene microspheres and fibrin clots are actively taken up by (brain) endothelial cells in vitro, and chart the dynamics of the actin cytoskeleton during this process using live cell imaging. Whereas microspheres were taken up through the formation of a cup structure by the apical endothelial membrane, fibrin clots were completely engulfed by the cells, marked by dense F-actin accumulation surrounding the clot. Both microspheres and fibrin clots were retained in the endothelial cells. Notably, fibrin clots were not degraded intracellularly. Using an in vivo microembolization rat model, in which microparticles are injected into the common carotid artery, we found that microspheres are transported by the endothelium from the microvasculature into the brain parenchyma. Microembolization with microspheres caused temporal opening of the blood-brain barrier and vascular nonperfusion, followed by microsphere extravasation and restoration of vessel perfusion over time. Taken together, angiophagy is accompanied by active cytoskeletal remodeling of the endothelium, and is an effective mechanism to restore perfusion of the occluded microvasculature in vivo.
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http://dx.doi.org/10.1186/s40478-020-01071-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7671188PMC
November 2020

Antibody-Mediated Inhibition of CTLA4 Aggravates Atherosclerotic Plaque Inflammation and Progression in Hyperlipidemic Mice.

Cells 2020 08 29;9(9). Epub 2020 Aug 29.

Department of Medical Biochemistry, Amsterdam Cardiovascular Sciences (ACS), Amsterdam University Medical Centers, University of Amsterdam, 1105AZ Amsterdam, The Netherlands.

T cell-driven inflammation plays a critical role in the initiation and progression of atherosclerosis. The co-inhibitory protein Cytotoxic T-Lymphocyte Associated protein (CTLA) 4 is an important negative regulator of T cell activation. Here, we studied the effects of the antibody-mediated inhibition of CTLA4 on experimental atherosclerosis by treating 6-8-week-old Ldlr mice, fed a 0.15% cholesterol diet for six weeks, biweekly with 200 μg of CTLA4 antibodies or isotype control for six weeks. F-fluorodeoxyglucose Positron Emission Tomography-Computed Tomography showed no effect of the CTLA4 inhibition of activity in the aorta, spleen, and bone marrow, indicating that monocyte/macrophage-driven inflammation was unaffected. Correspondingly, flow cytometry demonstrated that the antibody-mediated inhibition of CTLA4 did not affect the monocyte populations in the spleen. αCTLA4 treatment induced an activated T cell profile, characterized by a decrease in naïve CD44CD62LCD4 T cells and an increase in CD44CD62L CD4 and CD8 T cells in the blood and lymphoid organs. Furthermore, αCTLA4 treatment induced endothelial activation, characterized by increased ICAM1 expression in the aortic endothelium. In the aortic arch, which mainly contained early atherosclerotic lesions at this time point, αCTLA4 treatment induced a 2.0-fold increase in the plaque area. These plaques had a more advanced morphological phenotype and an increased T cell/macrophage ratio, whereas the smooth muscle cell and collagen content decreased. In the aortic root, a site that contained more advanced plaques, αCTLA4 treatment increased the plaque T cell content. The short-term antibody-mediated inhibition of CTLA4 thus accelerated the progression of atherosclerosis by inducing a predominantly T cell-driven inflammation, and resulted in the formation of plaques with larger necrotic cores and less collagen. This indicates that existing therapies that are based on αCTLA4 antibodies may promote CVD development in patients.
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http://dx.doi.org/10.3390/cells9091987DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7565685PMC
August 2020

The MARCH6-SQLE Axis Controls Endothelial Cholesterol Homeostasis and Angiogenic Sprouting.

Cell Rep 2020 08;32(5):107944

Department of Medical Biochemistry, Amsterdam University Medical Centers, Amsterdam Cardiovascular Sciences, Amsterdam Gastroenterology and Metabolism, University of Amsterdam, Meibergdreef 9, 1105AZ Amsterdam, the Netherlands. Electronic address:

The endothelial monolayer forms a barrier between the lumen of blood vessels and the underlying tissues. Stable VE-cadherin-based adherens junctions are essential for maintaining this barrier, whereas their remodeling is required for angiogenesis in health and disease. Here, we position the ERAD-associated ubiquitin ligase MARCH6 as a determinant of angiogenic sprouting and barrier integrity through its ability to promote the degradation of the rate-limiting cholesterol biosynthetic enzyme squalene epoxidase (SQLE). Accordingly, MARCHF6 ablation in endothelial cells increases SQLE protein and cholesterol load. This leads to altered membrane order, disorganized adherens junctions, decreased endothelial barrier function, and impaired SQLE-dependent sprouting angiogenesis. Akin to MARCHF6 silencing, the overexpression of SQLE impairs angiogenesis. However, angiogenesis is also attenuated when SQLE is silenced, indicating that fine-tuning cholesterol biosynthesis is a determinant of healthy endothelial function. In summary, we propose a mechanistic link between regulation of cholesterol homeostasis by the MARCH6-SQLE axis and endothelial integrity and angiogenesis.
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http://dx.doi.org/10.1016/j.celrep.2020.107944DOI Listing
August 2020

Long non-coding RNA LASSIE regulates shear stress sensing and endothelial barrier function.

Commun Biol 2020 05 26;3(1):265. Epub 2020 May 26.

Dept. of Physiology, Amsterdam Cardiovascular Sciences (ACS), Amsterdam UMC, VU University Medical Center, Amsterdam, The Netherlands.

Blood vessels are constantly exposed to shear stress, a biomechanical force generated by blood flow. Normal shear stress sensing and barrier function are crucial for vascular homeostasis and are controlled by adherens junctions (AJs). Here we show that AJs are stabilized by the shear stress-induced long non-coding RNA LASSIE (linc00520). Silencing of LASSIE in endothelial cells impairs cell survival, cell-cell contacts and cell alignment in the direction of flow. LASSIE associates with junction proteins (e.g. PECAM-1) and the intermediate filament protein nestin, as identified by RNA affinity purification. The AJs component VE-cadherin showed decreased stabilization, due to reduced interaction with nestin and the microtubule cytoskeleton in the absence of LASSIE. This study identifies LASSIE as link between nestin and VE-cadherin, and describes nestin as crucial component in the endothelial response to shear stress. Furthermore, this study indicates that LASSIE regulates barrier function by connecting AJs to the cytoskeleton.
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http://dx.doi.org/10.1038/s42003-020-0987-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7251106PMC
May 2020

Cell-cell junctions as sensors and transducers of mechanical forces.

Biochim Biophys Acta Biomembr 2020 09 28;1862(9):183316. Epub 2020 Apr 28.

Amsterdam UMC, University of Amsterdam, Location AMC, Department of Medical Biochemistry, Amsterdam Cardiovascular Sciences, Amsterdam, the Netherlands. Electronic address:

Epithelial and endothelial monolayers are multicellular sheets that form barriers between the 'outside' and 'inside' of tissues. Cell-cell junctions, made by adherens junctions, tight junctions and desmosomes, hold together these monolayers. They form intercellular contacts by binding their receptor counterparts on neighboring cells and anchoring these structures intracellularly to the cytoskeleton. During tissue development, maintenance and pathogenesis, monolayers encounter a range of mechanical forces from the cells themselves and from external systemic forces, such as blood pressure or tissue stiffness. The molecular landscape of cell-cell junctions is diverse, containing transmembrane proteins that form intercellular bonds and a variety of cytoplasmic proteins that remodel the junctional connection to the cytoskeleton. Many junction-associated proteins participate in mechanotransduction cascades to confer mechanical cues into cellular responses that allow monolayers to maintain their structural integrity. We will discuss force-dependent junctional molecular events and their role in cell-cell contact organization and remodeling.
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http://dx.doi.org/10.1016/j.bbamem.2020.183316DOI Listing
September 2020

Bosutinib prevents vascular leakage by reducing focal adhesion turnover and reinforcing junctional integrity.

J Cell Sci 2020 05 14;133(9). Epub 2020 May 14.

Amsterdam UMC, Vrije Universiteit Amsterdam, Department of Pulmonology, Amsterdam Cardiovascular Sciences, 1081 BT Amsterdam, The Netherlands

Endothelial barrier dysfunction leads to edema and vascular leak, causing high morbidity and mortality. Previously, Abl kinase inhibition has been shown to protect against vascular leak. Using the distinct inhibitory profiles of clinically available Abl kinase inhibitors, we aimed to provide a mechanistic basis for novel treatment strategies against vascular leakage syndromes. We found that the inhibitor bosutinib most potently protected against inflammation-induced endothelial barrier disruption. , bosutinib prevented lipopolysaccharide (LPS)-induced alveolar protein extravasation in an acute lung injury mice model. Mechanistically, mitogen-activated protein 4 kinase 4 (MAP4K4) was identified as important novel mediator of endothelial permeability, which signaled via ezrin, radixin and moesin proteins to increase turnover of integrin-based focal adhesions. The combined inhibition of MAP4K4 and Abl-related gene (Arg, also known as ABL2) by bosutinib preserved adherens junction integrity and reduced turnover of focal adhesions, which synergistically act to stabilize the endothelial barrier during inflammation. We conclude that MAP4K4 is an important regulator of endothelial barrier integrity, increasing focal adhesion turnover and disruption of cell-cell junctions during inflammation. Because it inhibits both Arg and MAP4K4, use of the clinically available drug bosutinib might form a viable strategy against vascular leakage syndromes.
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http://dx.doi.org/10.1242/jcs.240077DOI Listing
May 2020

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

J Cell Sci 2020 02 12;133(3). Epub 2020 Feb 12.

Amsterdam UMC, University of Amsterdam, location AMC, Department of Medical Biochemistry, Amsterdam Cardiovascular Sciences, 1105 AZ Amsterdam, The Netherlands

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.
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http://dx.doi.org/10.1242/jcs.239947DOI Listing
February 2020

Real-time imaging of multivesicular body-plasma membrane fusion to quantify exosome release from single cells.

Nat Protoc 2020 01 13;15(1):102-121. Epub 2019 Dec 13.

Amsterdam UMC, Vrije Universiteit Amsterdam, Department of Pathology, Cancer Center Amsterdam, de Boelelaan 1117, Amsterdam, The Netherlands.

Exosomes are small extracellular vesicles with a diameter of 40-150 nm, and are implicated in cellular homeostasis and cell-cell communication. They can be secreted in bulk in response to cell-extrinsic and cell-intrinsic signals that cause multivesicular body (MVB) fusion with the plasma membrane (PM). However, research on the regulation of exosome release is hampered by the failure of current methods to capture the dynamics of exosome release. Here we describe how live imaging with tetraspanin-based pH-sensitive fluorescent reporters can quantify the MVB-PM fusion rate of single cells. Our approach enables identification of exogenous stimuli, signaling pathways, and fusion complexes, and can map subcellular sites of fusion events. In addition, dual-color imaging can be used to assess simultaneous release of different cargo by MVB exocytosis. This protocol describes the complete imaging experiment, consisting of transient expression of tetraspanin reporters (2 d), live-cell (dual-color) total internal reflection fluorescence microscopy (30-60 min per condition), and semiautomatic image analysis by using a newly developed ImageJ macro (±30 min per condition).
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http://dx.doi.org/10.1038/s41596-019-0245-4DOI Listing
January 2020

Junction Mapper is a novel computer vision tool to decipher cell-cell contact phenotypes.

Elife 2019 12 3;8. Epub 2019 Dec 3.

National Heart and Lung Institute, National Institutes of Health, London, United Kingdom.

Stable cell-cell contacts underpin tissue architecture and organization. Quantification of junctions of mammalian epithelia requires laborious manual measurements that are a major roadblock for mechanistic studies. We designed Junction Mapper as an open access, semi-automated software that defines the status of adhesiveness via the simultaneous measurement of pre-defined parameters at cell-cell contacts. It identifies contacting interfaces and corners with minimal user input and quantifies length, area and intensity of junction markers. Its ability to measure fragmented junctions is unique. Importantly, junctions that considerably deviate from the contiguous staining and straight contact phenotype seen in epithelia are also successfully quantified (i.e. cardiomyocytes or endothelia). Distinct phenotypes of junction disruption can be clearly differentiated among various oncogenes, depletion of actin regulators or stimulation with other agents. Junction Mapper is thus a powerful, unbiased and highly applicable software for profiling cell-cell adhesion phenotypes and facilitate studies on junction dynamics in health and disease.
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http://dx.doi.org/10.7554/eLife.45413DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7034980PMC
December 2019

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

Nanoparticle-Aided Characterization of Arterial Endothelial Architecture during Atherosclerosis Progression and Metabolic Therapy.

ACS Nano 2019 12 10;13(12):13759-13774. Epub 2019 Jul 10.

Experimental Vascular Biology, Department of Medical Biochemistry, Amsterdam Cardiovascular Sciences (ACS) , Amsterdam University Medical Center , Amsterdam 1105 AZ , The Netherlands.

Atherosclerosis is associated with a compromised endothelial barrier, facilitating the accumulation of immune cells and macromolecules in atherosclerotic lesions. In this study, we investigate endothelial barrier integrity and the enhanced permeability and retention (EPR) effect during atherosclerosis progression and therapy in mice using hyaluronan nanoparticles (HA-NPs). Utilizing ultrastructural and plaque imaging, we uncover a significantly decreased junction continuity in the atherosclerotic plaque-covering endothelium compared to the normal vessel wall, indicative of disrupted endothelial barrier. Intriguingly, the plaque advancement had a positive effect on junction stabilization, which correlated with a 3-fold lower accumulation of administrated HA-NPs in advanced plaques compared to early counterparts. Furthermore, by using super-resolution and correlative light and electron microscopy, we trace nanoparticles in the plaque microenvironment. We find nanoparticle-enriched endothelial junctions, containing 75% of detected HA-NPs, and a high HA-NP accumulation in the endothelium-underlying extracellular matrix, which suggest an endothelial junctional traffic of HA-NPs to the plague. Finally, we probe the EPR effect by HA-NPs in the context of metabolic therapy with a glycolysis inhibitor, 3PO, proposed as a vascular normalizing strategy. The observed trend of attenuated HA-NP uptake in aortas of 3PO-treated mice coincides with the endothelial silencing activity of 3PO, demonstrated Interestingly, the therapy also reduced the plaque inflammatory burden, while activating macrophage metabolism. Our findings shed light on natural limitations of nanoparticle accumulation in atherosclerotic plaques and provide mechanistic insight into nanoparticle trafficking across the atherosclerotic endothelium. Furthermore, our data contribute to the rising field of endothelial barrier modulation in atherosclerosis.
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http://dx.doi.org/10.1021/acsnano.8b08875DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6933811PMC
December 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

Opening the vascular gate.

Nat Nanotechnol 2019 03;14(3):195-196

Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.

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http://dx.doi.org/10.1038/s41565-019-0372-7DOI Listing
March 2019

Endothelial cell rearrangements during vascular patterning require PI3-kinase-mediated inhibition of actomyosin contractility.

Nat Commun 2018 11 16;9(1):4826. Epub 2018 Nov 16.

Vascular Signalling Laboratory, ProCURE, Oncobell Program, Institut d´Investigació Biomèdica de Bellvitge (IDIBELL), Gran Via de l'Hospitalet 199, 08908, L´Hospitalet de Llobregat, Barcelona, Spain.

Angiogenesis is a dynamic process relying on endothelial cell rearrangements within vascular tubes, yet the underlying mechanisms and functional relevance are poorly understood. Here we show that PI3Kα regulates endothelial cell rearrangements using a combination of a PI3Kα-selective inhibitor and endothelial-specific genetic deletion to abrogate PI3Kα activity during vessel development. Quantitative phosphoproteomics together with detailed cell biology analyses in vivo and in vitro reveal that PI3K signalling prevents NUAK1-dependent phosphorylation of the myosin phosphatase targeting-1 (MYPT1) protein, thereby allowing myosin light chain phosphatase (MLCP) activity and ultimately downregulating actomyosin contractility. Decreased PI3K activity enhances actomyosin contractility and impairs junctional remodelling and stabilization. This leads to overstretched endothelial cells that fail to anastomose properly and form aberrant superimposed layers within the vasculature. Our findings define the PI3K/NUAK1/MYPT1/MLCP axis as a critical pathway to regulate actomyosin contractility in endothelial cells, supporting vascular patterning and expansion through the control of cell rearrangement.
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http://dx.doi.org/10.1038/s41467-018-07172-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6240100PMC
November 2018

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

Nuclear Receptor Nur77 Limits the Macrophage Inflammatory Response through Transcriptional Reprogramming of Mitochondrial Metabolism.

Cell Rep 2018 08;24(8):2127-2140.e7

Amsterdam UMC, University of Amsterdam, Medical Biochemistry, Amsterdam Cardiovascular Sciences, Meibergdreef 9, Amsterdam 1105 AZ, The Netherlands. Electronic address:

Activation of macrophages by inflammatory stimuli induces reprogramming of mitochondrial metabolism to support the production of pro-inflammatory cytokines and nitric oxide. Hallmarks of this metabolic rewiring are downregulation of α-ketoglutarate formation by isocitrate dehydrogenase (IDH) and accumulation of glutamine-derived succinate, which enhances the inflammatory response via the activity of succinate dehydrogenase (SDH). Here, we identify the nuclear receptor Nur77 (Nr4a1) as a key upstream transcriptional regulator of this pro-inflammatory metabolic switch in macrophages. Nur77-deficient macrophages fail to downregulate IDH expression and accumulate higher levels of succinate and other TCA cycle-derived metabolites in response to inflammatory stimulation in a glutamine-independent manner. Consequently, these macrophages produce more nitric oxide and pro-inflammatory cytokines in an SDH-dependent manner. In vivo, bone marrow Nur77 deficiency exacerbates atherosclerosis development and leads to increased circulating succinate levels. In summary, Nur77 induces an anti-inflammatory metabolic state in macrophages that protects against chronic inflammatory diseases such as atherosclerosis.
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http://dx.doi.org/10.1016/j.celrep.2018.07.065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6113932PMC
August 2018

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

Stem Cells Dev 2018 05 16;27(9):579-589. Epub 2018 Apr 16.

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

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.
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http://dx.doi.org/10.1089/scd.2017.0196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5934977PMC
May 2018

Sensing of Cytoskeletal Forces by Asymmetric Adherens Junctions.

Trends Cell Biol 2018 04 28;28(4):328-341. Epub 2017 Nov 28.

Department of Medical Biochemistry, Academic Medical Center, Amsterdam Cardiovascular Sciences, University of Amsterdam, Amsterdam, The Netherlands. Electronic address:

Within tissues, key cellular adaptations occur via mechanotransduction responses at cell-cell junctions. Adherens junctions (AJs) typically form between cells as a result of the binding of cadherin receptors of the same type (homotypic), and are linked to the force-propagating and -generating actomyosin cytoskeleton. Recent studies have found that AJs maintain monolayer integrity in dynamic tissues and drive collective cell behavior by converting into asymmetric remodeling entities. Here, we overview the molecular processes that may explain how asymmetric cell-cell junctions sense differences in cytoskeletal geometry between cells. We discuss the link between cadherin-complex dynamics and the actomyosin cytoskeleton at asymmetric cell-cell junctions. We then outline the role of Bin/Amphiphysin/Rvs (BAR) proteins, cytoplasmic regulators of endocytosis and cytoskeletal dynamics that sense force-induced membrane curvature, at AJs undergoing asymmetric remodeling. Lastly, we highlight the physiological importance of junctional asymmetry for epithelial and vascular tissue and discuss its potential role in disease.
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http://dx.doi.org/10.1016/j.tcb.2017.11.002DOI Listing
April 2018

Cell-cell junctional mechanotransduction in endothelial remodeling.

Cell Mol Life Sci 2017 01 9;74(2):279-292. Epub 2016 Aug 9.

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

The vasculature is one of the most dynamic tissues that encounter numerous mechanical cues derived from pulsatile blood flow, blood pressure, activity of smooth muscle cells in the vessel wall, and transmigration of immune cells. The inner layer of blood and lymphatic vessels is covered by the endothelium, a monolayer of cells which separates blood from tissue, an important function that it fulfills even under the dynamic circumstances of the vascular microenvironment. In addition, remodeling of the endothelial barrier during angiogenesis and trafficking of immune cells is achieved by specific modulation of cell-cell adhesion structures between the endothelial cells. In recent years, there have been many new discoveries in the field of cellular mechanotransduction which controls the formation and destabilization of the vascular barrier. Force-induced adaptation at endothelial cell-cell adhesion structures is a crucial node in these processes that challenge the vascular barrier. One of the key examples of a force-induced molecular event is the recruitment of vinculin to the VE-cadherin complex upon pulling forces at cell-cell junctions. Here, we highlight recent advances in the current understanding of mechanotransduction responses at, and derived from, endothelial cell-cell junctions. We further discuss their importance for vascular barrier function and remodeling in development, inflammation, and vascular disease.
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http://dx.doi.org/10.1007/s00018-016-2325-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5219012PMC
January 2017
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