Papers by G. Van Nieuw Amerongen
Blood, 2010
The shear stress-induced transcription factor Krüppel-like factor 2 (KLF2) confers antiinflammato... more The shear stress-induced transcription factor Krüppel-like factor 2 (KLF2) confers antiinflammatory properties to endothelial cells through the inhibition of activator protein 1, presumably by interfering with mitogen-activated protein kinase (MAPK) cascades. To gain insight into the regulation of these cascades by KLF2, we used antibody arrays in combination with time-course mRNA microarray analysis. No gross changes in MAPKs were detected; rather, phosphorylation of actin cytoskeleton-associated proteins, including focal adhesion kinase, was markedly repressed by KLF2. Furthermore, we demonstrate that KLF2-mediated inhibition of Jun NH(2)-terminal kinase (JNK) and its downstream targets ATF2/c-Jun is dependent on the cytoskeleton. Specifically, KLF2 directs the formation of typical short basal actin filaments, termed shear fibers by us, which are distinct from thrombin- or tumor necrosis factor-alpha-induced stress fibers. KLF2 is shown to be essential for shear stress-induced cell alignment, concomitant shear fiber assembly, and inhibition of JNK signaling. These findings link the specific effects of shear-induced KLF2 on endothelial morphology to the suppression of JNK MAPK signaling in vascular homeostasis via novel actin shear fibers.
American Journal of Respiratory and Critical Care Medicine, 2016
Altered pulmonary hemodynamics and high fluid shear stress (HSS) are characteristic hallmarks in ... more Altered pulmonary hemodynamics and high fluid shear stress (HSS) are characteristic hallmarks in the pathogenesis of Pulmonary Arterial Hypertension (PAH). However, the contribution of HSS to cellular and vascular alterations in PAH is unclear. We hypothesize that failing shear-adaptation is an essential part of the endothelial dysfunction in all forms of PAH and tested whether microvascular (MVEC) respectively arterial endothelial cells (PAEC) from PAH patient lungs adapt to HSS and if the shear-defect partakes in vascular remodeling in-vivo. PAH MVEC (n=7) and PAH PAEC (n=3) morphology, function, protein and gene expressions were compared to control MVEC (n=8) under static culture conditions and after 24, 72, and 120 hours HSS. PAH MVEC showed a significantly delayed morphological shear-adaptation (p=0.03) and evidence of cell injury at sites of non-uniform shear-profiles that are critical loci for vascular remodeling in PAH. In clear contrast, PAEC isolated from the same PAH lungs showed no impairments. PAH MVEC gene expression and transcriptional shear-activation were not altered, but showed significant decreased protein levels (p=0.02) and disturbed inter-endothelial localization of the shear-sensor PECAM-1. The decreased PECAM-1 levels were caused by caspase-mediated cytoplasmic cleavage, but not increased cell apoptosis. Caspase blockade stabilized PECAM-1 levels, restored endothelial shear-responsiveness in-vitro and attenuated occlusive vascular remodeling in the SuHx rat model of severe PAH. Delayed shear-adaptation, which promotes shear-induced endothelial injury, is a newly identified dysfunction specific to the microvascular endothelium in PAH. The shear-response is normalized upon stabilization of PECAM-1, which reverses intimal remodeling in-vivo.
Vascular Pharmacology, 2012
Furthermore, the absence of SMOC-1 leads to an impaired endothelial cell tube formation in vitro.... more Furthermore, the absence of SMOC-1 leads to an impaired endothelial cell tube formation in vitro. In vivo cell infiltration (of CD45+ cells) into Matrigel plugs was increased after 3 days in the presence of SMOC-1 and showed more prominent angiogenesis after another 7 days. The Xenopus and Drosophila homologues of SMOC-1 have been reported to interact with BMP signalling. Thus, we determined whether this is also the case for the human homologue. Coexpression of human SMOC-1 and BMP-response element luciferase-constructs in human embryonic kidney cells showed an attenuated luciferase signal.
Atherosclerosis Supplements, 2010
Aims
Endothelial cells (ECs) control vascular permeability by forming a monolayer that is sealed ... more Aims
Endothelial cells (ECs) control vascular permeability by forming a monolayer that is sealed by extracellular junctions. Various mediators modulate the endothelial barrier by acting on junctional protein complexes and the therewith con- nected F-actin cytoskeleton. Different Rho GTPases participate in this modulation, but their mechanisms are still partly resolved. Here, we aimed to elucidate whether the opening and closure of the endothelial barrier are asso-
ciated with distinct localized RhoA activities at the subcellular level.
Methods and results
Live fluorescence resonance energy transfer (FRET) microscopy revealed spatially distinct RhoA activities associated
with different aspects of the regulation of endothelial monolayer integrity. Unstimulated ECs were characterized by
hotspots of RhoA activity at their periphery. Thrombin receptor activation in the femoral vein of male wistar rats and
in cultured ECs enhanced RhoA activity at membrane protrusions, followed by a more sustained RhoA activity
associated with cytoplasmic F-actin filaments, where prolonged RhoA activity coincided with cellular contractility.
Unexpectedly, thrombin-induced peripheral RhoA hotspots were not spatially correlated to the formation of
large inter-endothelial gaps. Rather, spontaneous RhoA activity at membrane protrusions coincided with the
closure of inter-endothelial gaps. Electrical impedance measurements showed that RhoA signalling is essential for
this protrusive activity and maintenance of barrier restoration.
Conclusion
Spontaneous RhoA activity at membrane protrusions is spatially associated with closure, but not formation of inter- endothelial gaps, whereas RhoA activity at distant contractile filaments contributes to thrombin-induced disruption of junctional integrity. Thus, these data indicate that distinct RhoA activities are associated with disruption and re- annealing of endothelial junctions.
Keywords
Rho GTPase †FRET microscopy †Cytoskeleton †Endothelial function †Vasoactive agents
British journal of anaesthesia, 2016
The mechanisms causing increased endothelial permeability after cardiopulmonary bypass (CPB) have... more The mechanisms causing increased endothelial permeability after cardiopulmonary bypass (CPB) have not been elucidated. Using a bioassay for endothelial barrier function, we investigated whether endothelial hyperpermeability is associated with alterations in plasma endothelial activation and adhesion markers and can be attenuated by the use of pulsatile flow during CPB. Patients undergoing cardiac surgery were randomized to non-pulsatile (n=20) or pulsatile flow CPB (n=20). Plasma samples were obtained before (pre-CPB) and after CPB (post-CPB), and upon intensive care unit (ICU) arrival. Changes in plasma endothelial activation and adhesion markers were determined by enzyme-linked immunosorbent assay. Using electric cell-substrate impedance sensing of human umbilical vein endothelial monolayers, the effects of plasma exposure on endothelial barrier function were assessed and expressed as resistance. Cardiopulmonary bypass was associated with increased P-selectin, vascular cell adhesi...
Intensive Care Medicine, 2007
ABSTRACT
Atherosclerosis Supplements, 2010
Thorax, 2008
Angiopoietin-2 and vascular endothelial growth factor (VEGF) may impair vascular barrier function... more Angiopoietin-2 and vascular endothelial growth factor (VEGF) may impair vascular barrier function while angiopoietin-1 may protect it. It was hypothesised that circulating angiopoietin-2 is associated with pulmonary permeability oedema and severity of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) during septic or non-septic critical illness. Plasma levels of angiopoietin-1 and angiopoietin-2 were measured in mechanically ventilated patients (24 with sepsis, 88 without sepsis), together with the pulmonary leak index (PLI) for 67-gallium-labelled transferrin and extravascular lung water (EVLW) by transpulmonary thermal-dye dilution as measures of pulmonary permeability and oedema, respectively. ALI/ARDS was characterised by consensus criteria and the lung injury score (LIS). Plasma VEGF and von Willebrand factor (VWF) levels were assayed. Angiopoietin-2, VWF, PLI, EVLW and LIS were higher in patients with sepsis than in those without sepsis and higher in patients ...
The European respiratory journal, 2006
Cholesterol-lowering statins can ameliorate severe forms of vascular hyperpermeability in experim... more Cholesterol-lowering statins can ameliorate severe forms of vascular hyperpermeability in experimental studies, and may thereby ameliorate acute lung injury and sepsis. It is unknown whether this also applies to humans. This study aimed to define whether or not prior statin therapy reduces mild post-operative increases in pulmonary capillary protein permeability associated with acute lung injury after cardiac or major vascular surgery. A prospective observational study was performed in an intensive care unit of a university hospital on 64 patients, 37 after elective cardiac and 27 after major vascular surgery, of whom 68 and 44%, respectively, had received prior statin therapy. A mobile probe system was used to measure the pulmonary leak index (PLI), i.e. the transvascular transport rate of gallium-67-radiolabelled transferrin. For all of the patients together, the mean PLI did not differ between the statin and control groups (22.9 versus 24.4 x 10(-3) min(-1)). The prevalence of an...
Adhesion Protein Protocols, 1999
ABSTRACT
Fibrinolysis, 1996
Summary The plasminogen activation (PA) system is involved in vascular remodelling. Modulating it... more Summary The plasminogen activation (PA) system is involved in vascular remodelling. Modulating its activity in vascular cells might be a way to interfere in processes such as angiogenesis and restenosis. Adenoviral vectors have become a favourable tool for direct gene transfer into vascular cells. In the interest of using adenoviral vectors to modulate plasminogen activator activity and endothelial and smooth muscle cell migration, we studied the effects of endothelial and smooth muscle cell transduction in vitro and in the umbilical vein ex vivo with a replication-defective adenoviral vector containing the 13-galactosidase gene (AdCMVLacZ). Segments of the umbilical vein were infected with AdCMVLacZ (109-10~°pfu/ml). After 48 h strong 13-galactosidase expression could be observed in the vessel wall, which was restricted to the endothelial layer. Although some heterogeneity in the transduction throughout the vessel could be seen, 13-galactosidase expression was detectable for 21 days in explant. Infection of human vascular endothelial cells (HUVEC) with recombinant adenoviral vectors is a dose and time dependent process. To achieve 100% infection of cultured HUVEC after a 30 min infection period, adenovirus concentrations ranging from 10 ~° to 5.10 '° pfu/ml are required. To achieve a similar infection of HUVSMC a concentration of 2.109 to 10 '° is sufficient. Expression of I~-galactosidase can be detected for at least 14 days. Effects of transduction of HUVEC with AdCMVLacZ on proliferation, morphology and monolayer integrity were also studied. At high virus concentrations (> 10 '9 pfu/ml) an inhibitory effect on cell proliferation was detected and cell morphology displayed many giant cells, often polynuclear, in these cultures. Infection of confluent HUVEC monolayers had, apart from a rapid transient effect during the infection procedure, little effect on the permeability of these monolayers. Only when the monolayers were infected using concentrations of 10 '° pfu/ml or more, an increase in permeability of these monolayers could be observed.
The Journal of cell biology, Jan 5, 2015
The role of the RhoGTPase Rac1 in stabilizing mature endothelial adherens junctions (AJs) is not ... more The role of the RhoGTPase Rac1 in stabilizing mature endothelial adherens junctions (AJs) is not well understood. In this paper, using a photoactivatable probe to control Rac1 activity at AJs, we addressed the relationship between Rac1 and the dynamics of vascular endothelial cadherin (VE-cadherin). We demonstrated that Rac1 activation reduced the rate of VE-cadherin dissociation, leading to increased density of VE-cadherin at AJs. This response was coupled to a reduction in actomyosin-dependent tension across VE-cadherin adhesion sites. We observed that inhibiting myosin II directly or through photo-release of the caged Rho kinase inhibitor also reduced the rate of VE-cadherin dissociation. Thus, Rac1 functions by stabilizing VE-cadherin trans-dimers in mature AJs by counteracting the actomyosin tension. The results suggest a new model of VE-cadherin adhesive interaction mediated by Rac1-induced reduction of mechanical tension at AJs, resulting in the stabilization of VE-cadherin a...
Thrombosis and Haemostasis, 2009
In the past decade understanding of the role of the Rho GTPases RhoA, Rac1 and Cdc42 has been dev... more In the past decade understanding of the role of the Rho GTPases RhoA, Rac1 and Cdc42 has been developed from regulatory proteins that regulate specific actin cytoskeletal structures -stress fibers, lamellipodia and filopodia -to complex integrators of cytoskeletal structures that can exert multiple functions depending on the cellular context. Fundamental to these functions are three-dimensional complexes between the individual Rho GTPases, their specific activators (GEFs) and inhibitors (GDIs and GAPs), which greatly outnumber the Rho GTPases themselves, and additional regulatory proteins. By this complexity of regulation different vasoactive mediators can induce various cytoskeletal structures that enable the endothelial cell (EC) to respond adequately. In this review we have focused on this complexity and the consequences of Rho GTPase regulation for endothelial barrier function. The permeability inducers thrombin and VEGF are presented as examples of G-protein coupled receptor-and tyrosine kinase receptor-mediated Rho GTPase activation, respectively. These mediators induce complex but markedly different networks of activators, inhibitors and effectors of Rho GTPases, which alter the endothelial barrier function. An interesting feature in this regulation is that Rho GTPases often have both barrier-protecting and barrier-disturbing functions. While Rac1 enforces the endothelial junctions, it becomes part of a barrier-disturbing mechanism as activator of reactive oxygen species generating NADPH oxidase. Similarly RhoA is protective under basal conditions, but becomes involved in barrier dysfunction after activation of ECs by thrombin. The challenge and promise lies in unfolding this complex regulation, as this will provide leads for new therapeutic opportunities.
Proceedings of the National Academy of Sciences, 1996
Histamine H2 receptors transfected in Chinese hamster ovary (CHO) cells are time-and dosedependen... more Histamine H2 receptors transfected in Chinese hamster ovary (CHO) cells are time-and dosedependently upregulated upon exposure to the H2 antagonists cimetidine and ranitidine. This effect appears to be H2 receptor-mediated as no change in receptor density was observed after H1 or H3 antagonist treatment or after incubation with the structural analogue of cimetidine, VUF 8299, which has no H2 antagonistic effects. By using transfected CHO cells expressing different densities of wild-type H2 receptors or an uncoupled H2Leut24Ala receptor, the histamine H2 receptor was found to display considerable agonist-independent H2 receptor activity. Cimetidine and ranitidine, which both induce H2 receptor upregulation, actually functioned as inverse agonists in those cell lines displaying spontaneous agonistindependent H2 receptor activity. Burimamide, on the other hand, was shown to act as a neutral antagonist and did as expected not induce H2 receptor upregulation after long-term exposure. The displayed inverse agonism of H2 antagonists appears to be a mechanistic basis for the observed H2 antagonist-induced H2 receptor upregulation in transfected CHO cells. These observations shed new light on the pharmacological classification of the H2 antagonists and may offer a plausible explanation for the observed development of tolerance after prolonged clinical use.
Molecular Microbiology, 1996
Abf1p and Rap1p are global regulatory factors which play an essential role in the transcription a... more Abf1p and Rap1p are global regulatory factors which play an essential role in the transcription activation of yeast ribosomal protein genes. This functional link prompted us to investigate whether these factors may be functionally interchangeable. We focused on the indispensable C-terminal portions of both factors and performed mutual domain swaps. The functional capacity of the resulting hybrid proteins was subsequently examined using yeast strains conditionally expressing either the ABF1 or the RAP1 gene. Both the Abf1p-Rap1p and the Rap1p-Abf1p fusion proteins were found to be able to complement the growth defect of the respective strains. Furthermore, Abf1p and Rap1p are both able to promote transcription of a reporter gene through a combination of the respective binding site and a T-rich promoter element. These data strongly suggest that the C-terminal domains of Abf1p and Rap1p have, at least partially, identical functions. Finally, a deletion analysis of the so far largely uncharacterized C-terminal domain of Abf1p was performed, which revealed that two regions of 50 amino acids can perform all essential Abf1p functions.
Molecular Biology of the Cell, 2012
Endothelial nitric oxide synthase (eNOS)-mediated NO production plays a critical role in the regu... more Endothelial nitric oxide synthase (eNOS)-mediated NO production plays a critical role in the regulation of vascular function and pathophysiology. Caveolin-1 (Cav-1) binding to eNOS holds eNOS in an inactive conformation; however, the mechanism of Cav-1-mediated inhibition of activated eNOS is unclear. Here the role of Src-dependent Cav-1 phosphorylation in eNOS negative feedback regulation is investigated. Using fluorescence resonance energy transfer (FRET) and coimmunoprecipitation analyses, we observed increased interaction between eNOS and Cav-1 following stimulation of endothelial cells with thrombin, vascular endothelial growth factor, and Ca 2+ ionophore A23187, which is corroborated in isolated perfused mouse lung. The eNOS/Cav-1 interaction is blocked by eNOS inhibitor l-N Gnitroarginine methyl ester (hydrochloride) and Src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3, 4-d] pyrimidine. We also observe increased binding of phosphomimicking Y14D-Cav-1 mutant transduced in human embryonic kidney cells overexpressing eNOS and reduced Ca 2+ -induced NO production compared to cells expressing the phosphodefective Y14F-Cav-1 mutant. Finally, Src FRET biosensor, eNOS small interfering RNA, and NO donor studies demonstrate NO-induced Src activation and Cav-1 phosphorylation at Tyr-14, resulting in increased eNOS/Cav-1 interaction and inhibition of eNOS activity. Taken together, these data suggest that activation of eNOS promotes Src-dependent Cav-1-Tyr-14 phosphorylation and eNOS/Cav-1 binding, that is, eNOS feedback inhibition.
Journal of Crohn's and Colitis, 2013
Journal of Anatomy, 2002
The endothelium dynamically regulates the extravasation of hormones, macromolecules and other sol... more The endothelium dynamically regulates the extravasation of hormones, macromolecules and other solutes. In pathological conditions, endothelial hyperpermeability can be induced by vasoactive agents, which induce tiny leakage sites between the cells, and by cytokines, in particular vascular endothelial growth factor, which increase the exchange of plasma proteins by vesicles and intracellular pores. It is generally believed that the interaction of actin and non-muscle myosin in the periphery of the endothelial cell, and the destabilization of endothelial junctions, are required for endothelial hyperpermeability induced by vasoactive agents. Transient short-term hyperpermeability induced by histamine involves Ca 2+ /calmodulin-dependent activation of the myosin light chain (MLC) kinase.
Uploads
Papers by G. Van Nieuw Amerongen
Endothelial cells (ECs) control vascular permeability by forming a monolayer that is sealed by extracellular junctions. Various mediators modulate the endothelial barrier by acting on junctional protein complexes and the therewith con- nected F-actin cytoskeleton. Different Rho GTPases participate in this modulation, but their mechanisms are still partly resolved. Here, we aimed to elucidate whether the opening and closure of the endothelial barrier are asso-
ciated with distinct localized RhoA activities at the subcellular level.
Methods and results
Live fluorescence resonance energy transfer (FRET) microscopy revealed spatially distinct RhoA activities associated
with different aspects of the regulation of endothelial monolayer integrity. Unstimulated ECs were characterized by
hotspots of RhoA activity at their periphery. Thrombin receptor activation in the femoral vein of male wistar rats and
in cultured ECs enhanced RhoA activity at membrane protrusions, followed by a more sustained RhoA activity
associated with cytoplasmic F-actin filaments, where prolonged RhoA activity coincided with cellular contractility.
Unexpectedly, thrombin-induced peripheral RhoA hotspots were not spatially correlated to the formation of
large inter-endothelial gaps. Rather, spontaneous RhoA activity at membrane protrusions coincided with the
closure of inter-endothelial gaps. Electrical impedance measurements showed that RhoA signalling is essential for
this protrusive activity and maintenance of barrier restoration.
Conclusion
Spontaneous RhoA activity at membrane protrusions is spatially associated with closure, but not formation of inter- endothelial gaps, whereas RhoA activity at distant contractile filaments contributes to thrombin-induced disruption of junctional integrity. Thus, these data indicate that distinct RhoA activities are associated with disruption and re- annealing of endothelial junctions.
Keywords
Rho GTPase †FRET microscopy †Cytoskeleton †Endothelial function †Vasoactive agents
Endothelial cells (ECs) control vascular permeability by forming a monolayer that is sealed by extracellular junctions. Various mediators modulate the endothelial barrier by acting on junctional protein complexes and the therewith con- nected F-actin cytoskeleton. Different Rho GTPases participate in this modulation, but their mechanisms are still partly resolved. Here, we aimed to elucidate whether the opening and closure of the endothelial barrier are asso-
ciated with distinct localized RhoA activities at the subcellular level.
Methods and results
Live fluorescence resonance energy transfer (FRET) microscopy revealed spatially distinct RhoA activities associated
with different aspects of the regulation of endothelial monolayer integrity. Unstimulated ECs were characterized by
hotspots of RhoA activity at their periphery. Thrombin receptor activation in the femoral vein of male wistar rats and
in cultured ECs enhanced RhoA activity at membrane protrusions, followed by a more sustained RhoA activity
associated with cytoplasmic F-actin filaments, where prolonged RhoA activity coincided with cellular contractility.
Unexpectedly, thrombin-induced peripheral RhoA hotspots were not spatially correlated to the formation of
large inter-endothelial gaps. Rather, spontaneous RhoA activity at membrane protrusions coincided with the
closure of inter-endothelial gaps. Electrical impedance measurements showed that RhoA signalling is essential for
this protrusive activity and maintenance of barrier restoration.
Conclusion
Spontaneous RhoA activity at membrane protrusions is spatially associated with closure, but not formation of inter- endothelial gaps, whereas RhoA activity at distant contractile filaments contributes to thrombin-induced disruption of junctional integrity. Thus, these data indicate that distinct RhoA activities are associated with disruption and re- annealing of endothelial junctions.
Keywords
Rho GTPase †FRET microscopy †Cytoskeleton †Endothelial function †Vasoactive agents