Thrombin-induced endothelial barrier disruption in intact microvessels: role of RhoA/Rho kinase-myosin phosphatase axis

Amerongen, Geerten P. van Nieuw; Musters, René J.P.; Eringa, Etto C.; Sipkema, Pieter; Hinsbergh, Victor W.M. van

doi:10.1152/ajpcell.00551.2007

Cited by 59 publications

(66 citation statements)

References 46 publications

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“…Exposure of endothelial cells to mildly oxidized low density lipoprotein induced Rho kinase-dependent MLC phosphorylation, stress fiber formation, and intercellular gaps within minutes (38). Inhibition of MYPT-1 activity in intact microvessels also promoted a rapid phosphorylation of MLC, resulting in a contractile response and loss of barrier integrity (39). The pharmacologic inhibition of both isoforms would therefore be essential to block Rho kinase-mediated signaling through MLC and limit paracellular gap formation.…”

Section: Discussionmentioning

confidence: 99%

Rho Kinase-2 Activation in Human Endothelial Cells Drives Lysophosphatidic Acid-mediated Expression of Cell Adhesion Molecules via NF-κB p65

Shimada

Rajagopalan

2010

Journal of Biological Chemistry

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The trans-endothelial migration of activated leukocytes into inflamed tissue is an important mechanism in the pathogenesis of inflammatory diseases. Endothelial cells play a key role in this step by up-regulating the synthesis and secretion of chemokines and the cell surface expression of adhesion molecules (CAMs) 2 (1). The adhesion molecules ICAM-1 and VCAM-1 bind leukocytes expressing the major integrins lymphocyte function associated antigen-1 (LFA-1) and the VLA-4 (very late activation antigen-4), respectively, and are induced on endothelial cells by inflammatory stimuli (2, 3). Both are required for the attachment of circulating leukocytes to the endothelium and mediate not only transcellular migration but also the induction of intracellular signals resulting in transient junctional disruption (1) and the paracellular passage of leukocytes across the endothelial monolayer.Lysophosphatidic acid (LPA), a pleiotropic proinflammatory lipid mediator, is elevated in multiple disease states. High concentrations of the lipid have been detected in the bronchoalveolar lavage fluid of preclinical animal models of allergic asthma (4). LPA plays a role in regulating the secretion of pro-and anti-inflammatory mediators by airway epithelial cells and in modulating airway epithelial barrier integrity (5). Additionally, synovial fluid obtained from rheumatoid arthritis patients contain significant amounts of LPA and the LPA-synthesizing enzyme autotaxin driving cytokine secretion by and migration of resident synoviocytes (6, 7). The lipid mediator has also been implicated in the initiation of neuropathic pain (8). Blood platelets also store LPA and are an important source of the lipid. Serum and plasma contain nanomolar levels of LPA and de novo generation or release from platelet stores during inflammation may result in higher LPA levels at disease loci. LPA engages a family of at least five closely related G protein-coupled receptors LPA1-5 (9). It has been reported that HUVECs express LPA1 and LPA3 receptors and are responsive to LPA with induced expression of chemokines and cell adhesion molecules (CAMs) (10 -12). In vitro and in vivo studies employing Rho kinase inhibitors, isoform-specific LPA receptor inhibitors, and targeted receptor deletions link LPA acting through the LPA1 receptor, to the downstream activation of Rho kinase (8,13,14).Rho kinase, a serine/threonine kinase, was initially characterized as a mediator of the formation of RhoA-induced stress fibers and focal adhesions (15). Activation of the Rho kinase pathway leads to the phosphorylation of downstream substrates including the 20-kDa myosin light chain (MLC) (16), the myosin phosphatase subunit (MYPT-1) (17), and CPI-17A (18), which have been postulated to control a variety of fundamental cellular functions such as proliferation, survival, contraction, migration, and the transcriptional regulation of gene expression. However, abnormal activation of the pathway has been * This work was supported by Pfizer, Inc.

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Section: Discussionmentioning

confidence: 99%

Rho Kinase-2 Activation in Human Endothelial Cells Drives Lysophosphatidic Acid-mediated Expression of Cell Adhesion Molecules via NF-κB p65

Shimada

Rajagopalan

2010

Journal of Biological Chemistry

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“…Recent findings indicate that thrombin at high (Ͼ100 pM), but not at low (Ͻ50 pM) concentrations can disrupt the endothelial barrier via activation of PAR1 (15,93). Several downstream signaling events may be implicated, among which caveolin-1-dependent weakening of endothelial tight junctions (147), and G 12 /G 13 -dependent RhoA activation and concomitant actin stress fiber formation (303).…”

Section: H Roles Of Protease-activated Receptors In Coagulation Woumentioning

confidence: 99%

New Fundamentals in Hemostasis

Versteeg

Heemskerk

Levi

et al. 2013

Physiological Reviews

887

831

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Hemostasis encompasses the tightly regulated processes of blood clotting, platelet activation, and vascular repair. After wounding, the hemostatic system engages a plethora of vascular and extravascular receptors that act in concert with blood components to seal off the damage inflicted to the vasculature and the surrounding tissue. The first important component that contributes to hemostasis is the coagulation system, while the second important component starts with platelet activation, which not only contributes to the hemostatic plug, but also accelerates the coagulation system. Eventually, coagulation and platelet activation are switched off by blood-borne inhibitors and proteolytic feedback loops. This review summarizes new concepts of activation of proteases that regulate coagulation and anticoagulation, to give rise to transient thrombin generation and fibrin clot formation. It further speculates on the (patho)physiological roles of intra-and extravascular receptors that operate in response to these proteases. Furthermore, this review provides a new framework for understanding how signaling and adhesive interactions between endothelial cells, leukocytes, and platelets can regulate thrombus formation and modulate the coagulation process. Now that the key molecular players of coagulation and platelet activation have become clear, and their complex interactions with the vessel wall have been mapped out, we can also better speculate on the causes of thrombosis-related angiopathies.

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“…24 Moreover, inflammatory stimuli induce F-actin fiber formation in rat endothelial cells within the vascular wall in situ. 25 It is not yet known to what structures F-actin fibers attach in the endothelium of blood vessels, and it is also unclear if organization of the actin cytoskeleton relates to differences in endothelium of human arteries and veins.…”

Section: Arterioscler Thromb Vasc Biolmentioning

confidence: 99%

F-Actin–Anchored Focal Adhesions Distinguish Endothelial Phenotypes of Human Arteries and Veins

Geemen

Smeets

Stalborch

et al. 2014

ATVB

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Objective-Vascular endothelial-cadherin-and integrin-based cell adhesions are crucial for endothelial barrier function.Formation and disassembly of these adhesions controls endothelial remodeling during vascular repair, angiogenesis, and inflammation. In vitro studies indicate that vascular cytokines control adhesion through regulation of the actin cytoskeleton, but it remains unknown whether such regulation occurs in human vessels. We aimed to investigate regulation of the actin cytoskeleton and cell adhesions within the endothelium of human arteries and veins. Approach and Results-We used an ex vivo protocol for immunofluorescence in human vessels, allowing detailed en face microscopy of endothelial monolayers. We compared arteries and veins of the umbilical cord and mesenteric, epigastric, and breast tissues and find that the presence of central F-actin fibers distinguishes the endothelial phenotype of adult arteries from veins. F-actin in endothelium of adult veins as well as in umbilical vasculature predominantly localizes cortically at the cell boundaries. By contrast, prominent endothelial F-actin fibers in adult arteries anchor mostly to focal adhesions containing integrin-binding proteins paxillin and focal adhesion kinase and follow the orientation of the extracellular matrix protein fibronectin. Other arterial F-actin fibers end in vascular endothelial-cadherin-based endothelial focal adherens junctions. In vitro adhesion experiments on compliant substrates demonstrate that formation of focal adhesions is strongly induced by extracellular matrix rigidity, irrespective of arterial or venous origin of endothelial cells. Conclusions-Our data show that F-actin-anchored focal adhesions distinguish endothelial phenotypes of human arteries from veins. We conclude that the biomechanical properties of the vascular extracellular matrix determine this endothelial characteristic. (Arterioscler Thromb Vasc

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Thrombin-induced endothelial barrier disruption in intact microvessels: role of RhoA/Rho kinase-myosin phosphatase axis

Cited by 59 publications

References 46 publications

Rho Kinase-2 Activation in Human Endothelial Cells Drives Lysophosphatidic Acid-mediated Expression of Cell Adhesion Molecules via NF-κB p65

Rho Kinase-2 Activation in Human Endothelial Cells Drives Lysophosphatidic Acid-mediated Expression of Cell Adhesion Molecules via NF-κB p65

New Fundamentals in Hemostasis

F-Actin–Anchored Focal Adhesions Distinguish Endothelial Phenotypes of Human Arteries and Veins

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