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A Novel Microscopic Assay Reveals Heterogeneous Regulation of Local Endothelial Barrier Function.

ABSTRACT: Blood vessels are covered with endothelial cells on their inner surfaces, forming a selective and semipermeable barrier between the blood and the underlying tissue. Many pathological processes, such as inflammation or cancer metastasis, are accompanied by an increased vascular permeability. Progress in live cell imaging techniques has recently revealed that the structure of endothelial cell contacts is constantly reorganized and that endothelial junctions display high heterogeneities at a subcellular level even within one cell. Although it is assumed that this dynamic remodeling is associated with a local change in endothelial barrier function, a direct proof is missing mainly because of a lack of appropriate experimental techniques. Here, we describe a new assay to dynamically measure local endothelial barrier function with a lateral resolution of ?15 ?m and a temporal resolution of 1 min. In this setup, fluorescence-labeled molecules are added to the apical compartment of an endothelial monolayer, and the penetration of molecules from the apical to the basal compartment is recorded by total internal reflection fluorescence microscopy utilizing the generated evanescent field. With this technique, we found a remarkable heterogeneity in the local permeability for albumin within confluent endothelial cell layers. In regions with low permeability, stimulation with the proinflammatory agent histamine results in a transient increase in paracellular permeability. The effect showed a high variability along the contact of one individual cell, indicating a local regulation of endothelial barrier function. In regions with high basal permeability, histamine had no obvious effect. In contrast, the barrier-enhancing drug forskolin reduces the permeability for albumin and dextran uniformly along the cell junctions. Because this new approach can be readily combined with other live cell imaging techniques, it will contribute to a better understanding of the mechanisms underlying subcellular junctional reorganization during wound healing, inflammation, and angiogenesis.

SUBMITTER: Klusmeier N

PROVIDER: S-EPMC6486479 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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A Novel Microscopic Assay Reveals Heterogeneous Regulation of Local Endothelial Barrier Function.

Klusmeier Nadine N Schnittler Hans-Joachim HJ Seebach Jochen J

Biophysical journal 20190222 8

Blood vessels are covered with endothelial cells on their inner surfaces, forming a selective and semipermeable barrier between the blood and the underlying tissue. Many pathological processes, such as inflammation or cancer metastasis, are accompanied by an increased vascular permeability. Progress in live cell imaging techniques has recently revealed that the structure of endothelial cell contacts is constantly reorganized and that endothelial junctions display high heterogeneities at a subcel ...[more]

PMID: 30878197

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Project description:Recently we observed that endothelial cells cultured in tightly confluent monolayers display frequent local lamellipodia, and that thrombin, an agent that increases endothelial permeability, reduces lamellipodia protrusions. This led us to test the hypothesis that local lamellipodia contribute to endothelial barrier function. Movements of subcellular structures containing GFP-actin or VE-cadherin-GFP expressed in endothelial cells were recorded using time-lapse microscopy. Transendothelial electrical resistance (TER) served as an index of endothelial barrier function. Changes in both lamellipodia dynamics and TER were assessed during baseline and after cells were treated with either the barrier-disrupting agent thrombin, or the barrier-stabilizing agent sphingosine-1-phosphate (S1P). The myosin II inhibitor blebbistatin was used to selectively block lamellipodia formation, and was used to test their role in the barrier function of endothelial cell monolayers and isolated, perfused rat mesenteric venules. Myosin light chain (MLC) phosphorylation was assessed by immunofluorescence microscopy. Rac1 and RhoA activation were evaluated using G-LISA assays. The role of Rac1 was tested with the specific inhibitor NSC23766 or by expressing wild-type or dominant negative GFP-Rac1. The results show that thrombin rapidly decreased both TER and the lamellipodia protrusion frequency. S1P rapidly increased TER in association with increased protrusion frequency. Blebbistatin nearly abolished local lamellipodia protrusions while cortical actin fibers and stress fibers remained intact. Blebbistatin also significantly decreased TER of cultured endothelial cells and increased permeability of isolated rat mesenteric venules. Both thrombin and S1P increased MLC phosphorylation and activation of RhoA. However, thrombin and S1P had differential impacts on Rac1, correlating with the changes in TER and lamellipodia protrusion frequency. Overexpression of Rac1 elevated, while NSC23766 and dominant negative Rac1 reduced barrier function and lamellipodia activity. Combined, these data suggest that local lamellipodia, driven by myosin II and Rac1, are important for dynamic changes in endothelial barrier integrity.

Project description:Endothelial barrier function is tightly regulated by plasma membrane receptors and is crucial for tissue fluid homeostasis; its dysfunction causes disease, including sepsis and inflammation. The ubiquitous activation of Ca2+ signaling upon phospholipase C-coupled receptor ligation leads quite naturally to the assumption that Ca2+ signaling is required for receptor-regulated endothelial barrier function. This widespread hypothesis draws analogy from smooth muscle and proposes the requirement of G protein-coupled receptor (GPCR)-generated Ca2+ signaling in activating the endothelial contractile apparatus and generating interendothelial gaps. Notwithstanding endothelia being non-excitable in nature, the hypothesis of Ca2+-induced endothelial contraction has been invoked to explain actions of GPCR agonists that either disrupt or stabilize endothelial barrier function. Here, we challenge this correlative hypothesis by showing a lack of causal link between GPCR-generated Ca2+ signaling and changes in human microvascular endothelial barrier function. We used three endogenous GPCR agonists: thrombin and histamine, which disrupt endothelial barrier function, and sphingosine-1-phosphate, which stabilizes barrier function. The qualitatively different effects of these three agonists on endothelial barrier function occur independently of Ca2+ entry through the ubiquitous store-operated Ca2+ entry channel Orai1, global Ca2+ entry across the plasma membrane, and Ca2+ release from internal stores. However, disruption of endothelial barrier function by thrombin and histamine requires the Ca2+ sensor stromal interacting molecule-1 (STIM1), whereas sphingosine-1-phosphate-mediated enhancement of endothelial barrier function occurs independently of STIM1. We conclude that although STIM1 is required for GPCR-mediated disruption of barrier function, a causal link between GPCR-induced cytoplasmic Ca2+ increases and acute changes in barrier function is missing. Thus, the cytosolic Ca2+-induced endothelial contraction is a cum hoc fallacy that should be abandoned.

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A Novel Microscopic Assay Reveals Heterogeneous Regulation of Local Endothelial Barrier Function.

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A Novel Microscopic Assay Reveals Heterogeneous Regulation of Local Endothelial Barrier Function.

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