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Calcium Signaling Is Dispensable for Receptor Regulation of Endothelial Barrier Function.

ABSTRACT: 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.

SUBMITTER: Stolwijk JA

PROVIDER: S-EPMC5087712 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Calcium Signaling Is Dispensable for Receptor Regulation of Endothelial Barrier Function.

Stolwijk Judith A JA Zhang Xuexin X Gueguinou Maxime M Zhang Wei W Matrougui Khalid K Renken Christian C Trebak Mohamed M

The Journal of biological chemistry 20160913 44

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 Ca<sup>2+</sup> signaling upon phospholipase C-coupled receptor ligation leads quite naturally to the assumption that Ca<sup>2+</sup> signaling is required for receptor-regulated endothelial barrier function. This widespread hypothesis draws analogy from smooth muscle and propose ...[more]

PMID: 27624938

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Project description: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.

Project description:OBJECTIVE:Immune complex (IC) deposition activates polymorphonuclear neutrophils (PMNs), increases vascular permeability, and leads to organ damage in systemic lupus erythematosus and rheumatoid arthritis. The bioactive lipid sphingosine 1-phosphate (S1P), acting via S1P receptor 1 (S1P1 ), is a key regulator of endothelial cell (EC) barrier function. This study was undertaken to investigate whether augmenting EC integrity via S1P1 signaling attenuates inflammatory injury mediated by ICs. METHODS:In vitro barrier function was assessed in human umbilical vein endothelial cells (HUVECs) by electrical cell-substrate impedance sensing. Phosphorylation of myosin light chain 2 (p-MLC-2) and VE-cadherin staining in HUVECs were assessed by immunofluorescence. A reverse Arthus reaction (RAR) was induced in the skin and lungs of mice with S1P1 deleted from ECs (S1P1 EC-knockout [ECKO] mice) and mice treated with S1P1 agonists and antagonists. RESULTS:S1P1 agonists prevented loss of barrier function in HUVECs treated with IC-activated PMNs. S1P1 ECKO and wild-type (WT) mice treated with S1P1 antagonists had amplified RAR, whereas specific S1P1 agonists attenuated skin and lung RAR in WT mice. ApoM-Fc, a novel S1P chaperone, mitigated EC cell barrier dysfunction induced by activated PMNs in vitro and attenuated lung RAR. Expression levels of p-MLC-2 and disruption of VE-cadherin, each representing manifestations of cell contraction and destabilization of adherens junctions, respectively, that were induced by activated PMNs, were markedly reduced by treatment with S1P1 agonists and ApoM-Fc. CONCLUSION:Our findings indicate that S1P1 signaling in ECs modulates vascular responses to IC deposition. S1P1 agonists and ApoM-Fc enhance the EC barrier, limit leukocyte escape from capillaries, and provide protection against inflammatory injury. The S1P/S1P1 axis is a newly identified target to attenuate tissue responses to IC deposition and mitigate end-organ damage.

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Calcium Signaling Is Dispensable for Receptor Regulation of Endothelial Barrier Function.

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Calcium Signaling Is Dispensable for Receptor Regulation of Endothelial Barrier Function.

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