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.
Highlights
The endothelial layer of blood vessels is a highly regulated barrier between the bloodstream and the interstitial tissue, controlling transvascular passage of fluids, solutes, and cells
The barrier-disrupting response of human dermal microvascular endothelial cells (HDMECs) monolayers to thrombin and histamine differed in severity and kinetics; thrombin effects were more profound, and the barrier took over an hour to recover, whereas the histamine effects were modest in comparison, and recovery of the barrier to preagonist levels occurred within 10 min (Fig. 1, A and B)
Previous work from our laboratory showed that stromal-interacting molecule 1 (STIM1) and Orai1 are the molecular components of store-operated Ca2ϩ entry (SOCE) in several human endothelial cell types, including endothelial cells from the umbilical vein, the pulmonary artery, and the dermal microvasculature (HDMECs) [11, 23]
Summary
The endothelial layer of blood vessels is a highly regulated barrier between the bloodstream and the interstitial tissue, controlling transvascular passage of fluids, solutes, and cells. Disruption of endothelial barrier function causes increased vascular permeability and is associated with reorganization of the actin cytoskeleton and disassembly of adherens junctions which are contributed by vascular endothelial cadherin (VEcadherin)1⁄7catenin complexes [2, 4]. These events are under the control of various signaling pathways that are activated by diverse paracrine and autocrine mediators in blood and interstitial tissue, many of which act on heterotrimeric G proteincoupled receptors (GPCR) or receptor tyrosine kinase and play crucial roles in the control of vascular permeability, tone, angiogenesis and inflammation [2,3,4]. For the barrier-stabilizing agonist S1P, Ca2ϩ release from internal stores, but not Ca2ϩ entry, was proposed to induce Rac activation, promoting assembly of adherens junctions and strengthening of endothelial barrier function [22]
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