Abstract
During inflammation, the disruption of the endothelial barrier leads to increased microvascular permeability. Whether tension along cell junctions contributes to histamine-induced endothelial barrier disruption remains unknown. Rapid Ca2+ influx induced by both histamine and thrombin was accompanied by endothelial barrier breakdown revealed as drop of transendothelial electric resistance in primary human microvascular endothelial cells. Interestingly, GLISA measurements revealed activation of RhoA but not inactivation of Rac1 at the time-point of barrier breakdown. FRET measurements showed activation of RhoA at intercellular junctions after both thrombin and histamine exposure. Breakdown coincided with increased stress fiber formation but not with translocation of vinculin, which was located along junctions in the resting state similar to postcapillary venules ex vivo. Moreover, increased tension at AJs was indicated by immunostaining with a conformation-sensitive antibody targeting the α18-subunit of α-catenin. Ca2+ chelation by BAPTA-AM and ROCK1 inhibition by Y27632 abolished both increase of tension along AJs as well as barrier dysfunction. Moreover, BAPTA-AM decreased RhoA activation following histamine stimulation, indicating a key role of Ca2+ signaling in barrier breakdown. Taken together, in response to histamine, Ca2+ via RhoA/ROCK activation along endothelial adherens junctions (AJs) appears to be critical for barrier disruption and presumably correlated with enhanced tension. However, vinculin appears not to be critical in this process.
Highlights
The endothelium provides a selective barrier between the blood and the surrounding interstitial tissue providing nutrients to the tissues, regulating vascular homeostasis and the transmigration of leukocytes, preserving tissues through oncotic pressure control and participating in tumor neoangiogenesis and inflammatory reactions[1,2,3,4]
Barrier function in endothelial cells is controlled by adherens junctions (AJs) and tight junctions (TJs), which are disrupted during pathological states[29]
To elucidate the time course under which endothelial monolayers react to the short-term vasoactive mediators, histamine and thrombin, immunofluorescence analysis of vascular endothelial cadherin (VE-cadherin), filamentous actin (F-actin), and vinculin was performed at several time points
Summary
The endothelium provides a selective barrier between the blood and the surrounding interstitial tissue providing nutrients to the tissues, regulating vascular homeostasis and the transmigration of leukocytes, preserving tissues through oncotic pressure control and participating in tumor neoangiogenesis and inflammatory reactions[1,2,3,4]. Tight junctions (TJs) directly limit paracellular permeability whereas adherens junctions (AJs) mechanically couple neighboring cells[6] Both junction types are functionally linked with the cortical actin cytoskeleton through several adaptor molecules. RhoA was found to induce the disassembly of endothelial AJs in response to inflammatory conditions[23] Vasoactive mediators such as lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-α) inhibit cAMP/Rac[1] signaling and cause strong activation of RhoA5. We used the inflammatory mediator histamine, which has been shown to regulate endothelial properties through activation of the type I histamine receptor and Rho A signaling[27,28], to study the mechanisms involved in anaphylaxis in primary human microvascular endothelial cells in vitro, and microvascular rat mesentery vessels in vivo. We propose that histamine-mediated disruption of endothelial barrier function is Ca2+- and Rho kinase-dependent and correlates with enhanced tension at AJs
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