The role of the actin cytoskeleton in endothelial barrier function has been debated for nearly four decades. Our previous investigation revealed spontaneous local lamellipodia in confluent endothelial monolayers that appear to increase overlap at intercellular junctions. We tested the hypothesis that the barrier-disrupting agent histamine would reduce local lamellipodia protrusions and investigated the potential involvement of p38 mitogen-activated protein (MAP) kinase activation and actin stress fiber formation. Confluent monolayers of human umbilical vein endothelial cells (HUVEC) expressing green fluorescent protein-actin were studied using time-lapse fluorescence microscopy. The protrusion and withdrawal characteristics of local lamellipodia were assessed before and after addition of histamine. Changes in barrier function were determined using electrical cell-substrate impedance sensing. Histamine initially decreased barrier function, lamellipodia protrusion frequency, and lamellipodia protrusion distance. A longer time for lamellipodia withdrawal and reduced withdrawal distance and velocity accompanied barrier recovery. After barrier recovery, a significant number of cortical fibers migrated centrally, eventually resembling actin stress fibers. The p38 MAP kinase inhibitor SB203580 attenuated the histamine-induced decreases in barrier function and lamellipodia protrusion frequency. SB203580 also inhibited the histamine-induced decreases in withdrawal distance and velocity, and the subsequent actin fiber migration. These data suggest that histamine can reduce local lamellipodia protrusion activity through activation of p38 MAP kinase. The findings also suggest that local lamellipodia have a role in maintaining endothelial barrier integrity. Furthermore, we provide evidence that actin stress fiber formation may be a reaction to, rather than a cause of, reduced endothelial barrier integrity.
We tested the hypothesis that histamine disrupts the endothelial barrier by activation of the p38 MAP kinase and ERK‐1/2. Transendothelial electrical resistance (TER) of human umbilical vein endothelial cells (HUVEC) grown on small gold electrodes served as an index of barrier function, before and after addition of 10 μM histamine. The role of p38 MAP kinase was tested with 6 μM SB203580 and ERK‐1/2 with 10 μM PD98059 or 1 μM U0126. Specific, dual phosphorylation of p38 MAP kinase or ERK‐1/2 on their activation sites was detected by Western blotting. Dynamics of GFP‐actin and VE‐cadherin‐GFP expressed in HUVEC were also evaluated. The results show that histamine increased phosphorylation of both p38 MAP kinase and ERK‐1/2, which was blocked by pretreatment with SB203580, or PD98059/U0126, respectively. Histamine‐induced decreases in TER were inhibited by SB203580, but not affected by PD98059 or U0126 pretreatment. Histamine did not change VE‐cadherin‐GFP organization, but did briefly stop GFP‐actin‐rich edge protrusions. However, these protrusions were not decreased by histamine when the cells were pretreated with SB203580. The data suggest that p38 MAP kinase, but not ERK‐1/2, mediates histamine‐induced endothelial barrier disruption by reducing endothelial cell spreading motions. Supported by NIH grants R01HL098215 and P20GM103424, NSF grant HRD0928797, and the Louisiana Board of Regents Support Fund.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.