Histamine Activates p38 MAP Kinase and Alters Local Lamellipodia Dynamics, Reducing Endothelial Barrier Integrity and Eliciting Central Movement of Actin Fibers

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endothelial permeability, actin cables, inflammation, lamellipodia, p38 MAPK

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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.

the endothelium plays a critical role in cardiovascular function, containing a relatively high-pressure closed-loop circulation while also permitting diffusive exchange between the blood and tissues through the capillaries and postcapillary venules. The mechanisms that control the permeability of microvessels, which permit significant leakage of plasma proteins during inflammation, have been debated for nearly a century (15). Evidence collected over the past few decades has highlighted the active role of endothelial cells in response to inflammatory stimuli, including remodeling of the actin cytoskeleton (26, 42, 45). One of the current prevailing theories from this line of investigation has been that endothelial cells adopt a contractile state during inflammation, which favors opening of junctions between cells, permitting increased paracellular flux of fluids and solutes (32, 33).

The contractile theory is supported by evidence that certain agents that elevate permeability also cause the development of centripetal tension generated by the actin cytoskeleton, which can place stress on the junctions and limit their strength (26, 29). Several inflammatory mediators also promote development of actin stress fibers in endothelial cells (7, 32). Actin-mediated contraction in endothelial cells is promoted by phosphorylation of myosin regulatory light chains (MLC) on Thr-18/Ser-19, which is determined by the activities of MLC kinase (MLCK) and MLC phosphatase (MLCP). Inhibition of MLCK attenuates hyperpermeability caused by activated neutrophils (46), histamine (34), and ethanol (21), and shortens the time course of the thrombin-induced barrier dysfunction (29) in cultured endothelial cell monolayers. Inhibition of Rho kinase (ROCK), an upstream regulator of MLCP (7, 32), also attenuates hyperpermeability caused by activated neutrophils (11), thrombin (13, 38, 43), histamine (43), and vascular endothelial growth factor (35) in endothelial cell monolayer models. Inhibition of MLCK or ROCK also decreases actin stress fiber formation, typically observed in fixed cells by labeling F-actin with a fluorochrome-bound phalloidin (5, 6, 10, 11, 13, 19, 34, 35, 38, 43, 46).

However, there is also evidence of histamine-induced increases in microvascular permeability that do not quite fit this paradigm. Histamine increases the permeability of postcapillary venules in the absence of actin stress fiber formation in endothelial cells (4). Interestingly, it was also noted that actin stress fibers did form after permeability had returned to normal (4). It was also apparent in early studies with cultured endothelial cells that histamine did not cause actin stress fiber formation within the time frame of elevated permeability in cultured endothelial cells. Rather, histamine decreased actin cables, which was postulated to be a cause of histamine-induced permeability of the endothelium (42). Likewise, histamine caused only mild phosphorylation of MLC compared with thrombin, and did not elicit an increase in the isometric tension of cultured endothelial cell monolayers (2729). These findings indicate the importance of contraction-independent mechanisms in the control of the endothelial barrier (27, 43).

Recent work has highlighted the importance of cortical actin for maintaining endothelial barrier integrity (7, 33). The small GTPase Rac1 promotes cortical actin structures, thus stabilizing intercellular junctions (1, 40, 41), and RhoA activation localized to the cell periphery promotes barrier integrity (36). To better understand the dynamics of the actin cytoskeleton, we recently used time-lapse imaging of endothelial cells expressing green fluorescent protein (GFP)-actin. We discovered that endothelial cells had frequent, brief protrusions of the plasma membrane localized at intercellular junctions, termed local lamellipodia, which were reduced during increases in endothelial permeability and restored during the restoration of barrier function (12, 17). The local lamellipodia were dependent upon myosin II activity, and decreases in their protrusion frequency correlated with reduced Rac1 activity (12). These findings are relevant because maintenance of an optimal distance of the junctional cleft between endothelial cells is important for the normal permeability of postcapillary venules (14). Our previous study provided evidence that local lamellipodia may contribute to the maintenance of endothelial junctions. However, that study was limited to an investigation of thrombin and sphingosine-1-phosphate (S1P), and it remains unclear whether additional agents that alter microvascular permeability impact this mechanism.

To investigate the mechanism of action of histamine, we assessed the dynamic changes it induces in the actin cytoskeleton. We present evidence that histamine briefly reduced local lamellipodia formation. On the basis of our previous studies showing the importance of the p38 mitogen-activated protein (MAP) kinase in histamine-induced disruption of endothelial barrier integrity, we also determined the extent to which inhibition of p38 MAP kinase affects both histamine-induced changes in barrier function and lamellipodia protrusion/withdrawal. In addition, we developed a new understanding of the spatial mechanisms of stress fiber formation caused by histamine.

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American Journal of Physiology-Cell Physiology, v. 309, issue 1, p. C51-C59