Adderley Shaquria P, Lawrence Curtis, Madonia Eyong, Olubadewo Joseph O, Breslin Jerome W
Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida, Tampa, Florida; and.
Biology Unit, Department of Natural Sciences, Southern University at New Orleans, New Orleans, Louisiana.
Am J Physiol Cell Physiol. 2015 Jul 1;309(1):C51-9. doi: 10.1152/ajpcell.00096.2015. Epub 2015 May 6.
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.
近四十年来，肌动蛋白细胞骨架在内皮屏障功能中的作用一直存在争议。我们之前的研究发现，汇合的内皮细胞单层中会自发形成局部片状伪足，这似乎会增加细胞间连接的重叠。我们测试了以下假设：屏障破坏剂组胺会减少局部片状伪足的突出，并研究了p38丝裂原活化蛋白（MAP）激酶激活和肌动蛋白应力纤维形成的潜在参与情况。使用延时荧光显微镜对表达绿色荧光蛋白 - 肌动蛋白的人脐静脉内皮细胞（HUVEC）汇合单层进行了研究。在添加组胺之前和之后评估局部片状伪足的突出和缩回特征。使用细胞 - 基质阻抗传感测定屏障功能的变化。组胺最初降低了屏障功能、片状伪足突出频率和片状伪足突出距离。片状伪足缩回时间延长，缩回距离和速度降低伴随着屏障恢复。屏障恢复后，大量皮质纤维向中央迁移，最终类似于肌动蛋白应力纤维。p38 MAP激酶抑制剂SB203580减弱了组胺诱导的屏障功能和片状伪足突出频率的降低。SB203580还抑制了组胺诱导的缩回距离和速度的降低以及随后的肌动蛋白纤维迁移。这些数据表明，组胺可通过激活p38 MAP激酶来降低局部片状伪足的突出活性。研究结果还表明，局部片状伪足在维持内皮屏障完整性方面发挥作用。此外，我们提供的证据表明，肌动蛋白应力纤维的形成可能是内皮屏障完整性降低的一种反应，而非原因。
