Belvitch Patrick, Brown Mary E, Brinley Brittany N, Letsiou Eleftheria, Rizzo Alicia N, Garcia Joe G N, Dudek Steven M
Division of Pulmonary, Critical Care, Sleep, and Allergy, University of Illinois Hospital and Health Science System, Chicago, IL, USA.
Mercy Hospital and Medical Center, Chicago, IL, USA.
Pulm Circ. 2017 Feb 1;7(1):200-210. doi: 10.1086/690307. eCollection 2017 Mar.
Pulmonary endothelial cell (EC) barrier dysfunction and recovery is critical to the pathophysiology of acute respiratory distress syndrome. Cytoskeletal and subsequent cell membrane dynamics play a key mechanistic role in determination of EC barrier integrity. Here, we characterizAQe the actin related protein 2/3 (Arp 2/3) complex, a regulator of peripheral branched actin polymerization, in human pulmonary EC barrier function through studies of transendothelial electrical resistance (TER), intercellular gap formation, peripheral cytoskeletal structures and lamellipodia. Compared to control, Arp 2/3 inhibition with the small molecule inhibitor CK-666 results in a reduction of baseline barrier function (1,241 ± 53 vs 988 ± 64 ohm; < 0.01), S1P-induced barrier enhancement and delayed recovery of barrier function after thrombin (143 ± 14 vs 93 ± 6 min; < 0.01). Functional changes of Arp 2/3 inhibition on barrier integrity are associated temporally with increased intercellular gap area at baseline (0.456 ± 0.02 vs 0.299 ± 0.02; < 0.05) and thirty minutes after thrombin (0.885 ± 0.03 vs 0.754 ± 0.03; < 0.05). Immunofluorescent microscopy reveals reduced lamellipodia formation after S1P and during thrombin recovery in Arp 2/3 inhibited cells. Individual lamellipodia demonstrate reduced depth following Arp 2/3 inhibition vs vehicle at baseline (1.83 ± 0.41 vs 2.55 ± 0.46 µm; < 0.05) and thirty minutes after S1P treatment (1.53 ± 0.37 vs 2.09 ± 0.36 µm; < 0.05). These results establish a critical role for Arp 2/3 activity in determination of pulmonary endothelial barrier function and recovery through formation of EC lamellipodia and closure of intercellular gaps.
肺内皮细胞（EC）屏障功能障碍及恢复对急性呼吸窘迫综合征的病理生理学至关重要。细胞骨架及随后的细胞膜动力学在决定EC屏障完整性方面发挥关键机制作用。在此，我们通过跨内皮电阻（TER）、细胞间隙形成、外周细胞骨架结构和片状伪足的研究，对肌动蛋白相关蛋白2/3（Arp 2/3）复合物（一种外周分支肌动蛋白聚合的调节因子）在人肺EC屏障功能中的作用进行了表征。与对照组相比，用小分子抑制剂CK-666抑制Arp 2/3会导致基线屏障功能降低（1241±53对988±64欧姆；<0.01）、S1P诱导的屏障增强作用减弱以及凝血酶作用后屏障功能恢复延迟（143±14对93±6分钟；<0.01）。Arp 2/3抑制对屏障完整性的功能变化在时间上与基线时细胞间隙面积增加（0.456±0.02对0.299±0.02；<0.05）以及凝血酶作用30分钟后细胞间隙面积增加（0.885±0.03对0.754±0.03；<0.05）相关。免疫荧光显微镜显示，在Arp 2/3抑制的细胞中，S1P作用后及凝血酶恢复过程中片状伪足形成减少。与基线时（1.83±0.41对2.55±0.46微米；<0.05）及S1P处理30分钟后（1.53±0.37对2.09±0.36微米；<0.05）的载体组相比，Arp 2/3抑制后单个片状伪足的深度降低。这些结果表明，Arp 2/3活性在通过形成EC片状伪足和封闭细胞间隙来决定肺内皮屏障功能及恢复方面起关键作用。
