Rationale Disruption of the pulmonary endothelial barrier is a hallmark feature of sepsis and acute lung injury/ARDS. Cytoskeletal structures such as the peripheral protrusive structures lamellipodia and filopodia are hypothesized to be important determinants of endothelial barrier function. The actin related protein 2/3 complex (Arp 2/3) is a key regulator of branched actin polymerization and may play a role in the determination and recovery of endothelial cell (EC) barrier integrity. In the current study, we make detailed observations of actin structures and membrane formations in the presence of a specific Arp 2/3 inhibitor. In addition, we study the subcellular co-localization of Arp 2/3 and cortactin, another known protein regulator of peripheral actin dynamics.
Methods Cultured human lung microvascular endothelial cells (HLMVEC) were subjected to pre-treatment with the specific Arp 2/3 inhibitor (CK-666 50 µM) or vehicle (DMSO) x 1 hour. Cells were then treated with barrier enhancing sphingosine-1-phosphate (S1P 1 µM) or barrier disruptive thrombin (1 U/ml) and fixed at various time points (2–90 min) for subsequent imaging. Cells were permeabilized and treated with far-red phalloidin to stain actin, an anti-cortactin-GFP mAb as well as DAPI and imaged by confocal microscopy. Peripheral actin formations and associated membrane lamellipodia and filopodia were then measured and characterized. Additionally, the co-localization of Arp 2/3 and cortactin was determined.
Results Arp 2/3 inhibition markedly reduced lamellipodia formation in response to S1P (1 µM) over a range of time points (2–30 min). Lamellipodia depth was decreased in Arp 2/3 inhibited cells compared to control both at baseline (1.825 +/− 0.407 µM) vs. (2.545 +/− 0.459 µM) and following 30 min treatment with 1 µM S1P (1.534 +/− 0.365 µM) vs. (2.090 +/− 0.356 µM). Similarly, filopodia were shorter following Arp 2/3 inhibition (2.392 +/− 0.393 µM) vs. control (2.753 +/− 0.274 µM). Robust colocalization of Arp 2/3 and cortactin was observed very early (2–5 min) following S1P (1 µM) treatment in vehicle treated cells but was attenuated in the presence of the Arp 2/3 inhibitor. Following thrombin treatment (1 U/ml), peripheral lamellipodia were observed during the barrier recovery phase (30–60 min) but were markedly reduced following Arp 2/3 inhibition along with the persistence of intercellular gaps.
Conclusion These results further demonstrate the importance of the Arp 2/3 complex in pulmonary endothelial barrier integrity and recovery. These experiments also serve to relate the concept of altered peripheral actin and membrane dynamics leading to changes in EC barrier function.
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