Article Text

  1. P Belvitch1,
  2. S Dudek1,
  3. ME Brown2,
  4. JG Garcia3
  1. 1Pulmonary and Critical Care, University of Illinois at Chicago, CHICAGO, Illinois, United States
  2. 2University of Chicago, Chicago, Illinois, United States
  3. 3University of Arizona, Tucson, Arizona, United States


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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