Barrier-Protective Effects of Activated Protein C in Human Alveolar Epithelial Cells

Ferranda Puig,Ramon Farre,Antonio Artigas,Daniel Navajas,Mélanie Adda,Gemma Fuster,Lluís Blanch

doi:10.1371/journal.pone.0056965

Abstract

Acute lung injury (ALI) is a clinical manifestation of respiratory failure, caused by lung inflammation and the disruption of the alveolar-capillary barrier. Preservation of the physical integrity of the alveolar epithelial monolayer is of critical importance to prevent alveolar edema. Barrier integrity depends largely on the balance between physical forces on cell-cell and cell-matrix contacts, and this balance might be affected by alterations in the coagulation cascade in patients with ALI. We aimed to study the effects of activated protein C (APC) on mechanical tension and barrier integrity in human alveolar epithelial cells (A549) exposed to thrombin. Cells were pretreated for 3 h with APC (50 µg/ml) or vehicle (control). Subsequently, thrombin (50 nM) or medium was added to the cell culture. APC significantly reduced thrombin-induced cell monolayer permeability, cell stiffening, and cell contraction, measured by electrical impedance, optical magnetic twisting cytometry, and traction microscopy, respectively, suggesting a barrier-protective response. The dynamics of the barrier integrity was also assessed by western blotting and immunofluorescence analysis of the tight junction ZO-1. Thrombin resulted in more elongated ZO-1 aggregates at cell-cell interface areas and induced an increase in ZO-1 membrane protein content. APC attenuated the length of these ZO-1 aggregates and reduced the ZO-1 membrane protein levels induced by thrombin. In conclusion, pretreatment with APC reduced the disruption of barrier integrity induced by thrombin, thus contributing to alveolar epithelial barrier protection.

Highlights

The pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) involves pro-coagulant and pro-inflammatory mechanisms resulting in disrupted alveolar epithelium at cell-cell junctions, with the consequent infiltration of protein-rich edema fluid and inflammatory cells into the alveolar space [1,2]
The thrombin-induced acute decline in Cell impedance (CI) was significantly attenuated by pretreatment with activated protein C (APC) in all three human lung epithelial cell types (4.6% in A549 cells, 5.9% in human alveolar epithelial cells (HAECs), and 1.4% in H441 cells)
We measured the effect of APC on cell stiffness, cell contraction, and cell barrier integrity in human alveolar epithelial cells subjected to thrombin

Summary

Introduction

The pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) involves pro-coagulant and pro-inflammatory mechanisms resulting in disrupted alveolar epithelium at cell-cell junctions, with the consequent infiltration of protein-rich edema fluid and inflammatory cells into the alveolar space [1,2]. The physical integrity of the alveolar epithelial barrier plays an important role in these respiratory diseases. The integrity of this barrier depends on a dynamic balance between inward forces–cell mechanical tension generated by actomyosin contraction and cytoskeleton elastic recoil–and outward forces exerted by cell-cell and cell-matrix adhesions [3]. Thrombin can modulate the force balance in alveolar epithelial cells by increasing cell stiffness [8] and cell contraction [9] and by enhancing peripheral remodeling of the actin cytoskeleton [8,9,10] and of cellcell contacts, such as the tight junction ZO-1 [10]

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