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

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.

Effects of nebulized antithrombin and heparin on inflammatory and coagulation alterations in an acute lung injury model in rats

“…In our ALI model, although only AT alone was able to decrease lung weight and edema, nebulized AT alone as well as combined with heparin reduced protein concentration and cellular infiltration into the alveolar space. These results indicate a decrease of lung permeability that could be explained by the partial reestablishment of the alveolar capillary barrier being consistent with previous studies that showed the capacity of anticoagulants to reduce lung permeability . Also, when we determined the histological damage analyzing the hallmarks of ALI, both treated groups with anticoagulants slightly improved the lesion although it was nonsignificant.…”

“…These results indicate a decrease of lung permeability that could be explained by the partial reestablishment of the alveolar capillary barrier being consistent with previous studies that showed the capacity of anticoagulants to reduce lung permeability. 25,28,34,35 Also, when we determined the histological damage analyzing the hallmarks of ALI, both treated groups with anticoagulants slightly improved the lesion although it was nonsignificant. Our ALI model was proved to last at least for 72 hours; 48 hours was most likely too short time to find a more pronounced difference among groups.…”

“…23 Additionally, it is well known that the deregulation of coagulation and inflammatory pathways lead to increased permeability and edema, another characteristic of ARDS. [24][25][26] AT might enhance the alveolar epithelium restoration through its actions on the coagulant and the inflammatory cascades.…”

COVID‐19 hypothesis: Activated protein C for therapy of virus‐induced pathologic thromboinflammation

“…anti‐inflammatory signaling pathways initiated by APC acting on its receptors include those of endothelial cells, monocytes, neutrophils, dendritic cells, and T cells, allowing APC to control and regulate immune cell physiology 12,14,15 . APC and several of its recombinant mutants have well‐understood mechanisms of action on many cell types and in well‐characterized rodent injury models where its anti‐inflammatory, anti‐apoptotic, cytoprotective, neuroprotective, and mortality reduction activities are apparent 10‐12,14‐21 …”

Novel Functions of the Anticoagulant Activated Protein C in Maintaining Skin Barrier Integrity to Impact on Skin Disease

“…In addition, APC reduces the thrombin-induced disruption of alveolar epithelial barrier integrity via decreasing epithelial permeability, cell stiffening, cell contraction and enhancing ZO-1 aggregates at the cell-cell interface [42]. …”