Endothelial Cells and Maintenance of Barrier Integrity

Endothelial cells form the innermost layer of the blood vessels and mediate important interactions with the blood. The endothelial layer is a monolayer that acts as a barrier between the blood and internal environment. Maintenance of barrier integrity is critical for homeostasis and disruption of this barrier may lead to conditions where fluid inappropriately leaks out of the blood vessels. Barrier integrity is largely regulated by tight junctions, which are protein complexes found between adjacent cells that allow for the process of paracellular transport, or transport through the space between neighboring cells.

Endothelial monolayers can be studied in vitro using specialized culture systems in which endothelial cells form tight junctions and become a polarized monolayer, across which electrical resistance can be measured. This allows researchers to monitor changes in barrier integrity, as resistance decreases with increased barrier permeability. Importantly, changes in the expression of different tight junction proteins may affect permeability and have subsequent effects on barrier integrity.

Lifeline® Endothelial Cells in Lung Injury Research

Acute respiratory distress syndrome (ARDS) occurs when fluid enters the lungs and obstructs breathing, due to a breakdown between the alveoli—the air sacs of the lungs—and associated capillaries, the intersection of the blood and air where oxygen is exchanged. This space is maintained by an endothelial barrier, which becomes permeable in the later stages of ARDS, allowing fluid to enter the alveolar space and eventually leading to pulmonary edema.

Vascular leakage can also occur as a result of extracellular matrix dysfunction. In particular, increased expression of matrix metalloproteases (MMPs) have been associated with more severe acute lung injury (ALI). In a new study in Pharmacological Research, Artham and colleagues hypothesized that endothelial Akt and FoxO signaling impact stromelysin1 (MMP3) expression and activity and mediate pulmonary edema during ALI. The authors first used Lifeline® human primary pulmonary endothelial cells (HPAECs) to demonstrate that lipopolysaccharide (LPS), used to induce lung injury, decreased phosphorylation of Akt, FoxO1, and FoxO3a. The authors hypothesized that FoxO1 and FoxO3a are transcriptional repressors of claudin-5, an important tight junction protein that regulates tight junction integrity. To test this, they treated human microvascular endothelial cells (HMECs) and Lifeline® HPAECs with LPS and found that claudin-5 expression decreased in a manner partially dependent on FoxO activity.

Next, using a polarized monolayer culture system, the authors found that LPS treatment increased monolayer permeability, which improved following treatment with a FoxO inhibitor; treatment with an Akt inhibitor alone significantly increased monolayer permeability. To confirm the role of Akt signaling in LPS-induced barrier permeability, the authors used shRNA to knock down Akt in HMECs, which resulted in increased permeability and reduced claudin-5 expression; treatment with LPS had no additional effect on cells lacking Akt.

To investigate these effects in vivo, the authors used a mouse model in which Akt1 was knocked down only in endothelial cells. Following intra-tracheal instillation of LPS to induce ALI, the group found that mice lacking Akt1 had increased lung injury, lung edema, and vascular leakage compared with LPS-treated mice with intact Akt1. Additional knockdown of FoxO1/3a or treatment with a FoxO inhibitor ameliorated LPS-induced lung injury.

To confirm the role of stromelysin1 in Akt-FoxO regulation of endothelial barrier permeability in response to LPS, the authors found that treatment of endothelial cells with LPS or an Akt inhibitor increased stromelysin1 expression; LPS-induced stromelysin1 expression could be reversed following treatment with a FoxO inhibitor. Using mice with LPS-induced lung injury, the authors were able to demonstrate that FoxO or stromelysin1 inhibitors reduced LPS-induced lung injury. Additionally, the group illustrated that LPS treatment decreases claudin-5 expression in the lungs of mice, which was partially reversed by treatment with FoxO or stromelysin1 inhibitors. Finally, to determine whether stromelysin1 could be utilized as a therapeutic target, the authors treated mice with ALI and lacking endothelial Akt1 with a stromelysin1 inhibitor and found that lung injury and edema was improved.

Together, the results from this study identify an Akt1-FoxO-stromelysin1 signaling axis that regulates endothelial barrier integrity and lung edema during ALI, potentially through claudin-5 expression. Importantly, these findings also revealed that FoxO signaling and stromelysin1 are potential therapeutic targets in ARDS.

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