Cytosolic Active Caspase 3 Promotes Endothelial Barrier Integrity after Thrombin Stimulation

Abstract

BackgroundWe have previously shown that MK2 deficiency leads to cytoplasmic sequestration of active caspase 3, prevention of apoptosis and is associated with a preserved endothelial barrier function in response to endotoxin challenge. While caspase 3 is widely described as the executioner of apoptosis, it has numerous cytosolic substrates that are not related to execution of apoptosis. Therefore, we hypothesized that cytosolic caspase 3 promotes endothelial barrier function.MethodsHuman lung micro‐vascular endothelial cells (HMVECs) were exposed to a non‐apoptotic, thrombin, stimulus and endothelial barrier function was assessed using electric cell‐substrate impedance sensing (ECIS). After exposures, a subset of HMVECs were evaluated for actin cytoskeletal rearrangement using phalloidin staining. In addition, HMVEC cell lysates were harvested for protein analyses. Caspase 3 knockdown or inhibition was achieved using RNA interference or the pharmacological inhibitor q‐VD‐OPH, respectively.ResultsExposure of HMVECs to thrombin demonstrated a dose dependent transient decrease in endothelial barrier function as measured by a drop in electrical resistance followed by a rapid recovery on ECIS. Thrombin activation of HMVECs led to cytosolic caspase 3 activation, confirmed by fluorescent microscopy. Inhibition of caspases with q‐VD‐OPH in HMVECs led to a similar drop in thrombin‐induced endothelial barrier function but notably there was no subsequent barrier recovery. The lack of barrier recovery was not a result of endothelial cell death but rather a failure of de‐remodeling of thrombin‐induced actin stress fiber formation. Caspase 3 knockdown in HMVECs also resulted in lack of de‐remodeling after thrombin‐induced actin stress fiber formation.ConclusionTaken together, our results suggest cytosolic active caspase 3 promotes endothelial barrier integrity.