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Active Caspase 3 Plays a Role in the Recovery of the Endothelial Cell Barrier from Thrombin-Induced Disruption
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A5720 - Active Caspase 3 Plays a Role in the Recovery of the Endothelial Cell Barrier from Thrombin-Induced Disruption
Author Block: K. Carino, T. M. Nicholson, L. Johnston, K. Suresh, L. A. Shimoda, L. Servinsky, P. M. Hassoun, M. Damarla; Pulmonary and Critical Care, Johns Hopkins University, Baltimore, MD, United States.
Rationale: Studies have shown that activated caspase 3 remains in the cytosol in MK2-deficient mice. Cytosolic sequestration of caspase 3 is associated with protection from vascular permeability and apoptosis. Our objective is to investigate caspase 3's possible role in promoting endothelial cell barrier integrity.
Methods: We subjected human lung microvascular endothelial cells (HMVECs) to 1.25 U/mL of thrombin, an agonist that disrupts barrier integrity. Caspase 3 inhibition was achieved using the pan-caspase inhibitor, q-VD, the caspase 3 specific inhibitor, DEVD, or by si-RNA against caspase 3. Electric Cell-substrate Impedance Sensor (ECIS) measured barrier integrity. Hoecsht33342 staining of nuclear DNA determined apoptotic cell death. Protein expression was quantified through immunoblotting and cytoskeletal changes were visualized using fluorescent microscopy.
Results: Analysis of total cell lysates shows activation of caspase 3 within two minutes of thrombin stimulation. Hoecsht staining confirmed no change in total cell numbers nor in cell death following thrombin exposure. Exposure 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. Inhibition of caspases in HMVECs led to an exaggerated drop in thrombin-induced endothelial barrier function. Interestingly, inhibition of caspases also led to exaggerated actin polymerization and paracellular gap formation. Immunoblotting demonstrated an initial increase in phosphorylated myosin light chain (MLC) followed by a return to baseline by 120 minutes following thrombin stimulation. Immunoblotting also showed an exaggerated increase in phosphorylated MLC and a magnetic bead twist assay showed increased cell stiffness in caspase-inhibited cells.
Conclusion: Our results suggest that caspases, and likely caspase 3, play a role in promoting endothelial cell barrier integrity and its inhibition can lead to prolonged cell disruption.
Author Block: K. Carino, T. M. Nicholson, L. Johnston, K. Suresh, L. A. Shimoda, L. Servinsky, P. M. Hassoun, M. Damarla; Pulmonary and Critical Care, Johns Hopkins University, Baltimore, MD, United States.
Rationale: Studies have shown that activated caspase 3 remains in the cytosol in MK2-deficient mice. Cytosolic sequestration of caspase 3 is associated with protection from vascular permeability and apoptosis. Our objective is to investigate caspase 3's possible role in promoting endothelial cell barrier integrity.
Methods: We subjected human lung microvascular endothelial cells (HMVECs) to 1.25 U/mL of thrombin, an agonist that disrupts barrier integrity. Caspase 3 inhibition was achieved using the pan-caspase inhibitor, q-VD, the caspase 3 specific inhibitor, DEVD, or by si-RNA against caspase 3. Electric Cell-substrate Impedance Sensor (ECIS) measured barrier integrity. Hoecsht33342 staining of nuclear DNA determined apoptotic cell death. Protein expression was quantified through immunoblotting and cytoskeletal changes were visualized using fluorescent microscopy.
Results: Analysis of total cell lysates shows activation of caspase 3 within two minutes of thrombin stimulation. Hoecsht staining confirmed no change in total cell numbers nor in cell death following thrombin exposure. Exposure 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. Inhibition of caspases in HMVECs led to an exaggerated drop in thrombin-induced endothelial barrier function. Interestingly, inhibition of caspases also led to exaggerated actin polymerization and paracellular gap formation. Immunoblotting demonstrated an initial increase in phosphorylated myosin light chain (MLC) followed by a return to baseline by 120 minutes following thrombin stimulation. Immunoblotting also showed an exaggerated increase in phosphorylated MLC and a magnetic bead twist assay showed increased cell stiffness in caspase-inhibited cells.
Conclusion: Our results suggest that caspases, and likely caspase 3, play a role in promoting endothelial cell barrier integrity and its inhibition can lead to prolonged cell disruption.
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