Microvascular Endothelial Cell Hyperpermeability Induced by Endogenous Caspase 3 Activator Staurosporine

BACKGROUND: Microvascular hyperpermeability following conditions such as hemorrhagic shock occurs mainly owing to disruption of the adherens junctional protein complex in endothelial cells. The objective of this study was to examine the action of staurosporine, a potent activator of endogenous caspase 3 on the adherens junction and the cellular pathway through which it causes possible endothelial cell barrier dysfunction.METHODS: Rat lung microvascular endothelial cell (RLMEC) permeability was measured by fluorescein isothiocyanate-albumin flux across the monolayer in a Transwell plate. Integrity of the endothelial cell adherens junctions was studied using immunofluorescence of A-catenin and vascular endothelial-cadherin. Mitochondrial reactive oxygen species formation was determined by using dihydrorhodamine 123 and mitochondrial transmembrane potential by JC-1 fluorescent probe and flow cytometry. Caspase 3 enzyme activity was assayed fluorometrically. Cell death assay in RLMECs was performed using propidium iodide staining and analyzed by flow cytometry.RESULTS: Staurosporine (1 mu M)-treated RLMEC monolayers showed significant increase in permeability, which was decreased by pretreatment with caspase 3 specific inhibitor, Z-DEVD-FMK (p < 0.05). Immunofluorescence studies showed staurosporine induced disruption of the adherens junction, which was reversed by Z-DEVD-FMK. Staurosporine treatment led to an increase in mitochondrial reactive oxygen species formation and a decrease in mitochondrial transmembrane potential. Furthermore, staurosporine induced a significant increase in caspase 3 activity (p < 0.05) but not cell death in RLMECs (p < 0.05).CONCLUSION: Staurosporine-induced disruption of the adherens junction and microvascular endothelial cell hyperpermeability is associated with the activation of mitochondrial "intrinsic'' apoptotic signaling cascade but without causing endothelial cell death. Our results suggest that prevention of mitochondrial-mediated activation of caspase 3 has therapeutic potential against microvascular hyperpermeability. (J Trauma Acute Care Surg. 2013; 74: 516-523. Copyright (C) 2013 by Lippincott Williams & Wilkins)