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 β-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 µM)-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).


CONCLUSIONS
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.
Department of Surgery, Texas A&M Health Science Center College of Medicine and Scott and White Health Care, Temple, Texas, USA.
Devendra A
DA
Sawant
Binu
B
Tharakan
Richard P
RP
Tobin
John
J
Reilly
Felicia A
FA
Hunter
Martha Karen
MK
Newell
William Roy
WR
Smythe
Ed W
EW
Childs
D022005:Adherens Junctions / Q000187:drug effects / Q000502:physiology; D000818:Animals; D002199:Capillary Permeability / Q000187:drug effects* / Q000502:physiology; D053148:Caspase 3 / Q000378:metabolism*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000502:physiology; D004789:Enzyme Activation / Q000187:drug effects; D005434:Flow Cytometry; D008856:Microscopy, Fluorescence; D055806:Microvessels / Q000187:drug effects* / Q000502:physiology; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology*
D017382:Reactive Oxygen Species; D053148:Caspase 3; D019311:Staurosporine