© 1999 American Heart Association, Inc.
Cellular Biology |
Simulated Ischemia in Flow-Adapted Endothelial Cells Leads to Generation of Reactive Oxygen Species and Cell Signaling
From the Institute for Environmental Medicine, University of Pennsylvania Medical Center, Philadelphia, Pa.
Correspondence to Aron B. Fisher, MD, Institute for Environmental Medicine, University of Pennsylvania Medical Center, 1 John Morgan Building, Philadelphia, PA 19104-6068. E-mail abf{at}mail.med.upenn.edu
Abstract—We have previously
shown that increased reactive oxygen species (ROS) generation occurs
with ischemia in the oxygenated lung and have
hypothesized that mechanotransduction is the initiating event. In the
present study, we developed an in vitro model of
oxygenated ischemia by interrupting medium flow to
flow-adapted bovine pulmonary artery
endothelial cells in an artificial capillary system.
Cellular oxygenation during the "ischemic"
period was maintained by perfusing medium over the abluminal surface of
porous capillaries. Cells were assessed for ROS generation, nuclear
factor-
B (NF-B) and activator protein-1 (AP-1)
binding activities, and DNA synthesis using
dichlorofluorescein fluorescence by flow cytometry
and spectrofluorometry, electrophoretic mobility shift assay of nuclear
extracts with NF-B–specific or AP-1–specific
32P-labeled oligonucleotides, and
3H-thymidine incorporation into DNA. Cells that were flow
adapted for 2 to 7 days with 1 to 2 dyne/cm2 shear stress
exhibited a 1.6- to 1.9-fold increase in ROS generation during 1 hour
of simulated ischemia compared with continuously perfused
cells. This effect was abolished by diphenyleneiodonium chloride
(DPI), indicating a role for a flavoprotein such as NADPH oxidase. The
increase in ROS generation with ischemia was similar for cells
from low and high passages. With ischemia, flow-adapted cells
exhibited increases of 1.7-fold in nuclear NF-B and 1.5-fold in
nuclear AP-1; these changes were abolished by pretreatment with
N-acetylcysteine or DPI. Ischemia for 24 hours
resulted in a 1.8-fold increase of 3H-thymidine
incorporation into DNA and a significant increase of cells entering the
cell cycle, as indicated by flow cytometry with propidium iodide. We
conclude that flow-adapted endothelial cells generate
ROS with ischemia that results in activation of NF-B and
AP-1 and an increase of DNA synthesis. This effect is not mediated by
hypoxia, implicating a role for mechanotransduction in
ischemia-mediated cell signaling.
Key Words: shear stress • artificial capillary • reactive oxygen species • signal transduction
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