Abstract
Hydrogen peroxide (H2O2) plays an important role physiologically as the second messenger and pathologically as an inducer of oxidative stress in injury, ischemia and other conditions. However, it is unclear how H2O2 influences various cellular functions in health and disease differentially, particularly in the blood-brain barrier (BBB). We hypothesized that the change in cellular concentrations of H2O2 is a major contributor in regulation of angiogenesis, barrier integrity/permeability and cell death/apoptosis in BBB endothelial cells. Rat brain microvascular endothelial cells were exposed to various concentrations of H2O2 (1 nM to 25 mM). BBB tight junction protein (zonula ocludens-1; ZO-1) localization and expression, cytoskeletal organization, monolayer permeability, angiogenesis, cell viability and apoptosis were evaluated. H2O2 at low concentrations (0.001 μM to 1 μM) increased endothelial cell tube formation indicating enhanced angiogenesis. H2O2 at 100 μM and above induced monolayer hyperpermeability significantly (p < 0.05). H2O2 at 10 mM and above decreased cell viability and induced apoptosis (p < 0.05). There was a decrease of ZO-1 tight junction localization with 100 μm H2O2, but had no effect on protein expression. Cytoskeletal disorganizations were observed starting at 1 μm. In conclusion H2O2 influences angiogenesis, permeability, and cell death/apoptosis in a tri-phasic and concentration-dependent manner in microvascular endothelial cells of the blood-brain barrier.
Highlights
Hyperpermeability[8,13,17] studies from our lab as well as by others have shown oxidative stress by ROS is critical to endothelial cell barrier dysfunctions[8,17,18]
The results from trans-endothelial electrical resistance (TEER) measurement indicated that the lower concentrations of H2O2 (0.001 to 1 μM) showed no significant change in TEER compared to the untreated control group, whereas a significantly low resistance was observed (p < 0.05; n = 4; Fig. 1B) in cells treated with concentrations of H2O2 (10 μM) and above compared to control
The purpose of the study was to evaluate the relationship between angiogenesis, permeability and cell death/apoptosis in blood-brain barrier (BBB) endothelial cells following H2O2 exposure to understand why H2O2 behave differentially in these conditions
Summary
Hyperpermeability[8,13,17] studies from our lab as well as by others have shown oxidative stress by ROS is critical to endothelial cell barrier dysfunctions[8,17,18]. Reactive oxygen species have physiologic function and are known to be important in the regulation of angiogenesis, vessel growth from preexisting vessels. Reactive oxygen species are considered important in the physiologic regulation and pathophysiologic dysregulation of angiogenesis, hyperpermeability, and apoptosis, their relationship or role in the BBB has not been well-studied. The purpose of this study was to test the effect of varying concentrations of H2O2 on angiogenesis, barrier functions/permeability and cell viability/apoptosis to evaluate how it affects them differentially in BBB endothelial cells
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