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Alpha lipoic acid attenuates microvascular endothelial cell hyperpermeability by inhibiting the intrinsic apoptotic signaling

https://doi.org/10.1016/j.amjsurg.2007.09.028Get rights and content

Abstract

Background

This study examined whether alpha lipoic acid (ALA), an antioxidant with anti-apoptotic properties, synthesized in mitochondria of endothelial cells, would inhibit intrinsic apoptotic signaling and microvascular endothelial cell hyperpermeability.

Methods

Rat lung microvascular endothelial cells were transfected with BAK (BH3) peptide (5 μg/mL) or active caspase-3 (5 μg/mL) and were pretreated with ALA (10 and 100 μmol/L). Hyperpermeability was determined using fluorescein isothiocyanate albumin-flux across the cells grown as monolayer. Reactive oxygen species (ROS) formation was determined using 123 dihydrorhodamine and mitochondrial membrane potential using JC-1. Cytochrome c levels and caspase-3 activity were determined using an enzyme-linked immunosorbent assay and a fluorometric assay, respectively.

Results

ALA (100 μmol/L) pretreatment attenuated BAK (BH3)-induced hyperpermeability and ROS formation. ALA restored BAK (BH3)-induced collapse in mitochondrial membrane potential and decreased BAK (BH3)-induced cytochrome c release and caspase-3 activity.

Conclusions

These findings suggest that ALA attenuates BAK-induced monolayer hyperpermeability through the inhibition of ROS formation and intrinsic apoptotic signaling.

Section snippets

Chemicals and solutions

ALA and fluorescein isothiocyanate-bovine albumin (FITC-albumin) were obtained from Sigma (St. Louis, MO). JC-1 was obtained from Cell Technology Inc. (Mountain View, CA). JC-1 was prepared by reconstituting the lyophilized reagent with 500 μL of dimethyl sulfoxide to obtain a 100× stock solution. Immediately before the experiments the 100× solution was diluted 1:100 in 1× assay buffer. BAK (BH3) peptide and BAK L to A mutant peptide (R&D Systems, Minneapolis, MN), a 1 μg/μL stock, was mixed

Monolayer hyperpermeability

Transfection of BAK (BH3) peptide (5 μg/mL) on RLMEC monolayers induced hyperpermeability. The FITC-albumin fluorescent intensity of the control cells was taken as 100%. The FITC-albumin fluorescent intensity was significant in the BAK (BH3) peptide–transfected group compared with the control group (215.4% ± 8.4% of the control; P < .05; Fig. 1A), suggesting leakage of albumin across the endothelial monolayer. Cells pretreated with ALA (100 μmol/L) showed a decrease in BAK (BH3)-induced

Comments

In this study, we tested the ability of ALA, an endogenous antioxidant with anti-apoptotic properties, to attenuate microvascular endothelial cell hyperpermeability. BAK, a pro-apoptotic factor, was used to induce hyperpermeability. Our results showed that BAK (BH3) transfection increased microvascular endothelial cell hyperpermeability, which was attenuated by ALA pretreatment. In addition, ALA inhibited BAK (BH3)-induced mitochondrial ROS formation, prevented the collapse of mitochondrial

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