Journal List > J Korean Diabetes Assoc > v.30(3) > 1062373

Koh, Kim, Kim, Lee, Won, Kim, Lee, and Park: Protective Effect of PGC-1 on Lipid Overload-induced Apoptosis in Vascular Endothelial Cell

Abstract

Background

Fatty acids contribute to endothelial cell dysfunction and apoptosis by inducing accumulation of long chain fatty acyl CoA (LCAC), which increases oxidative stress in vascular endothelial cells. Forced expression of PGC-1 was shown to induce mitochondrial biogenesis and to control expression of mitochondrial enzymes involved in fatty acid oxidation. This study was undertaken to test the hypothesis that PGC-1 overexpression could prevent endothelial cell apoptosis by enhancing fatty acid oxidation and relieving oxidative stress in vascular endothelium.

Methods

Adenoviruses containing human PGC-1 (Ad-PGC-1) and β-galactosidase (Ad-β-gal) were transfected to confluent human aortic endothelial cells (HAECs). To investigate the effect of adenoviral PGC-1 gene transfer on apoptosis, combined treatment of linoleic acid (LA), an unsaturated fatty acid, was performed.

Results

PGC-1 overexpression inhibited the increase in ROS production and apoptosis of HAECs induced by LA. Also, PGC-1 led to a significant increase in fatty acid oxidation and decrease in triglyceride content in HAECs. LA caused the decrease of adenine nucleotide translocase (ANT) activity and transient mitochondrial hyperpolarization, which was followed by depolarization. PGC-1 overexpression prevented these processes.

Conclusion

In summary, PGC-1 overexpression inhibited mitochondrial dysfunction and apoptosis by facilitating fatty acid oxidation and protecting against the damage from oxidative stress in HAECs. The data collectively suggest that the regulation of intracellular PGC-1 expression might play a critical role in preventing atherosclerosis.

Figures and Tables

Fig. 1
Linoleic acid-induced apoptosis in HAECs. HAECs were maintained in media containing 0.5% FBS and exposed to 60 µM LA for the indicated periods of time. Cell viability was analyzed by viable cells using WST assay (A). Caspase-3, -8 and 9 activity were determined by the fluorescence of the cleaved substrate (B-D) (n = 6 per group).
*P < 0.05 vs 0 hr.
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Fig. 2
Effect of adenoviral overexpression of PGC-1 on LA-induced apoptosis. HAECs were infected with Ad-PGC-1 or Ad-β-gal. Two days after infection, cells were maintained in media containing 0.5% FBS and exposed to 60 µM LA for 24 h. Following treatment, caspase-3(A), -8(B) and -9(C) activation was analyzed by measuring activities using a caspase fluorescence assay kit (n = 6 per group).
*P < 0.05 vs control, #P < 0.05 vs Ad-β-gal.
jkda-30-151-g002
Fig. 3
Immunoblotting for active subunit of caspase-3 and PARP (A). Cells were exposed to 60 µM LA for 48 h. DNA fragmentation was decreased in PGC-1 overexpressing HAECs (B). Apoptosis was analyzed by measuring the levels of cytosolic histone-bound DNA fragments using a cell death ELISA assay kit (C). (n = 6 per group).
*P < 0.05 vs control, #P < 0.05 vs Ad-β-gal.
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Fig. 4
Triglyceride content and fatty acid oxidation in PGC-1 overexpressing HAECs. Triglyceride content (A) and Fatty acid oxidation with 3H-palmitate (B) (n=6 per group).
*P < 0.05 vs control, #P < 0.05 vs Ad-β-gal.
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Fig. 5
Effect of adenoviral overexpression of PGC-1 on mitochondrial function and intracellular reactive oxygen species formation. Mitochondria were isolated from PGC-1- and β-gal-overexpressing cells cultured in presence or absence of LA. The ANT-dependent ADP uptake of the mitochondria was assessed by incubating them in the presence of 14C-ADP (A). ΔΨM was assessed by loading cultured HAECs with JC-1 (10 µmol/L) for 15 min. JC-1 aggregation (red fluorescence) increases with increased ΔΨM (B). Cells were exposed to 60 µM LA for 6 h. ROS were measured with DCF-DA (10 µmol/L) for 15 min (C) (n=6 per group).
*P < 0.05 vs control, #P < 0.05 vs Ad-β-gal.
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