J Korean Soc Menopause.  2012 Dec;18(3):139-146.

Cytotoxic Effects of Lysophosphatidylcholine on Vascular Smooth Muscle Cells

• Lee DY
• Kang YH
• Choi DS
• Lee YJ
• Rhyu MR
• Yoon BK

Abstract

OBJECTIVES
To investigate the cytotoxic effects of lysophosphatidylcholine (lysoPC), an active component of oxidized low-density lipoproteins (LDL), on vascular smooth muscle cells (VSMCs).
METHODS
VSMCs were derived from rat aorta. Cell death was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay, lactic dehydrogenase (LDH) assay, and DNA fragmentation assay. Apoptosis was quantified by propidium iodide staining and fluorescent activated cell sorting (FACS) analysis, and intracellular free radical production was determined using 2',7'-dichlorofluorescin diacetate (DCF-DA). In addition, the changes in caspases, bcl-2 and bax proteins were evaluated by western blot analysis.
RESULTS
LysoPC over 25 microM induced more than 50% of the cell death at 10 hours on MTT assay with no change in the level of LDH. The DNA ladder pattern showed that cell death induced by lysoPC was caused by apoptosis, which was associated with increased free radical production. Vitamin E, a potent antioxidant and caffeic acid phenylethyl ester (CAPE), an inhibitor of nuclear factor-kappaB (NF-kappaB), blocked apoptosis. The casepase-3 precursor decreased and the active form of caspase-8 increased. Total bcl-2 and bax proteins did not change with lysoPC treatment, but translocation of bax from cytosole to the mitochondria membrane was observed.
CONCLUSION
LysoPC induces apoptosis in VSMCs via an oxidant mechanism, dependent on NF-kappaB.

Keyword

Apoptosis; Atherosclerosis; Lysophosphatidylcholines; Vascular smooth muscle cell

MeSH Terms

Animals
Aorta
Apoptosis
Atherosclerosis
bcl-2-Associated X Protein
Blotting, Western
Caffeic Acids
Caspase 8
Caspases
Cell Death
Cytosol
DNA
DNA Fragmentation
Fluoresceins
Lipoproteins, LDL
Lysophosphatidylcholines
Membranes
Mitochondria
Muscle, Smooth, Vascular
NF-kappa B
Oxidoreductases
Propidium
Rats
Vitamin E
Vitamins
Caffeic Acids
Caspase 8
Caspases
DNA
Fluoresceins
Lipoproteins, LDL
Lysophosphatidylcholines
NF-kappa B
Oxidoreductases
Propidium
Vitamin E
Vitamins
bcl-2-Associated X Protein

Figure

• 

  Fig. 1 Cytotoxic effects of lysophosphatidylcholine (lysoPC) on vascular smooth muscle cells (VSMCs). (A) Viability of VSMC treated with lysoPC, (B) cell necrosis induced by lysoPC, (C) lysoPC induced DNA fragmentation.

• 

  Fig. 2 Mechanisms for vascular smooth muscle cell (VSMC) apoptosis induced by lysophosphatidylcholine (lysoPC). (A) Involvement of reactive oxygen species and nuclear factor-kappaB (NF-κB), as assessed by propidium iodide (PI) staining, (B) kinetics of reactive oxygen species (ROS) production determined by 2',7'-dichlorofluorescin (DCF) staining. DSF: defined serum-free medium, CAPE: caffeic acid phenylethyl ester. *P < 0.001 vs. control.

• 

  Fig. 3 Mechanisms for vascular smooth muscle cell (VSMC) apoptosis induced by lysophosphatidylcholine (lysoPC), evaluated by western blot analyses. (A) expression of caspases, (B) expression of bcl-2, (C) expression of bax.

• 

  Fig. 4 Pathway of apoptosis.

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