J Korean Med Sci.  2012 Jul;27(7):803-810. 10.3346/jkms.2012.27.7.803.

Impact of Lysophosphatidylcholine on the Plasminogen Activator System in Cultured Vascular Smooth Muscle Cells

• Yoon BK
• Kang YH
• Oh WJ
• Park K
• Lee DY
• Choi D
• Kim DK
• Lee Y
• Rhyu MR

• KMID: 2157928
• DOI: http://doi.org/10.3346/jkms.2012.27.7.803

Abstract

The balance between tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) regulates fibrinolysis. PAI-1 expression increases in atherosclerotic arteries and vascular smooth muscle cells (VSMCs) are one of major constituents of atheroma. We investigated the impact of lysophosphatidylcholine (lysoPC), an active component of oxidized low-density lipoprotein, on the plasminogen activator system of the rat VSMCs. The lysoPC stimulated the protein and gene expressions of PAI-1 but did not affect the protein expression of t-PA. Fibrin overlay zymography revealed that lysoPC increased the activity of PAI-1 in the conditioned media, while concurrently decreasing that of free t-PA. Vitamin E inhibited the lysoPC-induced PAI-1 expression. Further, lysoPC increased the intracellular reactive oxygen species (ROS) formation. Caffeic acid phenethyl ester, an inhibitor of NF-kappaB, blocked this lysoPC effect. Indeed, lysoPC induced the NF-kappaB-mediated transcriptional activity as measured by luciferase reporter assay. In addition, genistein, an inhibitor of protein-tyrosine kinase (PTK), diminished the lysoPC effect, while 7,12-dimethylbenz[a]anthracene, a stimulator of PTK, stimulated PAI-1 production. In conclusion, lysoPC does not affect t-PA expression but induces PAI-1 expression in the VSMC by mediating NF-kappaB and the genistein-sensitive PTK signaling pathways via oxidative stress. Importantly, lysoPC stimulates the enzyme activity of PAI-1 and suppresses that of t-PA.

Keyword

Lysophosphatidylcholines; NF-kappa B; Oxidative Stress; Plasminogen Activator Inhibitor 1; Protein-Tyrosine Kinase; Muscle, Smooth, Vascular

MeSH Terms

Animals
Benz(a)Anthracenes/pharmacology
Caffeic Acids/pharmacology
Cells, Cultured
Genistein/pharmacology
Lipoproteins, LDL/metabolism
Lysophosphatidylcholines/*pharmacology
Muscle, Smooth, Vascular/cytology/*drug effects/metabolism
NF-kappa B/antagonists & inhibitors/metabolism
Oxidative Stress/drug effects
Phenylethyl Alcohol/analogs & derivatives/pharmacology
Plasminogen Activator Inhibitor 1/agonists/genetics/*metabolism
Protein Kinase Inhibitors/pharmacology
Protein-Tyrosine Kinases/antagonists & inhibitors/metabolism
Rats
Rats, Sprague-Dawley
Reactive Oxygen Species/metabolism
Signal Transduction/drug effects
Tissue Plasminogen Activator/*metabolism
Transcription, Genetic/drug effects
Up-Regulation/drug effects
Vitamin E/pharmacology
Benz(a)Anthracenes
Caffeic Acids
Lipoproteins, LDL
Lysophosphatidylcholines
NF-kappa B
Plasminogen Activator Inhibitor 1
Protein Kinase Inhibitors
Reactive Oxygen Species
Vitamin E
Genistein
Phenylethyl Alcohol
Protein-Tyrosine Kinases
Tissue Plasminogen Activator

Figure

• 

  Fig. 1 The effects of lysoPC on PAI-1 expression in VSMCs. (A), (B) and (C) Induction of PAI-1 protein as detected by Western blot analysis. Representative blots are shown in the upper section of each panel. The intensity of the bands was densitometrically determined and normalized to that of corresponding β-actin bands. Graphic data are depicted in the lower part of the panel. (A) Time-course effects of 10 µM lysoPC in the cell lysate. (B) Dose-response effects of lysoPC at 6 hr of treatment in the cell lysate. (C) Time-course effects of 10 µM lysoPC in the conditioned media normalized to the cell protein content. (D) Time-course effects of 10 µM lysoPC on gene expression of PAI-1 as evaluated by real-time RT-PCR. (E) Time-course effects of 10 µM lysoPC on tPA in the cell lysate and conditioned media as assessed by ELISA. *P < 0.05 vs vehicle-treated control; †P < 0.05 vs baseline.

• 

  Fig. 2 The effects of lysoPC on the activities of plasminogen activator and its inhibitors in the CM harvested from cultured VSMCs. Representative zymographs are shown in the upper section of each panel. The intensity of the bands was densitometrically determined, and graphic data are depicted in the lower part of the panel. (A) and (B) Time- and dose-dependent activity of PAI-1 after lysoPC treatment as assessed by reverse fibrin overlay zymography. (C) Time-course effects of 10 µM lysoPC on activity of t-PA as assessed by fibrin overlay zymography. *P < 0.05 vs vehicle-treated control.

• 

  Fig. 3 The involvement of oxidative stress and nuclear transcription factor NF-κB activation in the induction of PAI-1 in VSMCs stimulated with 10 µM lysoPC. (A) Effects of vitamin E (50 µM), a potent antioxidant, or CAPE (20 nM), an inhibitor of NF-κB, on PAI-1 expression induced by lysoPC for 6 hr as assessed by Western blot analysis. Representative blots are shown in the upper section of each panel. The intensity of the bands was densitometrically determined and normalized to that of corresponding β-actin bands. Graphic data are depicted in the lower part of the panel. (B) Intracellular ROS production detected by DCF fluorescence under confocal microscopy (× 100). (C) Kinetics of intracellular ROS production as assessed by flow cytometry analysis using DCF-DA. (D) Effects of lysoPC for 1 hr on NF-κB-mediated transcriptional activity as measured by luciferase reporter assay. *P < 0.05 vs baseline; †P < 0.05 vs DSF medium; ‡P < 0.05 vs vehicle-treated control.

• 

  Fig. 4 The effects of inhibitors of major cell signaling pathways on the expression of PAI-1 stimulated with lysoPC in vascular smooth muscle cells. Representative blots are shown in the upper panel. The intensity of the bands was densitometrically determined and normalized to that of corresponding β-actin bands. Graphic data are depicted in the lower panel. Western blot analysis of whole cell extracts from vascular smooth muscle cells treated for 6 hr with 10 µM lysoPC in the presence of calphostin C (10-7 M, an inhibitor of protein kinase C), KT-5720 (10-6 M, an inhibitor of protein kinase A) or genistein (10-6 M, an inhibitor of PTK). Effect of DMBA (20 mM, a stimulator of PTK) on PAI-1 expression is also shown. *P < 0.05 vs DSF medium; †P < 0.05 vs vehicle-treated control.

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