Yonsei Med J.  2011 May;52(3):413-419. 10.3349/ymj.2011.52.3.413.

Native Low-Density Lipoprotein-Dependent Interleukin-8 Production Through Pertussis Toxin-Sensitive G-Protein Coupled Receptors and Hydrogen Peroxide Generation Contributes to Migration of Human Aortic Smooth Muscle Cells

• Lim HK
• Ryoo S

• KMID: 1777012
• DOI: http://doi.org/10.3349/ymj.2011.52.3.413

Abstract

PURPOSE
Stimulation of human aortic smooth muscle cells (hAoSMCs) with native low-density lipoprotein (nLDL) induced the production of interleukin-8 (IL-8) that is involved in the pathogenesis of cardiovascular diseases. However, the process of signal transduction of nLDL was currently uncharacterized. Therefore, the aim of this study was to investigate the signal transduction pathway of nLDL-dependent IL-8 production and the effect of IL-8 on hAoSMCs migration.
MATERIALS AND METHODS
nLDL was prepared by ultracentrifugation with density-adjusted human serum of normocholesterolemia. In hAoSMCs, IL-8 secreted to medium was measured using ELISA assay, and Western blot analysis was performed to detect p38 MAPK activation as a key regulator of IL-8 production. nLDL-dependent H2O2 generation was determined by microscopic analysis using 2',7'-dichlorofluoroscein diacetate (DCF-DA). IL-8-induced migration of hAoSMCs was evaluated by counting the cell numbers moved to lower chamber using Transwell plates.
RESULTS
nLDL-induced IL-8 production was completely blocked by preincubation of hAoSMCs with pertussis toxin (PTX), which inhibited nLDL-dependent p38 MAPK phosphorylation. PTX-sensitive G-protein coupled receptor was responsible for nLDL-dependent H2O2 generation that was abrogated with preincubation of the cells with of polyethylene glycol-conjugated catalase (PEG-Cat). Pretreatment of PEG-Cat prevented nLDL-induced p38 MAPK phosphorylation and IL-8 production, which was partly mimicked by treatment with exogenous H2O2. Finally, IL-8 increased hAoSMCs migration that was completely blocked by incubation with IL-8 neutralizing antibody.
CONCLUSION
PTX-sensitive G-protein coupled receptor-dependent H2O2 generation by nLDL plays a critical role in IL-8 production in hAoSMC, and IL-8 may contribute to atherogenesis through increased migration of hAoSMCs.

Keyword

Native low-density lipoprotein; pertussis toxin-sensitive G-protein coupled receptor; hydrogen peroxide; interleukin-8; human aortic smooth muscle cells; migration

MeSH Terms

Cell Movement/*physiology
Cells, Cultured
Humans
Hydrogen Peroxide/*metabolism
Interleukin-8/*biosynthesis
Lipoproteins, LDL/*pharmacology
Muscle, Smooth, Vascular/cytology/*metabolism
Myocytes, Smooth Muscle/cytology/*metabolism
Pertussis Toxin/pharmacology
Phosphorylation/drug effects
Reactive Oxygen Species/metabolism
Receptors, G-Protein-Coupled/*physiology
Signal Transduction
p38 Mitogen-Activated Protein Kinases/metabolism

Figure

• 

  Fig. 1 Gαi/o-protein coupled receptor is responsible for IL-8 production in nLDL-stimulated hAoSMCs. (A) nLDL (100 µg/mL) significantly induced IL-8 production (*vs. untreated, p<0.01) that was completely abrogated with preincubation of the cells with PTX (100 ng/mL, **vs. nLDL, p<0.01), but CTX (100 ng/mL), heparin (2 mg/mL), and genistein (100 µmol/L) were not effective on nLDL-induced IL-8 production (#vs. nLDL, not significant). n=8 from 4 experiments. (B) hAoSMCs were incubated with nLDL (100 µg/mL) for 10 minutes in the presence or absence of PTX and CTX. PTX completely inhibited nLDL-induced phosphorylation of p38 MAPK, but CTX, heparin, and genistein had no effect (C). (D) Incubation of agonists (S1P, sphingosine-1-phosphate, 100 nmol/L; LPA, lysophosphatidic acid, 25 µmol/L) that stimulate Gαi/o-protein coupled receptors did not increase IL-8 production (*vs. nLDL, p<0.01; **vs. untreated, not significant, n=4). Representative blot from 3 experiments. Phosphate-buffered saline (PBS) was used as untreated control. nLDL, native low density-lipoprotein; hAoSMCs, human aortic smooth muscle cells; IL-8, interleukin-8; PTX, pertussis toxin; CTX, cholera toxin.

• 

  Fig. 2 PTX-sensitive Gαi/o-protein coupled receptor is involves in nLDL-dependent H2O2 generation. (A) DCF-fluorescence images to intracellular H2O2 in hAoSMCs were captured after treatment of nLDL (100 µg/mL) for 30 minutes in the presence or absence of PTX and PEG-Cat (500 U/mL). Treatment of nLDL led to an increase in H2O2 production that was abrogated with preincubation of PTX and PEG-Cat. Exogenous H2O2 (10 µmol/L, 10 minutes) was used as a control. Bar length is 10 µm. (B) Relative intensities of DCF fluorescences were presented as a bar graph (*vs. untreated, p<0.01; #vs. nLDL, p<0.01; ##vs. nLDL, p<0.01; **vs. untreated, p<0.01; n=3).

• 

  Fig. 3 Effect of nLDL-dependent H2O2 generation on IL-8 production. Preincubation of PEG-Cat (500 U/mL) blocked nLDL-induced phosphorylation of p38 MAPK (A) that was resulted in significant inhibition of IL-8 production (B)(*vs. untreated, p<0.01; #vs. nLDL, p<0.01; n=8 from 4 experiments). Exogenous H2O2 at 10 and 20 µmol/L markedly increased p38 MAPK phosphorylation at 10 minutes (C), resulting in induction of IL-8 production (D)(*vs. untreated, p<0.05; #vs. untreated, p<0.01, n=8 from 4 experiments).

• 

  Fig. 4 IL-8 stimulates the migration of hAoSMCs. For the migration assay, hAoSMCs suspensions were loaded into the upper wells of Transwell plates, whereas IL-8 (15 ng/mL) alone and IL-8 plus anti-IL-8 antiserum (0.5 µg/mL) were added into the lower wells. After 8 hours, cells that had migrated to the lower side of the filter were stained (A) and counted using an optical microscope at 400X magnification (B). IL-8 increased cell migration (*vs. untreated, p<0.01, n=4), but its neutralizing antibody reversed cell migration back to untreated control levels (#vs. IL-8, p<0.01, n=4). PDGF (PDGF-BB, 20 ng/mL) was used as a positive control. Bar length is 100 µm. PDGF, platelet-derived growth factor.

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