Published online before print March 2, 2006, doi: 10.1161/01.RES.0000216009.68958.e6
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© 2006 American Heart Association, Inc.
Report |
N-3 Fatty Acids Inhibit Vascular Calcification Via the p38-Mitogen-Activated Protein Kinase and Peroxisome Proliferator-Activated Receptor-
Pathways
From the Departments of Medicine and Physiology, University of California at Los Angeles.
Correspondence to Yin Tintut, PhD, Department of Medicine/Cardiology, CHS BH-307, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095. E-mail ytintut{at}mednet.ucla.edu
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Abstract |
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Fish oil supplementation is associated with lower risk of coronary artery disease in humans, and it has been shown to reduce ectopic calcification in an animal model. However, whether N-3 fatty acids, active ingredients of fish oil, have direct effects on calcification of vascular cells is not clear. In this report, we investigated the effects of eicosapentaenoic acid and docosahexaenoic acid (DHA) on osteoblastic differentiation and mineralization of calcifying vascular cells (CVCs), a subpopulation of bovine aortic medial cells that undergo osteoblastic differentiation and form calcified matrix in vitro. Results showed that N-3 fatty acids inhibited alkaline phosphatase (ALP) activity and mineralization of vascular cells, suggesting that they directly affect osteoblastic differentiation in vascular cells. By Western blot analysis, DHA activated p38-mitogen-activated protein kinase (MAPK) but not extracellular-regulated kinase (ERK) or Akt. An inhibitor of p38-MAPK partially reversed the inhibitory effects of DHA on osteoblastic differentiation and mineralization. Transient transfection experiments showed that DHA also activated peroxisome proliferator-activated receptor-
(PPAR-). Both p38-MAPK activator and PPAR- agonists reproduced the inhibitory effects of DHA on CVC mineralization. Pretreatment with DHA also inhibited interleukin-6–induced ALP activity and mineralization. Together, these results suggest that N-3 fatty acids directly inhibit vascular calcification, and that the inhibitory effects are mediated by the p38-MAPK and PPAR- pathways.
Key Words: fatty acids • vascular smooth muscle cells • calcification
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Vascular calcification, a clinically significant pathological process similar to endochondral osteogenesis,1 is positively regulated by developmental factors such as bone morphogentic protein-2 (BMP-2), Msx-2, and Wnt and negatively regulated by other factors such as osteopontin, matrix gamma-carboxyglutamic acid (GLA) protein, and nucleotide pyrophosphate/phosphodiesterase-1.1,2 Vascular calcification develops in parallel with atherosclerosis and is stimulated by inflammatory cytokines and lipid oxidation products.2 Fish oil consumption associates with reduced atherosclerosis.3 Its active ingredients, N-3 fatty acids, cis–5,8,11,14,17-eicosapentaenoic acid (EPA; C20:5n-3) and cis–4,7,10,13,16,19-docosahexaenoic acid (DHA; C22:6n-3), are potent anti-inflammatory peroxisome proliferator-activated receptor-
(PPAR-) agonists.4–5 N-3 fatty acid supplementation also reduces ectopic calcification in vivo.6–7 However, it is not known whether the effects are direct.
Several investigators identified vascular cells with osteogenic potential,8–10 including calcifying vascular cells (CVCs), which undergo osteoblastic differentiation and mineralization.11–12 In this study, we investigated whether N-3 fatty acids directly inhibit osteoblastic differentiation of CVCs. Results show that they inhibit both spontaneous and interleukin-6 (IL-6)–induced osteoblastic differentiation in these vascular cells, and that the effects are mediated by the p38-mitogen-activated protein kinase (MAPK) and PPAR- pathways.
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Materials and Methods |
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Agents were added 1 day after plating or as indicated. DHA and EPA were used at 25 µmol/L or as indicated. Mineralization (o-cresolphthalein) and alkaline phosphatase (ALP) activity were assayed as described.8,11 For luciferase assay, CVCs were transfected at day 2, treated with reagents the next day, and assayed 18 to 24 hours later. Means were compared by ANOVA and expressed as mean±SD. For details, see the online supplement, available at http://circres.aha.org.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Effect of N-3 Fatty Acids on Spontaneous Osteoblastic Differentiation
To determine whether N-3 fatty acids directly affect osteoblastic differentiation of vascular cells, CVCs were treated with DHA or EPA and assayed for ALP activity, an early marker, and mineralization, a functional marker of osteoblastic differentiation. Treatment with EPA for 4 days significantly inhibited ALP activity in a dose-dependent manner (Figure 1A). Dose-dependent inhibition was also observed with DHA (data not shown). Both EPA and DHA, but not the negative control proatherogenic oxidized PAPC, had similar inhibitory effects on ALP activity (Figure 1B). DHA also inhibited ALP activity in a time-dependent manner (Figure 1C). Treatment with either DHA or EPA for 11 days produced significantly less matrix calcium than vehicle alone (Figure 1D). An indirect effect of N-3 fatty acids via inhibition of cell growth was excluded by 3H-thymidine assay, which showed no significant effect of EPA or DHA on proliferation (data not shown).
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Effect of N-3 Fatty Acids on Intracellular Signaling Pathways
To determine the signaling pathway that mediates the inhibitory effects of N-3 fatty acids, Western analysis was performed. In response to DHA, p38-MAPK was phosphorylated but not extracellular-regulated kinase (ERK), MAPK kinase (MEK), or Akt (Figure 2A). EPA had similar effects (data not shown). To investigate whether p38-MAPK mediates the inhibitory effects of DHA, CVCs were pretreated with SB203580, a p38-MAPK inhibitor, for 30 minutes, followed by DHA treatment, and mineralization was assessed. As shown in Figure 2B, SB203580 partially reversed the DHA inhibitory effect.
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As an additional potential signaling pathway mediating DHA inhibition, we tested for transcriptional activation of PPAR-. As shown in Figure 2C, DHA activated the PPAR response element (PPRE)–luciferase construct in a similar manner to ciglitazone, a known PPAR- ligand. Treatment of CVCs with known PPAR- agonists inhibited CVC mineralization, suggesting that activation of PPAR- mediates DHA inhibitory effects (Figure 2D).
Effect of N-3 Fatty Acids on IL-6–Induced Osteoblastic Differentiation
To test whether DHA inhibits IL-6–induced osteoblastic differentiation, CVCs were pretreated with DHA for 2 days, followed by cotreatment with IL-6. ALP activity and mineralization were assessed 4 and 8 days after the IL-6 treatment, respectively. As shown in Figure 3A and 3B, IL-6–induced ALP activity and mineralization were significantly inhibited by the DHA pretreatment. Anisomycin (0.5 µg/mL), an activator of p38-MAPK, also inhibited both spontaneous and IL-6–induced ALP activity in a manner similar to DHA (Figure 3C).
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Next, we investigated whether IL-6–induced signal transducers and activators of transcription-3 (STAT-3) activation was affected by the DHA treatment. Cells were pretreated with DHA for 2 days followed by IL-6 treatment for 15 minutes. As we showed previously12 and in Figure 3D, IL-6 induced phosphorylation of STAT-3, which was partially attenuated by DHA. DHA alone had little effect on STAT-3 phosphorylation. IL-6 did not affect p38-MAPK activity either alone or induced by DHA (Figure 3D). Anisomycin, an activator of p38-MAPK, also inhibited IL-6–induced STAT-3 phosphorylation to a similar manner as DHA (Figure 3E).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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These findings indicate that omega-3 fatty acids directly inhibit both spontaneous and IL-6–induced osteoblastic differentiation of CVCs by activating both p38-MAPK and PPAR-
, which is known to inhibit osteoblastic differentiation.13 Known activators of both of these signaling pathways reproduced the inhibitory effects of DHA, suggesting that both pathways independently mediate DHA inhibitory effects on CVC differentiation. Cross-talk between the p38-MAPK and STAT-3 pathways has been reported.14–15 In CVCs, we observed that pretreatment with anisomycin or DHA to stimulate p38-MAPK partially blocked STAT-3 phosphorylation induced by IL-6. Interestingly, IL-6 had no effect on DHA-induced p38-MAPK, and unlike pretreatment, simultaneous cotreatment of IL-6 with DHA did not attenuate IL-6–induced mineralization in CVCs (data not shown). Thus, it appears that p38-MAPK may generate an inhibitor of STAT-3 over time.
Our results show that DHA inhibits IL-6–induced ALP activity and mineralization to a greater extent than it inhibits STAT-3 phosphorylation, suggesting that more than one signaling molecule may be targeted by DHA. IL-6 family members have been shown to upregulate Wnt5a,16 and recently, Shao et al have shown that Wnt and its downstream effector-molecule ß-catenin contribute to vascular calcification.17 Because PPAR- agonists inhibit Wnt/ß-catenin signaling,18 it is possible that N-3 fatty acids inhibit IL-6–induced osteoblastic differentiation and mineralization via targeting the Wnt/ß-catenin pathway.
The present results suggest potential mechanisms for the effects of fish oil consumption on vascular calcification. It appears that the anti-inflammatory role of active components of fish oil involve not only reduction of cytokine expression, as shown by Tappia et al,19 but also by interference with its downstream targets, such as STAT-3. It also raises the possibility that fish oil may affect vascular calcification, not simply by reducing atherosclerosis but through direct inhibition of osteoblastic differentiation of vascular cells.
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Acknowledgments |
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This work was supported in part by National Institutes of Health grant HL/AR69261.
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Footnotes |
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Original received January 11, 2006; revision received February 13, 2006; accepted February 20, 2006.
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References |
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