The benefit of docosahexanoic acid on the migration of vascular smooth muscle cells is partially dependent on Notch regulation of MMP-2/-9 - PubMed (original) (raw)

The benefit of docosahexanoic acid on the migration of vascular smooth muscle cells is partially dependent on Notch regulation of MMP-2/-9

Sandrine Delbosc et al. Am J Pathol. 2008 May.

Abstract

The Notch pathway is involved in the regulation of the migratory/proliferative phenotype acquired by vascular smooth muscle cells (VSMCs) in the pro-inflammatory context of vascular diseases. Here, we investigated whether docosahexaenoic acid (DHA), a polyunsaturated, omega-3 fatty acid, could reduce fibrinolytic/matrix-metalloproteinase (MMP) activity and whether this reduction occurs through the modulation of Notch signaling. Rat VSMCs were transdifferentiated with interleukin-1beta and then treated with DHA. Migration/proliferation was determined by performing a wound healing assay and measuring MMP-2/-9 activity, type 1 plasminogen activator inhibitor levels, and the expression of these proteins. The involvement of Notch in regulating the fibrinolytic/MMP system was evidenced using Notch pathway inhibitors and the forced expression of Notch1 and Notch3 intracellular domains. DHA significantly decreased VSMC migration/proliferation induced by interleukin-1beta as well as fibrinolytic/MMP activity. Prevention of Notch1 target gene transcription enhanced the interleukin-1beta effects on MMPs and on migration, whereas Notch3 intracellular domain overexpression reduced these effects. Finally, DHA increased Notch3 expression, Hes-1 transcription (a Notch target gene), and enhanced gamma-secretase complex activity. These results suggest that inhibition of the Notch pathway participates in the transition of VSMCs toward a migratory phenotype. These results also suggest that the beneficial inhibitory effects of DHA on fibrinolytic/MMP activity are related in part to the effects of DHA on the expression of Notch pathway components, providing new insight into the mechanisms by which omega-3 fatty acids prevent cardiovascular diseases.

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Figures

Figure 1

DHA inhibition of VSMC wound healing capacity. After 24 hours of serum starvation a linear wound was made in the center of culture dish and cells were treated by IL-1β (10 ng/ml) and/or DHA (50 μmol/L) during 48 hours (t48). The denuded area was visualized by using an inverted microscope (×40) and captured at t48. The surface of the colonized zone was calculated as described in Materials and Methods. For each condition, the results are expressed as % of colonized zone at t0 and represent mean ± SE for four independent experiments (shown to the right). ***P < 0.0001, vs control, and ††P < 0.001 IL-1β+DΗΑ vs IL-1β.

Figure 2

DHA effect on the fibrinolytic system. After 48 hours of IL-1β (10 ng/ml) and/or DHA (50 μmol/L) treatments, the culture medium was recovered for determination of MMP activity, PAI-1 secretion. mRNA were extracted as described in Materials and Methods. The lysis bands corresponding to the pro- or active form of MMP-2 and the pro-MMP-9 were visualized after Coomassie Blue coloration (A and B, respectively, left panels). The mRNA level of MMP-2 and -9 (A and B, respectively, right panels) were determined by real-time RT-PCR (LightCycler; Roche Diagnostics). The concentration of secreted PAI-1 (ng/ml of medium) (C upper panel) and mRNA level of t-PA and PAI-1 (C lower panel) were also determined. Amounts of mRNA expression were systematically normalized to HPRT transcripts level. Results are expressed as percentage of mRNA level of control cells. **P < 0.001, ***P < 0.0001 vs control ††P < 0.001, †††P < 0.0001 vs IL-1β.

Figure 3

γ-Secretase inhibition potentiates the effect of IL-1β on VSMC proliferation/migration. After serum starvation, a linear wound was made in the center of culture dish and cells were treated by IL-1β (10 ng/ml) and/or DAPT (0.5 μmol/L) during 48 (t48) hours. The denuded area was visualized and captured as described in Figure 1 legend. The percentage of the colonized zone is shown to the right. **P < 0.01, vs control.

Figure 4

Effect of NICD1 and NICD3 overexpression on VSMC migration/proliferation. VSMC were transfected as described in Materials and Methods with the constructs encoding NICD1, NICD3, or Mock. Twenty-four hours after, cells were starved. After serum starvation, a linear wound was made in the center of culture dish and cells were treated or not with IL-1β (10 ng/ml) during 48 (t48) hours. The denuded area was visualized and captured as described in Figure 1 legend. **P < 0.01, vs control.*P < 0.05, **P < 0.01, vs untreated cells.

Figure 5

γ-Secretase inhibition enhances the effects of IL-1β on the fibrinolytic system. After serum starvation, VSMCs were stimulated with IL-1β (10 ng/ml) for 48 hours and the inhibitor of γ-secretase DAPT (0.5 μmol/L) was added at the same time. The culture medium was recovered and mRNAs were extracted as described in Materials and Methods. The lysis bands corresponding to the pro- or active form of MMP-2 and the pro-MMP-9 were visualized after Coomassie Blue coloration (A and B, respectively, left panels). The mRNA level of MMP-2 and -9 (A and B, respectively, right panels) as well as that of t-PA and PAI-1 (C) were determined by real-time RT-PCR (LightCycler, Roche Diagnostics). Amounts of mRNA expression were systematically normalized to HPRT transcripts level. Results are expressed as percentage of mRNA level of control. ***P < 0.0001 vs control and †††P < 0.0001 vs IL-1β.

Figure 6

RBP-Jκ DN enhances the effects of IL-1β on MMP-2/-9 expression. VSMCs were transfected as described in Materials and Methods with the constructs encoding RBP-Jk DN or empty plasmid (mock). Twenty-four hours later, cells were starved and treated with IL-1β (10 ng/ml) for 48 hours. mRNA was extracted as described in Materials and Methods. mRNA levels of MMP-2 (left panel) and MMP-9 (right panel) were determined by real-time RT-PCR (LightCycler, Roche Diagnostics). Amounts of mRNA expression were systematically normalized to HPRT transcripts level. Results are expressed as percentage of mRNA level of control. ***P < 0.0001 vs untreated mock and ††P < 0.001, †††P < 0.0001 vs IL-1β.treated mock.

Figure 7

Effect of NICD1 and NICD3 over-expression on MMPs activity and expression. VSMCs were transfected as described in Mthods with the constructs encoding NICD1, NICD3,or Mock. Twenty-four hours later, cells were starved and treated with IL-1β (10 ng/ml) for 48 hours. The culture medium was then recovered and mRNA extracted as described in Materials and Methods. The lysis bands corresponding to the pro- or active form of MMP-2 (A, left panel) and the pro-MMP-9 (B, right panel) were visualized and their surfaces quantified as described in Materials and Methods and Figure 2 legend. The mRNA level of MMP-2 (A, right panel) and MMP-9 (B, right panel) were determined by real-time RT-PCR (LightCycler, Roche Diagnostics). Amounts of mRNA expression were systematically normalized to HPRT transcripts level. Results are expressed as percentage of mRNA level of control. *P < 0.05, **P < 0.01, ***P < 0.001 vs Mock IL-1β.

Figure 8

Effect of DHA treatment on Notch signaling pathway. After 48 hours of IL-1β (10 ng/ml) and/or DHA (50 μmol/L) treatments, mRNA or total protein were extracted as described in Materials and Methods. A: mRNA levels of Notch3 and Hes-1 were determined by real-time RT-PCR (LightCycler, Roche Diagnostics). Amounts of mRNA expression were systematically normalized to HPRT transcripts level. Results are expressed as percentage of mRNA level of control. B: Immuno-detections were performed as described in Materials and Methods. After blocking, membranes were probed with monoclonal mouse anti c-myc (1/1000). Apparent molecular mass of protein markers are given on the left side of the blot in kDa. The autoradiogram shown is representative of four independent experiments. The intensity of each band was quantified using Scion software (lower panel).

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The benefit of docosahexanoic acid on the migration of vascular smooth muscle cells is partially dependent on Notch regulation of MMP-2/-9 - PubMed (original) (raw)