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HDL counterbalance the proinflammatory effect of oxidized LDL by inhibiting intracellular reactive oxygen species rise, proteasome activation, and subsequent NF-B activation in smooth muscle cells ¹

INSERM U-466, Institut Louis Bugnard, CHU Rangueil, Toulouse, France

³Correspondence: INSERM U-466, CHU Rangueil, 31403 Toulouse Cedex 4, France. E-mail: anesalv@toulouse.inserm.fr or salvayre{at}toulouse.inserm.fr

SPECIFIC AIMS

In atherosclerosis lesions, oxidized low density lipoproteins (oxLDL) trigger inflammatory events potentially involved in plaque rupture and atherothrombosis. Reversibly, high density lipopoproteins (HDL) are anti-atherogenic and reduce the inflammatory response. The transcription factor NF-B plays a major role in the regulation of inflammatory gene expression. The aim of present work was to investigate the signaling pathways involved in oxLDL-induced NF-B activation and the mechanisms of the anti-inflammatory effect of HDL and to determine whether and how HDL modulate the oxLDL-induced NF-B activation.

PRINCIPAL FINDINGS

1. HDL inhibit NF-B activation and IB degradation induced by oxLDL
Gel-shift experiments from nuclear extracts of rabbit SMC indicated that mitogenic concentrations of oxLDL (100 µg/mL) elicit an early activation of NF-B (peaking at 30–60 min) (Fig. 1 A) correlated with degradation of the NF-B cytosolic inhibitor IB (Fig. 1B ). Preincubation of SMC with HDL (200 µg/mL) for 18 h reduced both the oxLDL-induced NF-B activation (Fig. 1A ) and IB degradation (Fig. 1B ). This protective effect was persistent since NF-B activation was still inhibited in SMC pretreated by HDL for 18 h, then washed and stimulated by oxLDL (Fig. 1C ). This led us to explore the oxLDL-activated signaling pathways potentially involved in NF-B activation and to study the inhibitory mechanism of HDL. We investigated the role of signaling pathways activated by oxLDL that have been shown to be involved in cytokine-induced NF-B activation, namely, the mitogenic sphingosine kinase pathway, the survival PI-3 kinase/Akt system, and the tyrosine kinase receptor pathway.

View larger version (20K): [in this window] [in a new window]   Figure 1. HDL inhibit the oxLDL-induced NF-B activation. A) Time course of EMSA experiments showing the NF-B activation induced by oxLDL (100 µg apoB/mL) and inhibitory effect of HDL (200 µg apoA/mL, preincubation for 18 h). B) Western-blot of IB (and ß-actin as a reference) after 1 h incubation with oxLDL ± HDL. C. Persistent effect of HDL against NF-B activation induced by oxLDL. SMC were preincubated for 18 h with HDL (200 µg/mL), then washed in PBS and stimulated for 1 h with oxLDL (in HDL-free medium).

2. Sphingosine kinase is not involved in the oxLDL-induced NF-B activation
In our experimental system, oxLDL trigger activation of Sm/Cer/S1P pathway, as shown by sphingomyelin hydrolysis (peaking at 120–150 min) and sphingosine kinase activation. Sphingosine-1-phosphate generation is not involved in the oxLDL-induced NF-B activation, because 1) its inhibition by D-MAPP (ceramidase inhibitor) and DMS (sphingosine kinase inhibitor) did not alter the oxLDL-induced NF-B activation, and 2) HDL did not inhibit this signaling pathway. It is therefore concluded that NF-B activation and the effect of HDL do not require sphingosine kinase activity. In contrast, ceramide could play a direct role in ROS generation and NF-B activation, both being inhibited by HDL.

3. The PI-3K/Akt and tyrosine kinase receptor pathways are not involved in oxLDL-induced NF-B activation
As PI-3K/Akt is known to be a potential pathway of NF-B activation, we investigated whether PI-3K (which is activated by oxLDL) plays a role in NF-B activation and is a target of HDL. The reported data show that, in our experimental system, NF-B is activated by oxLDL independent of PI-3K, as assessed by the use of PI-3K inhibitors LY294002 and wortmannin and by overexpression of a dominant negative form of the PI-3K p85 (which inhibit PI-3K triggered by oxLDL but not NF-B activation). Moreover, HDL inhibit NF-B, but not PI-3K, activation induced by oxLDL. These data suggest that PI-3K is not required for the oxLDL-induced NF-B activation and thus is not the target of HDL.

In our experimental model system, the oxLDL-induced NF-B activation was independent of any autocrine secretion of cytokines or growth factors in the culture medium and did not involve an activation of tyrosine kinase receptors EGFR and PDGFR (despite the fact that these receptors may directly be activated by oxLDL).

4. ROS and proteasome are involved in NF-B activation and are inhibited by HDL
As NF-B is a redox-sensitive transcription factor, we investigated whether 1) oxLDL may trigger a rise of intracellular ROS in a time course consistent with NF-B activation, 2) the possible link between ROS and NF-B activation, and 3) the mechanism by which HDL may act at this step.

Incubation of SMC with oxLDL triggered a rapid rise of intracellular ROS, peaking at 45 min, as shown by using the fluorogenic ROS-sensitive probe H2-DCFDA. HDL and the antioxidants NAC or PDTC inhibited both the ROS rise and NF-B activation triggered by oxLDL, suggesting that ROS mediate the oxLDL-induced NF-B activation. The antioxidant-like effect of HDL did not involve their vitamin E content, since 1) Cu-oxidized HDL depleted in vitamin E, were still able to inhibit NF-B activation, and 2) vitamin E tested at concentrations present in HDL (1–10 µM) was unable to block ROS increase and NF-B activation.

As the proteasome is generally involved in IB degradation and NF-B activation, we investigated the relationship between ROS and proteasome in oxLDL-induced NF-B activation and the site of inhibition by HDL. OxLDL elicited an initial rapid and transient activation of proteasome, peaking at 15 min, as shown by the hydrolysis of sLLVY-MCA. The oxLDL-induced activation of proteasome was completely inhibited by HDL and by antioxidants PDTC and NAC, thus pointing out the link between ROS and proteasome activation. In addition, PSI (a proteasome inhibitor) inhibited strongly the oxLDL-induced NF-B activation, thereby indicating that proteasome activity is required for NF-B activation. Moreover, the relationship between ROS and proteasome activation was mimicked in our experimental system by H₂O₂ (200 µM), which triggered proteasome activation, IB degradation, and NF-B activation. Again, this effect was blocked by HDL, which clearly exhibit an ‘antioxidant-like’ activity.

Altogether, these data suggest that oxLDL trigger intracellular ROS generation, with a subsequent activation of proteasome that degrades IB, thereby inducing NF-B activation. HDL acts by blocking the ROS rise (and subsequent events).

CONCLUSIONS AND SIGNIFICANCE

OxLDL are thought to trigger proinflammatory responses, but the signaling mechanisms are only partly understood. The findings of our paper point out 1) the sequence of signaling events triggered by oxLDL (like oxidative stress) and leading to NF-B activation, a major transcription factor involved in the regulation of the inflammatory response, and 2) the mechanism of the anti-inflammatory effect of HDL.

Our data show that oxLDL trigger the following sequence of signaling events: intracellular ROS generation, proteasome activation, IB degradation, and subsequent NF-B activation (Fig. 2 ). The oxLDL-induced rapid rise of intracellular ROS is required for the activation of the transcription factor NF-B, but little is known about the redox mechanisms and target molecules involved in the NF-B activation triggered by oxLDL. Our data show that the proteasome is a redox-sensitive cellular target, since it is activated by oxLDL through an antioxidant-sensitive mechanism and is required for IB degradation and subsequent NF-B activation triggered by oxLDL. Moreover, it may be involved in processing of the p105 and p100 precursors.

View larger version (40K): [in this window] [in a new window]   Figure 2. Schematic diagram. Sequence of events triggered by OxLDL in SMC and leading to NF-B activation. OxLDL trigger intracellular ROS generation and proteasome activation, which induce the degradation of IB and subsequent NF-B nuclear translocation. The effect of ROS on IKK complex activation is yet unknown. Concomitant and independent of NF-B activation, oxLDL activate the mitogenic Sm/Cer/S1-P pathway and survival of the EGFR/PI-3K/Akt pathway. HDL and antioxidants block NF-B activation by inhibiting both ROS increase and proteasome activation.

The data reported here also show that oxLDL-induced activation of the sphingosine kinase, tyrosine kinase receptors, and PI3-kinase pathways are concomitant lateral events, promoting proliferation and cell survival, but are not implicated in NF-B activation and subsequent proinflammatory response. These data suggest that the mechanism of oxLDL-induced NF-B activation differs from that mediated by cytokines (TNF- and IL1).

Another finding is the mechanism by which HDL antagonizes the proinflammatory effect of oxLDL by inhibiting the ROS/NF-B signaling pathway, more precisely, by inhibiting the ROS rise at the cellular level (cellular ‘antioxidant-like’ effect). The potent ‘antioxidant-like’ effect of HDL is not restricted to oxLDL-mediated redox signaling, but is a more general effect preventing oxidative stress (as shown by their inhibitory effect on H₂O₂-induced NF-B activation). This antioxidant effect of HDL is not related to their vitamin E content, and may result (at least in part) from associated antioxidant enzymes—namely, PAF-acetyl hydrolase or paraoxonase—but the precise mechanism remains to be elucidated. Finally, it may be noted that, in this experimental system, HDL do not inhibit the mitogenic (Sm/cer/S1P) and survival (PI-3K) pathways.

In conclusion, these findings establish a link between metabolic factors (lipoproteins) and the inflammatory processes of atherothrombosis. Oxidatively modified LDL are inducers of the transcription factor NF-B activation, which is a potent proinflammatory regulator. Such inflammatory events are thought to accelerate the progression of atherosclerotic lesions and to play a role in plaque rupture or erosion and subsequent thrombotic events. In contrast, our data show that the oxLDL and oxidant-induced NF-B activation is strongly inhibited by HDL, thereby explaining the mechanism of their ‘anti-inflammatory’ properties and their ability to prevent both lesion formation and complications.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0240fje; to cite this article, use FASEB J. (February 5, 2003) 10.1096/fj.02-0240fje

² Equal contributors.

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