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,1
UMR Physiologie et Physiopathologie, Université Pierre et Marie Curie, CNRS,
* Bâtiment A, Paris, France; and
Centre de recherches biomédicales des Cordeliers, Paris, France
1Correspondence: Centre de recherches biomédicales des Cordeliers, 15 rue de l’Ecole de Médecine, Paris 75270, CEDEX 06, France. E-mail: vincent.marechal{at}snv.jussieu.fr
SPECIFIC AIMS
Vascular smooth muscle cells (VSMCs) play a key role in the progression of atherogenesis. They proliferate and produce several proinflammatory molecules in atheroma plaques such as secretory type IIA phospholipase A2 (sPLA2-IIA) and prostaglandin E2 (PGE2). PGE2 is the major final product of sPLA2 activity in VSMCs. It mediates several functions in vascular biology including the regulation of vascular tone, thrombocyte function, and inflammatory responses.
High mobility group protein 1 (HMGB1) is an abundant and ubiquitous component of chromatin. Large amounts of HMGB1 can be released by necrotic cells as well as by activated immune cells. The soluble form of HMGB1 acts as a potent proinflammatory cytokine on various cells. Since HMGB1 is especially abundant in atheroma plaques, notably in macrophages, we hypothesized that it could stimulate sPLA2-IIA expression and PGE2 release from VSMCs. Our experiments were conducted on primary cultures of VSMCs isolated from rat aortas. These cells are especially suitable for studies of inflammatory processes.
PRINCIPAL FINDINGS
1. Purification and characterization of the recombinant HMGB1 protein (rHMGB1)
HMGB1 has been shown to exhibit various proinflammatory activities through interaction with RAGE, TLR-2, and/or TLR-4. Recombinant HMGB1 (rHMGB1) purified from E. coli acts as a cytokine similar to its mammalian counterpart, as has been demonstrated on rat VSMCs. Therefore, rHMGB1 was used in this study.
Polyhistidine-tagged rHMGB1 was synthesized by E. coli and purified under native conditions by Ni-NTA metal chromatography. LPS was removed by Triton X-114 extraction and the residual LPS content was <0.3 pg/µg rHMGB1. The resulting recombinant protein was >99% pure by SDS-PAGE. Before investigating the effect of rHMGB1 on VSMCs, we designed a series of assays with the aim of confirming that the rHMGB1 was biochemically and biologically active. The DNA binding ability of rHMGB1 was analyzed by electrophoresis mobility shift assay using hemicatenanes as the probe, and the proinflammatory activity of rHMGB1 was evaluated by measuring its ability to promote the release of TNF-
from primary human monocytes.
2. HMGB1 induces sPLA2-IIA gene expression in VSMCs
Secretory PLA2-IIA (sPLA2-IIA) is the most abundant isoform of secreted PLA2 in VSMCs and its role in atherogenesis is well documented. Experiments were conducted to evaluate the effects of rHMGB1 on sPLA2-IIA expression in primary rat VSMCs. The expression of the sPLA2-IIA gene in VSMCs incubated for 8 or 24 h with several concentrations of rHMGB1 was analyzed by RT- PCR. A modest but significant dose-dependent increase in sPLA2-IIA mRNA was observed when VSMCs were incubated for 24 h with 100 ng to 1 µg/ml rHMGB1. Higher concentrations of rHMGB1 did not lead to greater sPLA2-IIA gene expression. Therefore, all subsequent experiments were performed with 1 µg/ml rHMGB1. This concentration is compatible with HMGB1 concentrations that have been measured in vivo in various situations.
sPLA2 activity, used as an indicator of sPLA2-IIA release, was measured in the cell culture supernatant in the presence or absence of rHMGB1. There was a barely detectable increase in sPLA2 activity in VSMCs incubated with 1 µg/ml rHMGB1, even after 24 h.
3. HMGB1 enhances sPLA2-IIA gene expression in interleukin (IL) -1ßbeta; sensitized VSMCs
During atherogenesis, VSMCs are exposed to various proinflammatory cytokines that may act in concert to activate sPLA2-IIA production. We tested whether HMGB1 could act synergically with one of them, IL-1ßbeta;, to stimulate sPLA2-IIA expression. VSMCs were cultivated for 24 h with 10 ng/ml IL-1ßbeta;, then incubated for another 8 or 24 h with fresh medium containing 10 ng/ml IL-1ßbeta; and 1 µg/ml rHMGB1. IL-1ßbeta; alone induced a 7-fold increase in sPLA2-IIA gene expression compared to untreated cells. rHMGB1 induced a further 2-fold increase in these cells (Fig. 1
). Similar results were obtained when the sPLA2 enzymatic activity was measured. We concluded that the induction of sPLA2-IIA by rHMGB1 was potentiated by IL-1ßbeta;.
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4. HMGB1 induces PGE2 release by IL-1ßbeta;-treated VSMCs
PGE2 is the major final product of sPLA2 activity in VSMCs. Since rHMGB1 was shown to promote sPLA2-IIA gene expression in IL-1ßbeta;-treated VSMCs, we thought that PGE2 release might also be induced by HMGB1 under similar conditions. We tested this hypothesis by incubating IL-1ßbeta;-treated VSMCs with 1 µg/ml rHMGB1 for 8 or 24 h. Whereas IL-1ßbeta; alone induced a 3.5-fold increase in PGE2 release, rHMGB1 induced PGE2 accumulation up to 12-fold in VSMCs pretreated with 10 ng/ml IL-1ßbeta; (Fig. 2
A). Consequently, rHMGB1 dramatically increased the PGE2 released in VSMCs sensitized with IL-1ßbeta;.
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5. HMGB1 induces the expression of COX-2 and mPGES-1 genes in IL-1ßbeta;-treated cells
Arachidonic acid, a fatty acid generated by sPLA2-IIA, is metabolized into PGE2 by the cyclooxygenases (COX) and PGE synthases (PGES). Whereas the genes encoding COX-1, COX-3, cytosolic PGES, and type 2 microsomal PGES (mPGES-2) are constitutively expressed, COX-2 and mPGES-1 genes are highly regulated in response to several growth factors and cytokines. Since we previously observed that sPLA2-IIA was activated in response to rHMGB1, we wondered whether the other genes required for PGE2 synthesis, such as COX-2 and mPGES-1, could be regulated similarly by rHMGB1.
Incubation of cells with 1 µg/ml rHMGB1 for 24 h did not alter the expression of COX-2 or mPGES-1 genes. IL-1ßbeta; alone increased COX-2 transcription by 3-fold and mPGES-1 gene transcription by 2.5-fold. However, rHMGB1 induced a dramatic increase in COX-2 gene expression (11-fold) and mPGES-1 gene expression (7-fold) when the cells were pretreated with IL-1ßbeta; (Fig. 2B, C
). These data indicate that the induction of PGE2 release by rHMGB1 in IL-1ßbeta;-sensitized VSMCs is associated with the concomitant activation of three genes whose products are involved in PGE2 synthesis, namely sPLA2-IIA, COX-2, and mPGES-1.
6. IL-1ßbeta; induces the expression of RAGE, TLR-2, and TLR-4
RAGE, one of the main receptors of HMGB1, is both present and functional in VSMCs. To explain the role of IL-1ßbeta; in the response of VSMCs to rHMGB1, we asked whether IL-1ßbeta; might modulate RAGE expression and/or cell localization. Basal expression of the RAGE gene was analyzed by RT-PCR and Western blot using membrane proteins. VSMCs incubated with 10 ng/ml IL-1ßbeta; for 24 h had 4- to 5-fold more RAGE protein at the plasma membrane than control cells.
Two other known receptors for HMGB1, namely TLR-2 and TLR-4, are expressed in vascular smooth muscle cells. We found that IL-1ßbeta; enhanced TLR-2 gene expression >2-fold and enhanced TLR-4 gene expression to a lesser extent. Thus, the greater sensitivity of IL-1ßbeta;-treated VSMCs to HMGB1 is associated with an increased expression of the genes encoding RAGE, TLR-2, and possibly TLR-4.
CONCLUSIONS AND SIGNIFICANCE
Smooth muscle cells are recognized as key actors in atherogenesis. Recent work demonstrated that large amounts of extracellular HMGB1 (
40 pg/mg tissue/24 h) are produced by atherosclerotic plaques but not by normal arteries (M. Bianchi, personal communication). We have demonstrated that HMGB1 activates the expression of sPLA2-IIA, COX-2, and mPGES-1 in VSMCs sensitized with IL-1ßbeta;, a cytokine known to play an essential role in the progression of atherosclerotic plaques (Fig. 3
). This results in a dramatic increase in the release of PGE2 by VSMCs. Recent work from our lab indicates that sPLA2-IIA is involved in an autocrine activation loop in VSMCs. Taken together, these results suggest that HMGB1 activity might be important in atherosclerotic plaques because it could initiate an amplification loop that eventually leads to the accumulation sPLA2-IIA and PGE2.
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Our results indicate that IL-1ßbeta; acts by increasing the abundance of the three known receptors for HMGB1, namely RAGE, TLR-2, and TLR-4. RAGE was first recognized as a receptor for advanced glycation end products (AGE), the ultimate result of nonenzymatic glycation and the oxidation of protein and lipids. AGE accumulate in the blood plasma of diabetic patients and are important in diabetic vascular complications.
So far, the effects of AGE and HMGB1 on RAGE function are indistinguishable. Both AGE and HMGB1 stimulate the chemotaxis of VSMCs and activate NF-
B. Therefore, it is likely that the induction of RAGE by IL-1ßbeta; also sensitizes the VSMCs to the action of AGE.
In conclusion, this work sheds new light on the contribution of HMGB1 to the activation of VSMCs during the inflammation process associated with atherogenesis.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5514fje
This article has been cited by other articles:
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  B. Samuelsson, R. Morgenstern, and P.-J. Jakobsson Membrane Prostaglandin E Synthase-1: A Novel Therapeutic Target Pharmacol. Rev., September 1, 2007; 59(3): 207 - 224. [Abstract] [Full Text] [PDF]
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