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Smooth muscle cells in human atherosclerotic plaques secrete and proliferate in response to high mobility group box 1 protein

Annalisa Porto^*,,1, Roberta Palumbo^*,1, Maurizio Pieroni^*, Gianfranco Aprigliano^*, Roberto Chiesa, Francesca Sanvito^*, Attilio Maseri and Marco E. Bianchi^,2

^* San Raffaele Scientific Institute, Milan, Italy;

Institute of Cardiology, Catholic University, Rome, Italy; and

San Raffaele University, Milan, Italy

²Correspondence: San Raffaele University, via Olgettina 58, 20132 Milan, Italy. E-mail: bianchi.marco{at}hsr.it

SPECIFIC AIMS

There have been several suggestions that high mobility group box 1 protein (HMGB1), a nuclear protein that acts as a cytokine when released in the extracellular medium, may be involved in the pathophysiology of atherosclerosis. Here, we have specifically investigated whether HMGB1 is present within atherosclerotic plaques from human patients and whether it is passively released from necrotic cells or actively secreted by foam cells or other cell types.

Since HMGB1 promotes the proliferation of vessel-associated stem cells (mesoangioblasts), we also tested whether HMGB1 might promote the smooth muscle cell (SMC) proliferation that is associated with vessel stenosis.

PRINCIPAL FINDINGS

1. Atherosclerotic plaques secrete HMGB1
Fragments of human carotid atherosclerotic plaques (n=25) release extracellular HMGB1; fragments of nonatherosclerotic mammary arteries (n=10) do not release HMGB1. The release from atherosclerotic plaques is sustained in time, and due to active secretion; the contribution of necrotic cells is negligible, since we find no release of lactate dehydrogenase (LDH), a marker of dying cells.

2. SMCs in atherosclerotic plaques express HMGB1 in characteristic patterns
A fraction of all cell types in the plaque (foam cells, endothelial cells, and SMCs) contain HMGB1 in the cytoplasm. SMCs in atherosclerotic plaques showed three different patterns of HMGB1 expression: undetectable expression, only in the nuclei, and in both nuclei and cytosol. SMCs in normal arteries express a low level of HMGB1 and only in nuclei. Secretion of HMGB1 follows a nonclassical pathway that involves its relocation from the nucleus to the cytosol; the presence of HMGB1 in the cytosol suggests that it is being secreted.

3. Cholesterol-stimulated SMCs secreted HMGB1
To prove that HMGB1 was actually being secreted by SMCs, we exposed primary human SMCs in culture to a simple atherogenetic stimulus, cholesterol. Cholesterol complexed to methyl-ßbeta;-cyclodextrin (10 µg/ml) had no toxic effect on SMCs but induced the accumulation of Oil Red O staining lipid droplets throughout the cytosol, the expression of the cholesterol-responsive CD68 marker, and the secretion of HMGB1 into the medium.

4. SMCs respond to HMGB1
Human SMCs also respond to extracellular HMGB1 (Fig. 1 ). HMGB1 is a chemoattractant, but it also a mitogen that promotes SMC proliferation in serum-free medium. Moreover, extracellular HMGB1 promotes further HMGB1 secretion.

View larger version (16K): [in this window] [in a new window]   Figure 1. Effects of HMGB1 stimulation on human SMCs. A) Human SMCs migrate in response to HMGB1. SMCs were subjected to chemotaxis assays with 30 ng/ml HMGB1, 10 ng/ml PDGFBB, or 10% FCS. Data are average ± SD of 4 experiments performed in duplicate; effect of HMGB1 is highly significant (P<0.001) compared with control. Migration of human SMCs was abrogated by antibodies against HMGB1 or receptor for advanced glycation end-products (P<0.001) compared with HMGB1 alone. B) Human aorta SMCs proliferate in response to HMGB1. Cells were grown in F12K medium alone, or containing 30 ng/ml of HMGB1, or 10% FCS. HMGB1 induced cell proliferation, and SMCs in medium alone died. Each point is mean ± SD of 3 experiments in duplicate, and result is highly statistically significant (P<0.001). C) Human SMCs secrete HMGB1 after stimulation with HMGB1. Subconfluent SMCs were starved for 16 h and exposed for 48 h to a fragment of HMGB1 (Box B) that retains properties of full-length HMGB1 but is not recognized by the monoclonal antibody used here for HMGB1 detection. The absence of LDH in supernatant indicates that HMGB1 is secreted and not released by necrotic cells.

CONCLUSIONS AND SIGNIFICANCE

A growing number of reports describe the presence of extracellular HMGB1 during various inflammatory conditions, both acute and chronic. We show here that a significant amount of HMGB1 is secreted in human carotid atherosclerotic plaques, both by inflammatory cells and, unexpectedly, by SMCs (Fig. 2 ). Normally, SMCs contain a much lower amount of HMGB1 than other cell types, but we show that HMGB1 expression and secretion are promoted by cholesterol loading. In turn, extracellular HMGB1 acts as mitogenic and chemotactic factor for human SMCs, and itself promotes more HMGB1 expression and secretion.

View larger version (43K): [in this window] [in a new window]   Figure 2. Schematic diagram depicts a section of a human artery during atherotic plaque formation (adapted from Ross, N. Engl. J. Med., vol. 340, pp. 115–126). We find that some SMCs express very little HMGB1 (in brown), some express it conspicuously in the nucleus, and some express it both in nucleus and cytoplasm. The latter are secreting HMGB1, which in turn can promote SMC migration, SMC proliferation, and further HMGB1 secretion. Thus, HMGB1 secretion can support an autocrine loop of SMC activation leading to intimal and neointimal hyperplasia.

Our findings identify an autocrine/paracrine loop based on HMGB1 that can act as a mechanism of neointima hyperplasia during the progression of atherosclerosis and during restenosis after coronary angioplasty.

FOOTNOTES

¹ These authors contributed equally to this work.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-5867fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-5867fjev1 20/14/2565    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Porto, A. Articles by Bianchi, M. E. Search for Related Content PubMed PubMed Citation Articles by Porto, A. Articles by Bianchi, M. E.