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,1
,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
ABSTRACT
High mobility group box 1 protein (HMGB1) is a chromatin component leaked out by necrotic cells and actively secreted by activated myeloid cells. The extracellular protein is a potent mediator of tissue remodeling. We show here that human atherosclerotic plaques, but not normal arteries, produce extracellular HMGB1. Secreted HMGB1 originates from endothelial cells, by neointimal foam cells, and also smooth muscle cells (SMCs). SMCs are an unexpected source for secreted HMGB1, since they normally express much lower amounts of HMGB1 than other cells types, and they do not secrete it. However, cultured SMCs actively secrete HMGB1 after cholesterol loading. In turn, in response to HMGB1, SMCs proliferate, migrate, and secrete more HMGB1. Thus, SMCs are both a source and a target of HMGB1; blocking HMGB1 secretion by SMCs can be an important strategy for treatment of atherosclerotic disease and in particular restenosis.—Porto, A., Palumbo, R., Pieroni, M., Aprigliano, G., Chiesa, R., Sanvito, F., Maseri, A., Bianchi, M. E. Smooth muscle cells in human atherosclerotic plaques secrete and proliferate in response to high mobility protein box 1.
Key Words: atherosclerosis • cholesterol • cytokine • inflammation
ATHEROSCLEROSIS INVOLVES MULTIPLE processes including endothelial dysfunction, inflammation, vascular proliferation, and extracellular matrix (ECM) alterations (1)
. Recent studies emphasized the role of inflammation in mediating all stages of atherosclerosis from initial atherosclerotic vascular lesion to plaque destabilization leading to thrombus formation and vessel occlusion (2)
. Besides inflammation, migration and proliferation of vascular smooth muscle cells (SMCs) are important responses to proinflammatory cytokines and peptide growth factors and promote both atheroma formation and restenosis after angioplasty (1
, 3)
.
High mobility group box 1 protein (HMGB1) is a very abundant chromatin protein residing in the eukaryotic cell nucleus and acting in the assembly of nucleoprotein complexes (4
5
6)
. When cells die in a traumatic, unprogrammed way, they release HMGB1 in the medium together with many soluble proteins (7)
. Nearby cells detect the presence of extracellular HMGB1 and react to death of their neighbors thanks to a number of receptors, including receptor for advanced glycation end-products (RAGE) and Toll-like receptors 2 and 4 (8)
. Significantly, HMGB1 is not released by apoptotic cells; thus, extracellular HMGB1 represents a signal of necrosis and tissue damage (9)
.
In addition to being passively released by necrotic cells, HMGB1 can also be actively secreted by activated macrophages and myeloid cells (10)
. The secretion of HMGB1 by myeloid cells does not involve the endoplasmic reticulum and the Golgi apparatus but requires the relocalization of the protein from the nucleus to specific vesicles, the secretory lysosomes (11)
. The translocation to cytosol requires the acetylation of HMGB1 on several specific lysines (12)
.
Both passively released and actively secreted HMGB1 convey the same signal: extracellular HMGB1 attracts and activates inflammatory cells (13
, 14)
, enhances the expression of vascular adhesion molecules in endothelial cells (15)
, impairs the barrier function of intestinal epithelia (16)
, and attracts stem cells into damaged tissue (17)
.
We and others (18)
have suggested that HMGB1 may be involved in the pathophysiology of atherosclerosis: HMGB1 promotes the migration of rat arterial SMCs and foam cells in early atherosclerotic lesions express HMGB1 and probably secrete it locally (19)
. Here, we show that cells in human carotid atherosclerotic plaques secrete HMGB1 in the medium. Remarkably, much of this secretion is due to SMCs themselves. SMCs were considered as unlikely sources of HMGB1, since in normal vessel walls or in normal culture conditions, they contain much less HMGB1 than most other cell types (18)
. However, SMCs challenged with cholesterol synthesize HMGB1 in large amounts and secrete it. Moreover, SMCs also respond to HMGB1 by migrating, proliferating, and secreting more HMGB1. Thus, SMCs can maintain an autocrine loop possibly responsible for neointimal hyperplasia and for focal SMCs proliferation leading to in-stent restenosis after angioplasty.
MATERIALS AND METHODS
Materials
Endotoxin-free HMGB1 and Box B were provided by HMGBiotech (Milan, Italy). Rabbit polyclonal anti-HMGB1 antibody (Ab) raised against peptide 166–181 was purchased from BD Biosciences; mouse monoclonal antibody (mAb) KS1 against HMGB1 Box A from Medical and Biological Laboratories (Nagoya, Japan); rabbit polyclonal against human RAGE from Santa Cruz Biotechnology (Santa Cruz, CA, USA); goat polyclonal anti-lactate dehydrogenase (LDH) Ab from Chemicon (Temecula, CA, USA); mouse monoclonal anti-CD68 and anti-
-actin antibodies from DAKO (Glostrup, Denmark); goat polyclonal anti-rabbit and anti-mouse antibodies conjugated to Alexa Fluor 488 or 594 from Molecular Probes (Eugene, OR, USA); and goat polyclonal anti-mouse Ab conjugated to Cy5 from Jackson ImmunoReserch Laboratories (West Grove, PA, USA). The cholesterol-methyl-ß-cyclodextrin complex was purchased from Sigma (St. Louis, MO, USA).
Carotid endoarterectomy specimens
Human carotid endoarterectomy specimens (n=25) were obtained from 25 patients (16 males, mean age 75±5 yr; 9 women, mean age 71±8) with carotid stenosis of >70% as demonstrated by digital subtraction angiography and Doppler ultrasonography. The specimens were cut along their transversal axis: one-half was fixed in 10% neutral buffered formalin within 2 min after surgical removal, the other half was put in culture for isolation of SMCs and collection of secreted proteins. Serial 3 µm thick sections were cut from paraffin blocks and mounted on slides previously treated with poly-L-lysine solution (Sigma). For histology, serial sections of carotid plaques were stained with hematoxylin & eosin and Movat pentachrome to evaluate the morphological damage at light microscope (i55, Nikon, Tokyo, Japan), according to the American Heart Association classification scheme (20)
. The histological examination revealed that all specimens contained ruptured lipid-rich plaques; thin fibrous cap atheromata were alternated with other stages of atherosclerosis (fibrous cap atheromata and intimal xanthomata) in the same specimen. For the experiments on proteins secreted by the plaque, we defined as complicated the regions characterized by thin fibrous cap, large necrotic core, and thrombotic material, while adjacent atherosclerotic regions were defined as noncomplicated.
Nonatherosclerotic internal mammary arteries specimens were obtained from patients (n=10, mean age=65±5 yr, all men) undergoing coronary artery by-pass surgery; these were used as negative control samples and were manipulated like the atherosclerotic plaque specimens. All mammary artery segments showed adaptive intimal thickening and duplication of the internal elastic membrane.
All samples were obtained from vascular surgeons as surgical residues in accordance with ethical committee regulations, and all patients gave informed consent.
Secreted proteins of the plaque
Surgical pieces (arterial wall and atherosclerotic plaques, 
1 cm length and 500 mg wt) were washed three times in PBS as described previously (21)
. We separated out the noncomplicated region from ruptured and thrombotic regions of carotid plaque material with a scalpel, using optical stereomicroscopy. We used only the noncomplicated regions in order to detect the secretion of HMGB1 and avoid the passive release of HMGB1 from thrombotic and necrotic materials. The samples were put into culture plate inserts and incubated in protein-free culture medium (RPMI, 1% HEPES, 1% penicillin, streptomycin, and amphotericin) for 3 days at 37°C. The culture medium, containing the secreted proteins, was collected every 24 h and analyzed by immunoblot to detect HMGB1 and LDH; the tissue was lysed in SDS-PAGE loading buffer and analyzed similarly. As a control, fresh samples were incubated for 16 h in protein-free culture medium containing 10 mM sodium azide and 6 mM deoxyglucose to promote necrosis (7)
; the samples were washed and further incubated in protein-free culture medium, which was collected every 24 h.
Cell culture
SMCs were obtained from human carotid specimens after digestion with collagenase type II (22)
. Cells were grown in F-12 Nutrient Mixture (Ham) Kaighn’s Modification (F12K) containing 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin, and incubated at 37°C with 5% CO2. Human aorta SMCs were obtained from American Type Culture Collection (Rockville, MD, USA) and cultured according to the supplier’s instructions. More than 99% of the cells expressed -actin. All experiments were performed with SMCs with a passage number 
4.
Cholesterol loading
Cholesterol was delivered to cells by using Chol:Mß CD complex as "water-soluble cholesterol." Chol:MßCD contains 
50 mg of cholesterol/g solid (molar ratio, 1:6 cholesterol/MßCD). All concentrations are indicated as cholesterol weight. Subconfluent human SMCs from aorta and atherosclerotic carotid artery were incubated with or without Chol:MßCD (10 µg/ml) in 0.2% BSA for 72 h.
In vitro studies of HMGB1 effects on SMCs
Chemotaxis and proliferation assays and FACS analysis were performed as described (17)
. The percentage of cells incorporating bromodeoxyuridine (BrdU) was estimated using the Anti-Bromodeoxyuridine + Nuclease kit, following the manufacturer’s instructions (GE Healthcare, Chalfont St. Giles, UK). The percentage of cells in a terminal state of apoptosis was estimated using the dead/end fluorometric apoptosis detection system (Promega, Madison, WI, USA); FACS analysis was performed on a FACSCalibur instrument.
HMGB1 secretion by human SMCs
The secretion of HMGB1 in response to HMGB1 was tested on subconfluent SMCs. Cells were seeded in 6-well plates (105 cells/well) and grown in F12K medium supplemented with 10% FCS. After 24 h, the medium was replaced with serum-free F12K medium for 16 h. Subsequently, the cells were incubated in serum-free F12K medium alone or with the addition of different concentrations of HMGB1 Box B. Cell lysates and supernatants were collected after 48 h and analyzed by Western blotting for HMGB1 and LDH, as described previously (18)
.
Histology, immunohistochemistry, and immunofluorescence
Cells
After cholesterol loading, human SMCs in Lab-Tek chamber slides (Nalge Nunc, Rochester, NY, USA) were fixed in 4% paraformaldehyde in PBS and stained with Oil Red O for neutral lipids. Immunofluorescence was done as described previously (18)
using polyclonal anti-HMGB1 Ab (1:1000), monoclonal anti-CD68 (1:200) and anti-Ab smooth muscle -actin (1:2000), or phalloidin-FITC. Cells were imaged using a Delta Vision Restoration Mycroscopy System (Applied Precision, Issaquah, WA, USA) built around an Olympus IX70 microscope equipped with mercury-arc illumination.
Tissue
Serial sections of carotid atherosclerotic arteries were permeabilized, saturated, and processed for immunohistochemistry or immunofluorescence (18)
. Rabbit IgG staining as nonimmune control and hematoxylin & eosin staining (data not shown) were performed at the same time.
Digital images
Digital images were elaborated using Adobe Photoshop 7.0 and included in figures using Adobe Illustrator 11.0.
RESULTS
Extracellular HMGB1 production by human atherosclerotic plaques
We tested whether HMGB1 is in the secreted proteome of arterial walls from patients affected by atherosclerosis. We analyzed carotid endoarterectomies; in order to avoid as much as possible the presence of HMGB1 released by necrotic cells, we analyzed the noncomplicated regions of atherosclerotic lesions identified as described previously. Endoarterectomies samples were cut in small pieces and kept in RPMI medium without serum. We found a large amount (40 pg/mg tissue) of HMGB1 released in the supernatants every 24 h (Fig. 1
) but no LDH, indicating that no necrotic cells were present; in contrast, similar samples necrotized with sodium azide and deoxyglucose released HMGB1 immediately, but no more HMGB1 was detected in the supernatant on prolonged incubation (data not shown). Taken together, these data indicate that HMGB1 was actively secreted by living cells rather than released by dead cells or washed away from the ECM. As a control, we tested whether the wall of nondiseased arteries also secreted HMGB1. Internal mammary arteries obtained from patients undergoing coronary artery bypass surgery were manipulated in the same way of endoarterectomies; no HMGB1 was found in the supernatants (Fig. 1)
.
|
These data show that a significant amount of extracellular HMGB1 is present in atherosclerotic lesions; this amount, if not washed out continuously by the circulating blood, is certainly within the biologically active range on endothelia and SMCs in the arterial wall. We found no detectable HMGB1 in the plasma of patients from whom the plaques were derived, possibly because the dilution into the bloodstream reduced its levels below detection limits (5 ng/ml blood, results not shown).
Expression of HMGB1 in cells within the atherosclerotic plaque
To discover the origin of the secreted HMGB1, we investigated HMGB1 expression in the cells of human atherosclerotic plaques by immunohistochemistry (Fig. 2
A-F). We found that HMGB1 is contained in both nuclei and cytosol of endothelial cells and is abundant in both nuclei and cytosol of inflammatory cells infiltrating the neointima. The morphological features and the distribution of infiltrating inflammatory cells resembled those usually associated with macrophagic foam cells. Surprisingly, human SMCs of neointima and media showed three different patterns of HMGB1 expression: cells expressing HMGB1 in both nuclei and cytosol, cells containing HMGB1 only in the nuclei, and cells not expressing HMGB1. SMCs presenting HMGB1 in both nuclei and cytosol were mostly localized in the neointima and to a lesser extent in the media of the atherosclerotic vessel.
|
As expected, in human normal arteries HMGB1 was abundant only in the nuclei of endothelial cells and present at a low level in the nuclei (but not in the cytoplasm) of most SMCs (Fig. 2G, H, I
).
To extend these observations, we double-labeled arterial tissue sections for HMGB1 and specific markers of endothelial cells (CD31), foam cells (CD68), and SMCs (-actin). HMGB1 was found in the nuclei and cytoplasm of endothelial, foam cells, and a substantial fraction of SMCs (results not shown). The presence of HMGB1 in the cytoplasm of some SMCs is remarkable and suggests that HMGB1 can be secreted not only by endothelial and foam cells but by SMCs as well.
Cholesterol promotes HMGB1 expression and secretion by human SMCs
Whereas the findings that endothelial and foam cells can secrete HMGB1 were expected, the finding that HMGB1 is present in the cytosol of plaque SMCs, and therefore potentially secreted by them, was completely unexpected. We therefore tested directly whether SMCs can indeed secrete HMGB1 and whether atherogenic stimuli can promote HMGB1 secretion by SMCs.
We then exposed SMCs isolated from normal arteries or from atherosclerotic plaques to cholesterol for 72 h. At concentrations up to 10 µg/ml, cholesterol complexed to methyl-ß-cyclodextrin had no toxic effect: LDH was absent in the supernatants of treated cells (data not shown). After cholesterol loading, all SMCs isolated from atherosclerotic human arteries showed Oil Red O staining lipid droplets throughout the cytosol (Fig. 3
A), whereas only a few SMCs stained positively before loading. Cholesterol-loaded cells also expressed strongly the CD68 marker, which is up-regulated in SMCs in response to cholesterol (23)
(fig. 3B
). No difference in morphology and -actin expression between treated and untreated cells was found.
|
After challenge with cholesterol, SMCs also secreted HMGB1 in the medium (Fig. 3C
). We noticed a few SMCs from the atherosclerotic plaque that showed HMGB1 in the cytosol before cholesterol loading (results not shown), but very little HMGB1 was secreted by the population of cells as a whole (Fig. 4
C). Most likely, a few cells had been already stimulated to assume a secretory phenotype in the atherosclerotic lesion from which they were obtained.
|
Similar results were obtained with human SMCs derived from normal aorta, except that no cell was positive to Oil Red O staining or contained HMGB1 in the cytoplasm before cholesterol challenge (results not shown).
Taken together, these data demonstrate that an atherogenic stimulus like cholesterol can promote the secretion of HMGB1 from SMCs in vitro. We then checked whether in the atherosclerotic plaques the SMCs expressing HMGB1 also express the CD68 marker. Indeed, this was always the case (Fig. 4)
.
Effects of HMGB1 on human SMCs: SMCs proliferate, migrate, and secrete HMGB1
Previously, we have shown that HMGB1 is a chemoattractant for rat SMCs (18)
. As expected, HMGB1 also stimulated the migration of human SMCs (Fig. 5
A). This effect is completely dependent on the HMGB1/RAGE interaction, since both antibodies against RAGE and HMGB1 suppress the migration of human SMCs.
|
We have also recently demonstrated that HMGB1 stimulates the proliferation vessel-associated stem cells, the mesoangioblasts (17)
. Since mesoangioblasts can differentiate into SMCs (24)
, we checked whether SMCs can also respond to HMGB1 with proliferation. Human SMCs were starved for 16 h, and then HMGB1 was added to the serum-free medium. Figure 5B
shows that there is a significant increase in the number of cells after stimulation with HMGB1 for 3 days. Proliferating cells had a normal morphology and excluded trypan blue up to the end of the experiment, whereas cells in control cultures without HMGB1 were dying (data not shown).
We also analyzed by FACS samples of cells incubated for 1 day in the presence of FCS, in the absence of FCS (serum-free), and in the absence of FCS but in the presence of HMGB1 (Fig. 5C
). The number of cells with more than a diploid content of DNA increased when HMGB1 was added to the serum-free medium, when compared with cells incubated in serum-free medium. Likewise, we counted the cells incorporating BrdU (Fig. 5D
): again, the percentage of positive cells was high for FCS-incubated cells, low for cells incubated in serum-free medium, and intermediate for cells exposed to HMGB1.
Finally, we assessed the level of apoptosis in cells cultured in the presence of FCS, in serum-free medium with and without HMGB1, and in serum-free medium to which 2 ng/ml tumor necrosis factor (TNF) had been added (Fig. 5E
). Whereas a significant number of cells incubated in the absence of FCS were already terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) positive after 1 day, the presence of HMGB1 reduced this number considerably.
Taken together, these data indicate that SMCs incubated in the presence of HMGB1 enter the cell cycle and proliferate, whereas cells incubated in serum-free medium exit the cell cycle and start to apoptose. Thus, HMGB1 acts as a mitogenic factor for SMCs, as it does for mesoangioblasts.
The ability of SMCs to be a source and a target of extracellular HMGB1 creates the conditions for a possible autocrine loop. We tested whether in response to extracellular HMGB1 SMCs can secrete HMGB1 themselves. SMCs were starved for 16 h in the absence of serum, and then increasing concentrations of HMGB1 Box B were added to the medium without serum. Box B retains the cytokine properties of full-length HMGB1 (15)
but is not recognized by the KS1 mAb, which recognizes an epitope within Box A. After 48 h, we found that SMCs significantly increased HMGB1 expression and secretion in response to HMGB1 Box B in a dose-dependent manner (Fig. 5F
). The absence of LDH in the supernatant of stimulated SMCs demonstrated that HMGB1 was secreted and not released by necrotic cells.
DISCUSSION
A growing number of reports describe the presence of extracellular/cytoplasmic HMGB1 during various inflammatory conditions, both acute and chronic (25
, 26)
. In the present study, we show that a large amount of HMGB1 is secreted by human carotid atherosclerotic plaques, both by inflammatory cells and, unexpectedly, by SMCs. Normally, SMCs contain a much lower amount of HMGB1 than other cell types (27)
, but we show that HMGB1 expression and secretion are promoted by atherogenic stimuli like cholesterol loading. In turn, extracellular HMGB1 acts as mitogenic and chemotactic factor for human SMCs, and itself promotes more HMGB1 expression and secretion. Our findings identify an autocrine/paracrine loop based on HMGB1, which can act as a mechanism of neointima hyperplasia during the progression of atherosclerosis and during restenosis after coronary angioplasty.
Atherosclerotic plaques produce and secrete HMGB1
Our first observation is that HMGB1 is present in much larger quantities in atherosclerotic plaques as compared with normal arterial walls; plaque fragments in culture keep secreting a significant amount of HMGB1. In principle, plaques can contain extracellular HMGB1 derived from its abundant necrotic cells; our results do not disprove a necrotic origin of HMGB1 but were designed to detect specifically the actively secreted protein. Continued and sustained release of HMGB1 in the medium in the absence of necrosis is a clear indication of active secretion.
We then investigated the cellular origin of the secreted HMGB1. HMGB1 secretion is associated with the translocation from the nucleus to the cytoplasm and eventual exocytosis (11
, 12)
. In cells of myeloid origin, like monocytes, macrophages, and dendritic cells, HMGB1 is accumulated from the cytoplasm into secretory vesicles, whereas other cell types show no sign of vesicular accumulation. In any event, HMGB1 cytoplasmic localization, whether vesicular or not, is taken as a marker of its secretion, which cannot be easily demonstrated in vivo because the extracellular protein is diluted in the extracellular fluid and possibly eliminated. In the case of atherosclerotic plaques, all three cells types, endothelial cells, SMCs, and foam cells, contained cytoplasmic HMGB1. In the case of SMCs, this finding was unexpected, as normal SMCs are among the cell types that contain a very low level of HMGB1 (18
, 27)
.
SMCs secrete HMGB1 in response to atherogenic stimuli
We next investigated whether the secretion of HMGB1 is an integral part of the array of responses displayed by SMCs exposed to atherogenic stimuli. In fact, SMCs from normal arteries shift HMGB1 from the nucleus to the cytoplasm, and eventually secrete it, when they are loaded with cholesterol. Interestingly, a few SMCs isolated from plaques already showed a similar phenotype even before cholesterol loading. Arguably, these SMCs had already accumulated cholesterol while in the plaque or were exposed to some other atherogenic stimuli. These results, although obtained in vitro, are clearly paralleled by the phenotype of SMCs in situ: in the plaque, SMCs that contain cytoplasmic HMGB1 also express CD68.
In secreting SMCs, cytoplasmic HMGB1 is apparently not localized in vesicles. This is contrary to what is seen in myeloid cells, which accumulate HMGB1 and other cytokines like interleukin (IL)-1ß in a specific population of lysosomes, called secretory lysosomes (11
, 28)
. Secretory lysosomes have only been found in hematopoietic cells (29)
and are apparently absent both in SMCs and in neurons that also secrete HMGB1 in specific conditions (27)
. Cytoplasmic, nonvesicular HMGB1 has also been found in skeletal muscle fibers in biopsies from chronic myositis patients (30)
. HMGB1 translocation, and potentially secretion, may be a common property of cells of the muscular lineage undergoing stress, although cardiomyocytes have not been investigated yet.
SMCs at the center of an HMGB1 loop
Our finding that SMCs can secrete HMGB1 is intriguing because SMCs also respond to extracellular HMGB1. We confirmed our previous finding that HMGB1 stimulates the migration of SMCs (18)
; we also show now that HMGB1 can serve as a mitogenic stimulus for SMCs. In the absence of serum, SMCs in culture slowly die, whereas the addition of HMGB1 promotes cell proliferation. The mitogenic and chemoattractant effects exerted by HMGB1 appear to be mainly mediated by RAGE, which is widely expressed by all cellular types of atherosclerotic plaque, in particular activated SMCs of the expanding neointima (31)
.
The ability of SMCs to produce and respond to extracellular HMGB1 creates the conditions for an autocrine/paracrine loop. We demostrated that SMCs stimulated with HMGB1 increase the expression of HMGB1 in a dose-dependent manner and secrete it. Very recently Jaulmes et al. (32)
showed that HMGB1 can also promote the production of PGE2 by vascular SMCs. A loop of SMC activation and proliferation is logically a mechanism that can lead to neointimal hyperplasia and in-stent restenosis. Blocking HMGB1 from SMCs may be a new and specific therapeutic strategy for the local control of SMC proliferation and migration, especially for the prevention of in-stent restenosis.
ACKNOWLEDGMENTS
We thank F. Crea (Institute of Cardiology, Catholic University, Rome) for continuing support. We also thank Dr. S. Benussi for providing internal mammary artery specimens, Dr. C. Foglieni for assistance in histological examination of carotid plaques, and N. Collu, J. Hering, and F. De Marchis for excellent technical assistance. This work was supported by the Italian Ministry of Health and Fondazione Monte dei Paschi di Siena.
FOOTNOTES
1 These authors contributed equally to this work. 
Received for publication February 26, 2006. Accepted for publication July 24, 2006.
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