Published online before print September 7, 2007
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* Inserm, U698, Univ Paris 7, CHU X-Bichat, Paris, France; and
Centre Cardiologique du Nord, Saint-Denis, France
1Correspondence: INSERM U698, Hôpital X. Bichat, 46, rue Henri Huchard, 75877 Paris Cedex 18, France. E-mail: meilhac{at}bichat.inserm.fr
ABSTRACT
The pathological remodeling of the arterial wall in atherosclerosis involves protease activities, which play a major role in complications via plaque rupture. Circulating leukocytes and particularly neutrophils have been shown to be an independent predictor of recurrent ischemic events. However, neutrophils are poorly documented within atherosclerotic plaques. We hypothesized that intraplaque hemorrhage could convey neutrophils into the lesion, spreading into the necrotic core, thus participating in its protease enrichment. One hundred human carotid endarterectomy specimens were dissected into culprit-stenosing plaques (CPs) and adjacent noncomplicated plaques. Half of CPs exhibited hemorrhage, which was confirmed by the release of hemoglobin. Pro- and active forms of matrix metalloproteinase-9 (MMP-9) were increased in media conditioned by hemorrhagic plaques. Higher levels of lipocalin [neutrophil gelatinase-associated lipocalin (NGAL)]/MMP-9 complexes, specifically released by neutrophils, were also found in conditioned media from plaques with hemorrhage. Immunohistochemical analysis of the corresponding carotid samples showed that neutrophil markers such as elastase, NGAL/MMP-9, CD66b, and proteinase 3 colocalized with blood constituents (i.e., hemoglobin, plasminogen). All markers of neutrophil degranulation were positively correlated in CP-conditioned media (
1-antitrypsin/elastase complexes, myeloperoxidase, and -defensins), and higher levels came from CPs containing intraplaque hemorrhages. Addition of an elastase inhibitor at the time of incubation led to a decrease in the proMMP-9 activation in CPs, suggesting cross-talk between proteases released by neutrophils. Finally, we found that neovessels observed at the interface between cap and core exhibit an activated endothelium, which may favor leukocyte diapedesis. Our study thus provides evidence for the involvement of neutrophils in plaque vulnerability.
Key Words: human leukocyte elastase • proteinase 3 • neovessels
INTRODUCTION
The involvement of intraplaque hemorrhages in atherothrombotic plaque vulnerability was proposed as early as 1936 [1
, 2
] in human coronary arteries and has been confirmed regularly through histopathological approaches in carotid endarterectomy samples [3
]. Kolodgie et al. [4
] have shown that accumulation of unesterified cholesterol within the lesion was in part linked to the presence of erythrocyte membranes. Follow-up studies over a period of 18 months using magnetic resonance imaging have shown that the presence of hemorrhages increased plaque progression [5
] and that symptomatic plaques had a higher incidence of intraplaque hemorrhage [6
]. In parallel, genotype/phenotype of haptoglobin has been shown to influence the coronary risk in diabetic patients in relation to intraplaque hemorrhage [7
]. These are associated with the presence of neovessels in the plaque shoulder at the interface between the cap and the necrotic core [8
9
10
]. As intraplaque hemorrhages convey all the blood components into the plaque, which then spread into the necrotic core, including erythrocytes, circulating leukocytes (of which 
60% are PMNs, 30% are lymphocytes, and less than 10% are monocytes), plasma proteins, and platelets, we hypothesized that carotid culprit lesions are enriched in PMNs and associated proteases.
Until now, the role of PMNs in the evolution of atherothrombosis has retained little attention [11 , 12 ]. Despite the fact that epidemiological studies have shown that PBL count, and above that, all neutrophil count is a risk prediction of cardiovascular events [13 , 14 ], their presence within the lesions has been largely eluded [15 , 16 ]. However, Naruko et al. [17 ] have reported recently the presence of neutrophils in culprit lesions in cases of acute coronary syndrome. In parallel, several biomarkers, more or less specific of PMNs, have been shown recently to have prognostic value in atherothrombosis, including myeloperoxidase (MPO) [18 , 19 ], matrix metalloproteinase-8 (MMP-8) [20 ], MMP-9 [21 ], and elastase.
In the present study, we tested the hypothesis that PMNs are present in culprit plaques (CPs) containing hemorrhages and can release proteases, which are potentially involved in plaque fragilization. For this purpose, we first explored the concomitant release of MMP-9, neutrophil gelatinase-associated lipocalin (NGAL), elastase, MPO, and -defensins as markers of PMN activation by carotid endarterectomy samples, with or without intraplaque hemorrhage. Second, we colocalized these markers of PMNs and intraplaque hemorrhage by immunohistochemistry. We propose that PMN diapedesis across activated neovessels could represent another route whereby PMNs may infiltrate the lesions. Our results, taken together, suggest that PMNs may contribute to the evolution of atherosclerosis toward plaque vulnerability.
MATERIALS AND METHODS
Tissue sampling
Human carotid endarterectomy samples (n=102) and nonatherosclerotic endarteries (mammary; n=31) were obtained from patients undergoing carotid surgery and coronary bypass using internal mammary artery, respectively, at the Centre Cardiologique du Nord (Saint-Denis, France). Tissue samples were collected in cold RPMI (4°C) containing antibiotics and antimycotic (penicillin-streptomycin and amphotericin) and processed within 2 h after surgery. No antiprotease treatment was used to prevent inhibition of proteolytic activity, which was to be assessed subsequently. These tissues are considered as surgical waste in accordance with French ethical laws (L.1211-3–L.1211-9) and the INSERM Ethics Committee.
Dissection into culprit and noncomplicated plaques (NPs and CPs)
Carotid endarterectomy samples were dissected as described previously [22
], separating the culprit zone from the adjacent plaque. CPs, which are the cause of surgery, correspond to the stenosing segment. CPs were compared with nonstenosing, adjacent plaques (NPs, i.e., the remaining part of the proximal common and the external and internal carotid) and control nonatherosclerotic mammary endarteries.
In accordance with the classification established by Stary et al. [11 ], histological characteristics of our samples showed that CPs represented advanced type V or VI atherosclerotic lesions. The adjacent area, considered as NP, corresponded to type III or IV lesions. Nonatherosclerotic control endarteries were obtained by removal of the adventitia from whole mammary arterial segments.
Macroscopical evaluation of CPs revealed sample heterogeneity for the presence of intraplaque hemorrhage but the constant presence of fibrous cap and extracellular lipid deposits. CPs were separated, according to macroscopical criteria: the presence of hemorrhage (H; n=63) or not (NH; n=39). This separation was confirmed by biochemical assays.
Conditioned media
Small pieces of tissue (2 mm3) were weighed and incubated for 24 h in RPMI culture medium containing 1% L-glutamine, 1% penicillin, streptomycin, and amphotericin at 37°C (5% CO2) and without serum. For standardization, the volume of medium was adjusted to sample wet weight (6 mL per gram). Conditioned media from CPs and NPs were collected, then centrifuged (14,000 g for 15 min at 4°C), and stored at –80°C until further analysis.
Tissue extraction and protein assay
After incubation for obtaining conditioned media, the tissue samples were stored at –80°C and then extracted under more stringent conditions following a protocol similar to Choudhary et al. [23
]. Briefly, 50 tissue samples [10 carotids with 10 without hemorrhage (CPs and NPs) and 10 mammary arteries] were homogenized in 0.05 M Tris-HCl, pH 7.5, containing 1% Triton X-100 and 0.03% sodium azide using a Polytron and centrifuged (14,000 g for 10 min at 4°C). The supernatant was taken for analysis. Protein concentration was measured using the Bradford assay (Bio-Rad, Hercules, CA, USA).
Immunoblot analysis and substrate zymography
Proteins were separated by SDS-PAGE after normalization to tissue wet weight. After electrophoresis, samples were transferred onto a nitrocellulose membrane (Amersham, Piscataway, NJ, USA), which was probed with a goat polyclonal antibody against hemoglobin at 0.1 µg/mL (Abcam, Cambridge, MA, USA, ab8575) or a monoclonal anti-NGAL (Antibody Shop, Denmark, HYB 211-01) and a HRP-conjugated secondary antibody. Zymography was performed in duplicate as described previously [24
]. Protease activities were quantified by densitometry using the National Institutes of Health Image 1.60 ppc software. Results were expressed as OD per mg tissue (OD/mg tissue) and percentage of active MMPs [active/(active+proform)].
Determination of hemoglobin/heme content
Heme content was assessed by addition of formic acid to the conditioned media (v/v 10/90) and was considered as being proportional to hemoglobin release. Hemoglobin from bovine erythrocytes was used as a standard. After binding of formate to the heme, OD was monitored at 405 nm [25
].
ELISA
Concentrations of 1-antitrypsin/elastase complex, MPO, and -defensins were determined using ELISA kits (Calbiochem, San Diego, CA, USA, QIA96; and Hycult, The Netherlands, HK324 and HK317, respectively) following the manufacturers’ instructions.
Immunohistochemistry
Immunohistochemistry was performed on cryosections of 10 representative carotid specimens using various antibodies (Hycult) as primary antibodies diluted in TBS/0.2% Tween, 0.6% caseine, pH 6, and a peroxidase LSAB-Dako kit (Dako, Denmark) for detection. The binding reaction was detected by 3'-diaminobenzidine tetrahydrochloride. Slides were then counterstained with Mayer’s hematoxylin. Control irrelevant antibodies (Dako) were applied at the same concentration to assess nonspecific staining.
Statistical analysis
Differences between CPs and NPs from the same endarterectomy sample were evaluated by the Wilcoxon-paired nonparametric test. Differences between endarterectomy samples and controls or between carotid CPs and NPs, with or without intraplaque hemorrhage, were assessed by the Mann-Whitney nonparametric test. Correlations were assessed with the nonparametric Spearman test (Statview software, Version 4.57). Statistical significance was accepted for P < 0.05. Results are expressed as box plots, in which the median is shown. Upper and lower limits of boxes represent interquartiles (25th and 75th), whereas upper and lower bars show percentiles (10th and 90th).
RESULTS
Hemorrhage accounts for plaque enrichment in active MMP-9
Characterization of CPs with or without hemorrhage
Some plaque characteristics can be determined during macroscopic dissection, such as the presence of an intraplaque hemorrhage, lipid deposition, or calcification (Fig. 1A
). The carotid plaques (n=91) were thus separated into two groups: those with visible hemorrhage (H, n=54) and those without (NH, n=37). Each carotid sample was divided into CP and NP and incubated with culture medium. The hemoglobin levels were assessed in conditioned media from CP and the corresponding NP by a chromogenic assay and by Western blot (Fig. 1B
and 1C)
. The macroscopic evaluation fitted well with the results of the biochemical determination of hemoglobin content (in 98% of cases). The first criteria, belonging to the hemorrhagic sample group, was a visible hemorrhage, whatever its size and hemoglobin content. The threshold chosen to ascertain the presence of a hemorrhage biochemically was 3.5 µg hemoglobin/mg tissue (Fig. 1B)
. A few NH CPs (n=2) were above this level, and so these plaques were allotted to the hemorrhage group. There was significantly more hemoglobin in conditioned media from H CP and NP than NH CP and NP. The data were confirmed by Western blot (Fig. 1C)
. There was a good correlation (P<0.01 and 
=0.4) between the hemoglobin content remaining in the tissue versus that found in the corresponding conditioned media (Fig. 1D)
. The results obtained in conditioned media are thus a good reflection of the total content of hemoglobin within the tissue.
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Figure 1. Characterization of plaques with (H) or without hemorrhage (NH). Macroscopic images (A) of CPs, H (two left panels) and NH (two right panels). (Left-most panel) We can see a surgical incision of the cap, mimicking what happens in plaque rupture. Verification of the hemoglobin (Hb) content of CP versus NP-conditioned media, H or NH, by a chromogenic assay (B) and by Western blot (C). Correlation between the protein content of the conditioned media and the corresponding tissue extract (D).
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Figure 2. The release of MMP-9 is increased with intraplaque hemorrhage. Detection of the different forms of MMP-9 in conditioned media of CPs and NPs, H or NH, by gelatin zymography. Representative zymogram (A) from two typical patients, H and NH. Lysis bands corresponding to proMMP-9, MMP-9, and NGAL/MMP-9 were quantified and expressed as described in Materials and Methods (B–D). Detection of NGAL/MMP-9 was performed by Western blot (D).
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Gelatin zymography allowed the detection of two other forms of MMP-9 (Fig. 2A) , specifically released by PMNs: the complex NGAL/MMP-9 (120 kDa) and the homodimer of proMMP-9 (200 kDa). Both were detected mostly in conditioned media from CP. Above all, higher levels of NGAL were present in conditioned media from H CP compared with paired NP and compared with NH CP (Fig. 2D) . This result was confirmed by Western blot (Fig. 2D) . These results support the hypothesis of the PMN being a cellular source of MMP-9 in carotid plaques with hemorrhage.
Hemorrhage-derived cells within atheromatous plaque: the PMN involvement
Colocalization of PMN markers with hemorrhage- derived constituents
The approach by immunohistochemistry was focused on the presence of hemorrhage-derived constituents within the plaque: hemoglobin; plasminogen; a platelet marker (GPIIIa or CD61); PMN-specific markers, a marker of neutrophil maturation (CD66b), proteinase 3 (PR3), and NGAL/MMP-9; and markers expressed predominantly in neutrophils: human leukocyte elastase (HLE) and MMP-9 (Fig. 3
). The hemorrhage-derived constituents were all colocalized in the shoulder area at the interface of the cap, the core, and the media, spreading into the necrotic core within the CP. For plasminogen, the immunostained region was more extensive (in the cap and core, as described previously [22
]) than for hemoglobin and CD61; the same trend was observed for MMP-9 throughout the core. Neutrophils were colocalized with these blood constituents as well as with the PMN markers: HLE, NGAL/MMP-9, PR3, and CD66b. Progression through the tissue thickness by examining serial sections allowed us to identify neovessels in the shoulder area (Fig. 3
, PR3) which were probably at the origin of the observed intraplaque hemorrhage.
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Figure 3. Immunocolocalization of PMN and hemorrhage markers in a CP. Sections of a CP immunostained for CD66b (PMN marker), HLE, PR3, CD68 (macrophage marker), MMP-9, NGAL, plasminogen (Pg), hemoglobin, and CD61 (platelet marker). Immunostaining of hemorrhage markers (hemoglobin, Hb, and plasminogen, CD61) is colocalized with the PMN markers (CD66b, HLE, PR3, NGAL, and MMP-9). MMP-9, a common marker of PMN and macrophages, colocalized with both cell types. Orientation: Intima left; media, bottom; and necrotic core, top and right. The staining of PR3 (objective original magnification, x4) showed the topology of the plaque; objective original magnification, x10, for all other pictures.
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-defensins, stored in neutrophil azurophil granules, were assessed by ELISA. They were found predominantly in hemorrhagic plaques (CP and NP), which released greater amounts than nonhemorrhagic ones (Fig. 4B)
. All the markers assessed followed the same trend in conditioned media and in tissue extracts (Fig. 4B)
. It is interesting that results obtained for MPO and -defensins in conditioned media correlate strongly with those obtained in tissue extract (Fig. 4B)
. It shows once again that conditioned media are a reliable reflection of their total tissue content, thus validating their use for the rest of the study. The same trend, as observed for MPO and -defensins, was found for the 1-antitrypsin/elastase complexes (Fig. 4C)
. This result shows that elastase has been released by leukocytes and subsequently bound by its physiologic inhibitor 1-antitrypsin. We then verified whether total elastase content determined by ELISA paralleled its active part. Elastase proteolytic capacity was determined by zymography (Fig. 4D)
, and the same trend was observed for antigenic- and activity-based detection of elastase.
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Figure 4. PMN markers predominate in conditioned media from plaque with hemorrhage. PMN markers were assessed in conditioned media (A) and tissue extracts (B) from CPs and NPs, H or NH, and from mammary endarteries (mam). (A) Statistical analysis. (B, left) MPO and -defensins; (C, left) 1-antitrypsin/HLE complexes were quantified in conditioned media by ELISA. (B, middle) Average of the content of MPO and -defensins in 10 representative plaque samples were determined in tissue extracts (darkest fraction on the bottom) and corresponding conditioned media (lightest fraction above). (B, right) Correlation between the MPO and -defensin content of conditioned media and the corresponding tissue extracts. (D) HLE activity was assessed by zymography. (E) Correlations were practiced among MMP-9, NGAL/MMP-9, elastase, MPO, and -defensins in conditioned media. NS, Not significant.
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Activation, inhibitory and chemoattractive capacities of CP in relation to hemorrhage
Elastase activates MMP-9 ex vivo
Elastase is an in vitro activator of MMP-9 [26
] and thus, may be involved in MMP activation in human atheromatous plaques. CPs and NPs were divided into two similar segments, each incubated with or without exogenous elastase (10 nM, n=15) or 1-antitrypsin (200 nM, n=30). 1-antitrypsin is an inhibitor of serine proteases, including elastase. Activated MMP-9 and 1-antitrypsin/HLE complexes generated during the 24-h tissue-incubation period were assessed in conditioned media. We compared levels generated by tissue incubated with exogenous proteins (HLE and 1-antitrypsin) with those of paired tissues incubated with RPMI alone (Fig. 5
). Addition of exogenous elastase exhibited a greater elastase activity (detected by zymography) in samples concerned (CP and NP), as expected. Moreover, the exogenous elastase was able to form complexes with 1-antitrypsin, present within CP and NP tissues (Fig. 5A)
. After addition of exogenous elastase, significantly more 1-antitrypsin/HLE complexes were generated by NP than by CP (P<0.05, data not shown). This suggests a more effective elastase inhibitory capacity in NP than in CP. Elastase, added at the time of incubation, led to an increase in active MMP-9 levels in CP and NP (Fig. 5B)
but exhibited the same proMMP-9 level. The active MMP-9 generated did not modify the proMMP-9 level significantly. The inhibitor 1-antitrypsin, added at the time of incubation, led to a decrease in the activation of proMMP-9 in CP (Fig. 5C)
.
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Figure 5. Effectiveness of elastase in MMP-9 activation. CPs and NPs were divided into two segments and separately incubated in RPMI medium, with or without exogenously added HLE (A) or 1-antitrypsin (B) for 24 h. Conditioned media were then sampled and analyzed for 1-antitrypsin/HLE complexes (A) and active MMP-9 (B) generated. Representative zymograms are shown (A–C).
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Figure 6. Immunocolocalization of PMN markers and activated neovessels in a CP. (A) Sections of the shoulder region of a CP immunostained for von Willebrand factor (vW), hemoglobin, CD66b (PMN marker), CD68 (macrophage marker), NGAL, MMP-9, HLE, and MMP-8. No hemorrhage was detected in this shoulder region. The von Willebrand factor staining highlights the presence of numerous neovessels. Numerous cells are present around the neovessels, including macrophages (CD68-positive) and PMNs (CD66b-positive). (B) Higher magnification of the same area focused on a neovessel immunostained with PMN markers (CD66b, HLE, PR3, MPO, NGAL, and P-selectin). The neovessel is P-selectin-positive, possibly activated. *, Neovessel. Objective original magnification A, x10; B, x40.
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In the present study, we report that intraplaque hemorrhages are present in more than 50% of the carotid endarterectomy samples, which we collected. The macroscopic evaluation performed during dissection was validated by the measurement of heme release and by Western blotting, detecting free hemoglobin in the conditioned media. The presence of hemoglobin in CPs was also evidenced by immunohistochemistry. These results are in accordance with those from Virmani’s group obtained on coronary samples, showing that erythrocytes may participate in plaque vulnerability and progression [4 ]. In most of the samples, which we collected, a fibrous cap was present, suggesting that intraplaque hemorrhages were not the result of a recent plaque rupture, fissure, or erosion. The presence of neovessels within CPs supported the hypothesis that blood components could be conveyed into the plaque via neovascularization [27 ].
As hemoglobin from erythrocytes is detected at the interface of the shoulder region and the necrotic core, we hypothesized that polymorphonuclear neutrophils could also be present in atherothrombotic lesions, as they represent 60% of the circulating leukocytes. We detected PMN markers chiefly in association with intraplaque hemorrhages, in the shoulder of culprit lesions, spreading into the necrotic core. Several markers were assessed in the conditioned medium of CPs, H or NH, versus NPs. As demonstrated earlier [28
] and more recently confirmed by our group [22
], human atherothrombotic lesions contain MMP-9. A spatial distribution of MMPs was published recently by Choudhary et al. [23
], providing quantification of MMP-9 and -2 in the different areas of representative carotid endarterectomy samples. It is interesting that they described that MMP-9 was abundant in segments with intraplaque hemorrhage. Concomitantly, we reported that MMP-9 content correlates with plaque complexity in carotid samples [22
]. It can be activated and released from the necrotic core and may thereby participate in plaque vulnerability. In parallel, plasma MMP-9 levels have been demonstrated to have prognostic value in human atherosclerosis [21
]. The cellular origin of this MMP-9 remained to be explored, as it is expressed by various cells potentially present in atherosclerotic plaques, including macrophages [29
], smooth muscle cells [28
], and PMNs [30
]. In the present study, we show that this increased MMP-9 release is correlated with hemoglobin release, suggesting a link between MMP-9 and intraplaque hemorrhages. Moreover, MMP-9 is released, a least in part, as high molecular weight forms, MMP-9 homodimers, and NGAL/MMP-9 [25
] heterodimer, a specific feature of PMN gelatinase activity, whereas macrophages and smooth muscle cells secrete only monomeric forms [30
]. In addition to MMP-9, which is contained in gelatinase and specific granules, we assessed another proteolytic marker of PMN degranulation, HLE, whose activity was detected by zymography. Elastase activity was increased in culprit lesions relative to noncomplicated and normal endartery conditioned media. Moreover, significantly more elastase was released by culprit lesions containing hemorrhages, where 1-antitrypsin/elastase complexes were similarly increased. Plasma levels of elastase (free or complexed with 1-antitrypsin) were shown to be correlated with the occurrence of carotid plaques [31
, 32
], and atherosclerosis was reported to be associated with genetically determined, low levels of plasma 1-antitrypsin [33
]. The specificity of HLE as a marker of neutrophils has been questioned recently in a study by Dollery et al. [34
], who reported that a subset of macrophages can produce elastase in vitro and that immunostaining for elastase colocalized with CD68 in atheromatous plaques. However, data from Naruko et al. [17
], obtained by immunohistochemistry on coronary artery samples, revealed a large number of neutrophils stained for elastase, MPO, and CD66b. The involvement of neutrophils in plaque progression is also suggested by a recent study from Exner et al. [18
], showing that MPO predicts progression of carotid stenosis in states of low high-density lipoprotein cholesterol.
As in our study elastase activity was associated with the presence of intraplaque hemorrhages, we hypothesized that PMNs, conveyed by the blood into the plaque or trapped within the intraplaque hematoma, could be a source of elastase. Neutrophil elastase is a potent protease, capable of degrading most proteins to which it is exposed. Whereas elastase is effective in innate antibacterial immune responses, in some situations, it can be deleterious by degrading the extracellular matrix directly [35
] or indirectly, via proteolytic activation of proMMPs [36
]. Addition of the elastase inhibitor 1-antitrypsin to CPs or NPs during incubation decreased the percentage of active MMP-9 released. Conversely, addition of exogenous elastase led to an increase in the percentage of active MMP-9, suggesting that elastase contained within the plaque can participate in proteolytic conversion of pro- to active MMP-9.
It is often assumed that neutrophils have a short lifespan [37 ], especially after activation within the tissues. This is probably why, in most histological studies of human CPs, PMNs are rarely identified via their characteristic nuclear morphology. The release of PMN content can occur upon activation (degranulation) or when senescent PMNs are not removed appropriately by surrounding phagocytes, i.e., macrophages. Postapoptotic necrosis could be a major source of inflammation by the release into the extracellular space of proteases, cytokines, and oxidative enzymes. It has been shown recently that phagocytosis of apoptotic cells by macrophages is impaired in atherosclerosis [38 ], which may favor leakage of proteases from PMNs in the tissue.
The presence of intraplaque hemorrhage can result from plaque rupture, subsequently surrounded by fibrous tissue as a result of the healing process, and/or from newly formed vessels, which bring blood components into the plaque. These neovessels appear immature and thus, are probably fragile and susceptible to rupture, principally in the shoulder regions of the plaque [39 ]. Several lines of evidence support the hypothesis that neovascularization of the atherosclerotic plaque originates mainly from the adventitia [40 41 42 ]. Our results suggest that these neovessels become more fragile in the proteolytic context of the intima and the core, thus favoring leakage of plasma-borne molecules and leukocyte diapedesis. The expression of adhesion molecules by these newly formed microvessels promotes and maintains the influx of inflammatory cells and thus, may contribute to plaque fragilization and rupture [43 ]. In our study, we also found that most of the neovessels were present in the shoulder area and that the neutrophils were chiefly localized in the vicinity of these neovessels, as observed by Naruko et al. [17 ] in coronary arteries. Expression of P-selectin (a PMN adhesion molecule necessary for their diapedesis) is increased on endothelial cells of plaques from patients with unstable angina compared with those with stable disease [44 ]. Accordingly, we found that neovessels observed in the shoulder areas are P-selectin-positive and surrounded by PMNs, suggesting that this endothelium may favor the diapedesis of leukocytes. This may represent another route of PMN recruitment, explaining why PMN markers could be detected in spite of the absence of macroscopically detectable, intraplaque hemorrhage in carotid samples.
In conclusion, through an experimental approach using human tissue, we have explored the impact of PMN-released proteases and established for the first time a link between enrichment in these proteases and the presence of intraplaque hemorrhage in atherothrombotic culprit lesions. This suggests a biological relationship connecting intraplaque hemorrhage, protease conveyance and retention, and plaque fragility.
ACKNOWLEDGEMENTS
A. L. was financed by the Groupe d’Etudes sur l’Hémostase et la Thrombose et the Institut d’Athérothrombose. This study was supported by INSERM and by the Leducq Foundation (Leducq Transatlantic Network on Atherothrombosis). The authors thank Dr. Mary Osborne-Pellegrin for editing this manuscript and Dr. Martine Jandrot-Perrus and Dr. Marie-Anne Gougerot-Pocidalo for their scientific advice. We thank Dr. Andreassian and Dr. Palombi from the Centre Cardiologique du Nord for providing us with carotid and mammary artery samples.
Received November 10, 2006; revised July 24, 2007; accepted August 14, 2007.
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