Atherosclerosis and Lipoproteins |
From the Division of Molecular Medicine, Faculty of Medicine and Health Science, University of Auckland, Auckland, New Zealand.
Correspondence to Geoffrey Krissansen, PhD, Division of Molecular Medicine, Faculty of Medicine and Health Science, 85 Park Rd, University of Auckland, PO Box 92019, Auckland, NZ. E-mail gw.krissansen{at}auckland.ac.nz
| Abstract |
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Key Words: atherosclerosis heat shock proteins 60 and 70 apoE-deficient mice immunohistochemistry aorta
| Introduction |
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Given the widespread acceptance of the apoE-deficient (apoE-/-) mouse1215 as a model of human atherosclerosis, the present study sought to determine whether atherogenesis in apoE-/- mice correlates with arterial expression of mammalian hsp60 and hsp70 (stress-inducible form).
| Methods |
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Tissue Sample Preparations and Histology
After euthanasia of mice by CO2 asphyxiation and in situ fixation of tissues by perfusion with 4% paraformaldehyde (pH 7.4), the heart and ascending aorta up to iliac bifurcation were dissected. Aortic segments were embedded in OCT compound (Tissue Tek) and snap-frozen, and cryosections (8 µm) that were collected onto poly-L-lysinecoated slides were stored at -80°C.
Immunohistochemistry
Aortic hsp60 and hsp70 expression was detected by staining frozen sections with 2 different mouse monoclonal antibodies (mAbs) specific for either mammalian hsp60 (SPA-806, clone LK-1; 1:150 dilution) or the stress-inducible form of mammalian hsp70 (SPA-810, clone C92F3A-5; 1:200 dilution; Stressgen, which can be accessed online at http://www.stressgen.com). The SPA-810 mAb does not react with the constitutively expressed form of hsp70 (Hsc70), and the SPA-806 mAb does not cross-react with bacterial hsp60. Sections were developed with a mouse-on-mouse immunodetection kit (Vector Laboratories), an avidin-biotin immunoperoxidase ABC complex (Vector Laboratories), and Sigma fast 3,3'-diaminobenzidine (Sigma Chemical Co) and counterstained in Gills hematoxylin.
Double Immunohistochemical Staining of SMCs and ECs
To characterize hsp expression by smooth muscle cells (SMCs), sections were immunostained with either SPA-806 or SPA-810 with the use of a Vector mouse-on-mouse kit and Sigma fast 3,3'-diaminobenzidine. They were subsequently incubated for 90 minutes with an alkaline phosphataseconjugated anti
-SMC actin antibody (1:100, Sigma), and antibody binding was visualized with the use of Vector red (Vector Laboratories). To determine hsp expression by endothelial cells (ECs), sections from 3-week-old apoE-/- mice were immunostained with the anti-hsp mAbs, as described above, and then with a rabbit polyclonal antihuman factor VIII-related antigen antibody (1:10, Biomeda). Immunoreactivity was visualized by use of a Vector ABCalkaline phosphatase kit (rabbit IgG) and Vector red substrate.
Double Immunofluorescence Staining and Confocal Microscopy
Double immunofluorescence staining of aortic arch sections from 3- and 20-week-old apoE-/- mice was used to colocalize hsps with markers of ECs (CD31), monocyte/macrophages (MOMA-2), and T lymphocytes (CD3). The antibodies used were biotinylated rat anti-mouse CD31 mAb (1:50, Pharmingen), FITC-conjugated rat anti-mouse MOMA-2 mAb (1:10, Serotec), and FITC-conjugated rat anti-mouse CD3 (1:10, Serotec). Immunoreactivity of anti-hsp mAbs SPA-806 and SPA-810 was detected with Texas redconjugated horse anti-mouse IgG (1:200, Vector Laboratories). Biotinylated anti-mouse CD31 was detected with streptavidin-FITC (1:150, Sigma). Fluorescently stained sections were examined with a Leica TCS 4D confocal microscope equipped with an argon/krypton laser.
Western Blotting
Common aortic segments were pooled and homogenized in protein lysate buffer at 4°C, and after centrifugation at 10 000g, protein supernatants (100 µg per well) were resolved on 10% SDS-polyacrylamide gels. Proteins were transferred to Hybond C Extra (Amersham Life Science England) and Western-blotted with SPA-806 (1:500) and SPA-810 (1:1000) mAbs.
Supplementary information for the Methods section is available online at http://atvb.ahajournals.org.
| Results |
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Hsp Expression Becomes Extensive as Atherogenesis Progresses in 8-Week-Old ApoE-/- Mice
The earliest cellular change in 8-week-old apoE-/- mice was the adherence of mononuclear cells to endothelia throughout the arterial tree at lesion-prone sites. Expression of hsp60 and hsp70 became intense at aortic valve commissures and on endothelial, intimal, and adventitial regions of early fatty streaks of the free aortic wall (Figure 1e, 1g, and 1h) in the sinus region. Oil red O staining of the latter regions revealed multiple foam cell deposits on valve commissures and pockets and also in the subendothelial region in the form of multilayered foam cell deposits in the wall of the aortic root region (Figure 1i). No obvious cellular changes in the medial layers of the free aortic wall were observed. There was no staining when primary antibodies were omitted (Figure 1f). Nascent plaques/early fatty streaks strongly expressing hsp60 were beginning to develop (Figure 1j), and recently developed intimal thickenings in the proximal ascending aorta had attached mononuclear cells highly expressing hsp70 (Figure 1k) and hsp60 (data not shown). Neighboring ECs and medial layers occasionally expressed both hsps. The majority of hsp-positive mononuclear cells adhering to aortic endothelium were monocytes/macrophages (Figure 1l).
Hsp60 and Hsp70 Expression Is Heterogeneous in Advanced Plaques of 20-Week-Old ApoE-/- Mice
In mice aged 20 weeks, lesions had progressed from intermediate fibrofatty plaques containing multiple layers of lipid-filled macrophages and SMCs to advanced lesions displaying a heterogenous pattern of hsp60 and hsp70 expression. Hsp60 and hsp70 expression patterns were identical. Both hsps were strongly expressed in the subendothelial region, fibrous caps, and areas surrounding necrotic cores, with expression extending to the medial layer underlying necrotic cores and shoulder regions of advanced plaques in the aortic sinus (Figure 2a; data for hsp60 are not shown). Lesional expression in the remainder of the aorta was similar to that of the aortic root. Staining of tissue sections detected extracellular hsp60 and hsp70 components (Figure 2a, 2c, and 2d), particularly in necrotic regions, in agreement with the fact that hsps can be secreted by cells and that human hsp70 has extracellular and intracellular locations in human plaques.7 Staining was specific; there was no staining after omission of the primary antibody (Figure 2b). The medial layer of the abdominal aorta was not always positive for hsps (Figure 2c and 2d). Advanced lesion areas, characterized by calcification foci and osteoclast-like cells, lacked hsp expression, whereas hsp positivity was seen in adventitia (Figure 2e; data for hsp70 are not shown).
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Downregulation of Hsp Expression in Advanced Plaques of 40- and 69-Week-Old ApoE-/- Mice
The aortas of Western dietfed 40-week-old and chow dietfed 69-week-old apoE-/- mice had advanced complicated plaques containing large necrotic cores and acellular areas that were collagenous and highly calcified. Hsp60 and hsp70 could not be detected in the developed calcium foci of 40-week-old mice (data not shown), and there was only weak and patchy expression in lesions of the aortic sinus and remainder of the aorta (Figure 2f and 2g). The aortic sinus lesions of 69-week-old mice similarly lacked hsp expression (Figure 2h and 2i), and there was only weak to patchy expression in lesions of the abdominal aorta (Figure 2j; data for hsp60 are not shown). There was a general geographic coincidence between intimal/plaque lipid accumulation and hsp60 and hsp70 expression in the aortic lesions of 20-, 40-, and 69-week-old-mice. Rarely was there intraplaque necrosis or lipid accumulation without hsp immunostaining (data not shown).
Control aortae from normocholesterolemic, wild-type apoE+/+ mice were lesion free, had a normal histology, and lacked expression of hsp60 and hsp70 at all ages examined, as illustrated for 20-week-old mice in Figure 2k.
Western Blotting of Aortic Tissue Homogenates Confirms Hsp Levels
Western blot analysis of aortic tissue homogenates of pooled lesion-prone and lesioned sites of 3-, 8-, and 20-week-old apoE-/- mice revealed strong single bands of 60 and 70 kDa (Figure 3, lanes b; top and bottom panels, respectively) after staining with the hsp-specific mAbs. In contrast, neither hsp60 nor hsp70 was detected in nonlesioned distal abdominal aortic tissue of 3- and 8-week-old mice (Figure 3, lanes d). In accord, expression of hsp60 and hsp70 was downregulated in the lesions of 40- and 69-week-old mice (Figure 3, lanes c). A lysate of heat-shocked EL-4 T-lymphoma cells served as a positive control, generating strong bands of 60 and 70 kDa (Figure 3, lanes a), whereas unstressed EL-4 cells lacked expression (Figure 3, lanes e). Hsp60 and hsp70 were not detected in aortic tissue homogenates pooled from the aortas of normocholesterolemic wild-type apoE+/+ mice (Figure 3, lanes f).
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Lesional ECs, SMCs, Monocytes/Macrophages, and CD3+ T Lymphocytes Express Hsp60 and Hsp70
Endothelia
Double staining of aortic sections of 3-week-old mice with the different anti-hsp mAbs and an anti-CD31 mAb revealed that hsp70 (Figure 4a and 4b) and hsp60 (data not shown) were expressed by ECs. This result was confirmed by using a mAb against factor VIIIrelated antigen (Figure 4c; data for hsp70 are not shown). As evident from Figure 4c, endothelial hsp expression precedes cellular attachment/infiltration. Lesion-prone sites such as the lesser curvature of the aortic arch displayed strong endothelial hsp60 expression, whereas nonlesion-prone sites of the distal abdominal aorta lacked hsp expression (Figure 4d).
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SMCs
Migratory SMCs were occasionally detected at developing lesion sites of 20-week-old mice, and some of these cells (stained orange) expressed hsp70 (Figure 4; compare panels e and f) and hsp60 (data not shown). Small numbers of SMCs were detected in and around the necrotic core of advanced lesions, with several (stained orange) expressing hsp60 (Figure 4g and 4h) and hsp70 (data not shown). Certain SMC-rich areas underlying the necrotic core and fibrous cap regions of advanced lesions exhibited increased expression of hsp60 and hsp70.
Monocytes/Macrophages
Monocytes/macrophages expressing hsp70 (Figure 5a through 5c) and hsp60 (data not shown) were the most prominent cell type in lesions. Cells doubly positive for the hsps and MOMA-2 were seen distributed around the necrotic core, shoulder, fibrous cap, and subendothelial regions of advanced plaques.
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T Cells
Small numbers of hsp60 (Figure 5d through 5f) and hsp70 (Figure 5g through 5i) expressing CD3+ T cells were seen preferentially located, often in clusters, in the subendothelium/fibrous cap, necrotic core (macrophage-rich area with intense hsp expression), and shoulder regions of plaques of 20-week-old mice.
| Discussion |
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The expression patterns of hsp60 and hsp70 were identical irrespective of lesion stage, as in human lesions.7 Atherosclerosis is an immune-based disease,18 and in accord, lesions were infiltrated with leukocytes. Aortic ECs, infiltrating and resident monocyte/macrophages, CD3+ T lymphocytes, and intimal and medial SMCs expressed hsps, indicating that all major lesional cell types were susceptible to the toxic environment of the atherosclerotic plaque. In murine models of atherosclerosis, atherogenesis preferentially starts at the aortic root,15 and lesions indicative of atherosclerosis have been described for human aortic and mitral valves.19 This could be due to mechanical stresses from turbulent blood flow patterns at this site,20 at which lipid is first deposited. In accord, areas within the vicinity of aortic valve commissures of 3-week-old apoE-/- mice were the first sites to express both hsps. Although hsp expression often correlated with oil red Odetectable lipid deposits on aortic valves, it also extended to the apparently lipid-free aortic wall adjacent to valve commissures. That lipid deposition necessarily precedes hsp upregulation cannot be discounted without sensitive methods to colocalize lipids with hsps. Rarely was there lipid accumulation without intense hsp60 and hsp70 staining; however, there were plaque areas displaying intense hsp60 and hsp70 staining without notable lipid deposition. Both hsps were heterogeneously expressed in intermediate fibrofatty and advanced necrotic fibrous-capped plaques of 20-week-old apoE-/- mice. Large, relatively acellular, collagenous areas of the lesions stained lightly for both hsps, whereas calcified foci and occasionally the medial layer adjacent to developing plaques lacked hsp expression. Lesions at various stages of evolution were observed in a single 69-week-old mouse, in which lesion development started at the aortic root and progressed distally. Hence, hsps represent disease markers that can be used to trace the disease as it progresses within the aortic tree. The mechanisms responsible for the decline of hsp expression in late-stage aortic lesions of aged mice are unknown. The acellular nature of large necrotic cores due to apoptosis21 and the cytolytic effects of anti-hsp65/60 and hsp70 autoantibodies22,23 provide an obvious explanation. Hsp70 expression also diminishes with aging in acute hypertension.24,25
The cellular stressors responsible for the induction of hsp60 and hsp70 in the aortas of apoE-/- mice are not known, but the initial insult is likely to be lipid deposition. ApoE-/- mice lack apoE, a glycoprotein ligand that mediates LDL receptor clearance of serum lipoproteins,26 and spontaneously develop hyperlipidemia, as in humans expressing dysfunctional apoE.27 LDL oxidized in the arterial wall is chemotactic for and enhances the adhesiveness of monocytes,28 activates T cells,29 and is cytotoxic.30 It induces the expression of hsp60 and hsp70 in several cell types.31,32
Hsp expression differs between different species, making comparisons complicated. The stress-inducible form of hsp70 investigated in the present study is not constitutively expressed in most species, except primates.33 In humans, unlike mice, it is homogeneously distributed throughout normal-appearing areas of the intima and media.7,8 Hence, studies of aortic hsp70 expression in apoE-/- mice are advantageous because they are not complicated by having to distinguish the constitutive versus inducible expression of hsp70. The expression of hsp70 in mice should directly mark aortic cells that are under stress. Nevertheless, the expression pattern of hsp70 in early fatty streaks of 8-week-old mice and in advanced fibroproliferative lesions of 20-week-old apoE-/- mice is largely in agreement with the expression pattern of hsp70 in human lesions. In humans, there appear to be major changes in the localization of hsp70 during atherosclerotic evolution rather than quantitative changes.7,8 Increased expression in diseased aortic regions is balanced by decreases in regions in which hsp70 is normally found. Hsp70 is poorly expressed in complicated, acellular, collagenous plaques,7,9 in accord with the present data. In humans, expression of hsp60 is correlated positively with atherosclerotic severity, with the highest levels of expression seen in the shoulder regions and around the necrotic core of atherosclerotic plaques,5 in accord with the present data.
Hsp60 and hsp70 have been incriminated in triggering several autoimmune/chronic inflammatory diseases.11 Data suggest a multifaceted role for hsps in atherosclerosis,4,31 a condition in which hsp70 is likely to be involved in cytoprotection9 and, conversely, in which hsp60 possibly acts as an autoantigen.3436 Circulating soluble hsp60 is associated with cardiovascular disease,37 and anti-hsp60 antibodies are associated with established human atherosclerosis, mediating endothelial cytotoxicity38 and being predictive of mortality.39 Autologous hsp60 has been identified as a danger signal to the innate immune system.40 Bacterial and autologous hsp60 stimulates human and mouse macrophages to release proinflammatory cytokines6,40 and to activate cell types found in aortic lesions, leading to the upregulation of cell adhesion molecules.41 Hsp70 potentially plays a dual role in atherosclerosis, serving as a chaperone and a cytoprotector to enhance the survival of arterial SMCs9 and acting as a novel cytokine to cause monocytes to express interleukin-1ß, interleukin-6, and tumor necrosis factor-
.42 Hence, as with hsp60, hsp70 released from necrotic lesional cells may stimulate the innate immune response to promote inflammation during atherogenesis. Serum titers of anti-hsp70 antibodies are raised in patients with cardiovascular diseases.23
In conclusion, hsp60 and hsp70 are disease markers that can be used to stage the progression of atherosclerosis. Modulating the expression of hsp60 and hsp70 may allow the roles of these hsps in atherogenesis to be identified. In this regard, the present study suggests that the apoE-/- mouse will be a useful model in which such a study could be undertaken.
| Acknowledgments |
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Received May 26, 2001; accepted September 6, 2001.
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