This Article Abstract Full Text (PDF) Data Supplement Alert me when this article is cited Alert me if a correction is posted Citation Map 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 Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kanwar, R. K. Articles by Krissansen, G. W. Search for Related Content PubMed PubMed Citation Articles by Kanwar, R. K. Articles by Krissansen, G. W. Related Collections Mechanism of atherosclerosis/growth factors Animal models of human disease Pathophysiology Other arteriosclerosis

(Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:1991.)
© 2001 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Temporal Expression of Heat Shock Proteins 60 and 70 at Lesion-Prone Sites During Atherogenesis in ApoE-Deficient Mice

Rupinder K. Kanwar; Jagat R. Kanwar; Dongmao Wang; Douglas J. Ormrod; Geoffrey W. Krissansen

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

Top Abstract Introduction Methods Results Discussion References

 
In the study, we investigate whether the expressions of heat shock protein (hsp)60 (a potential autoantigen) and the stress-inducible form of cytoprotector hsp70 are correlated with the development of atherosclerotic lesions in the aortic tree of apolipoprotein E–deficient (apoE^-/-) mice. The apoE^-/- mouse model is advantageous because the stress-inducible form of hsp70 is not constitutively expressed in mice, unlike primates; hence, tissues under stress can be clearly defined. Both mammalian hsps were detected newly expressed (before mononuclear cell infiltration) on aortic valves and endothelia at lesion-prone sites of 3-week-old apoE^-/- mice. In 8- and 20-week-old mice, they were strongly and heterogeneously expressed in early to advanced fibrofatty plaques, with levels correlating with lesion severity. Expression was markedly downregulated in advanced collagenous, acellular, calcified plaques of 40- and 69-week-old mice and was absent in control aortas of normocholesterolemic wild-type (apoE^+/+) mice. Western blot analysis of tissue homogenates confirmed the temporal expression of the hsps. Double immunostaining revealed that both hsps were expressed by lesional endothelial cells, macrophages, smooth muscle cells, and CD3⁺ T lymphocytes. This study provides evidence that hsp60 and hsp70 are temporally expressed on all major cell types in lesion-prone sites during atherogenesis, suggesting that few cells escape the toxic environment of the atherosclerotic plaque.

Key Words: atherosclerosis • heat shock proteins 60 and 70 • apoE-deficient mice • immunohistochemistry • aorta

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Heat shock proteins (hsps) have been implicated as antigenic targets for the activation of lesional T cells in atherosclerotic plaques.^1,2 Hsps are a family of highly conserved ubiquitous proteins that function as molecular chaperones and aid cells in coping with stressful environments.³ Atherosclerosis is a multifactorial disease characterized by inflammation and endothelial injury arising from infection, hemodynamic forces, oxidized LDL, dietary factors, toxins, and chemical insults, all of which can activate the stress response and induce hsps.⁴ Expression of mammalian hsp60^5,6 and hsp70^7–9 is increased in atherosclerotic lesions of humans. Antibodies against hsp60 are increased in the serum of patients with atherosclerosis and are associated with disease severity.¹⁰ Because of their inducibility, strong immunogenicity, and high interspecies homology, hsp60 and hsp70 have been incriminated in triggering autoimmune disease.¹¹

Given the widespread acceptance of the apoE-deficient (apoE^-/-) mouse^12–15 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

Top Abstract Introduction Methods Results Discussion References

 
Mice and Diets
ApoE^-/- mice were the progeny of breeding colonies described earlier^13,16; they were maintained on a Western-type diet (No. TD88137, Harlan Teklad). Retired breeders (69 weeks old) and normocholesterolemic wild-type (apoE^+/+) mice were maintained on a normal mouse chow diet (3.8% fat, diet 86, NRM Auckland). Experimental protocols were approved by the University of Auckland Animal Ethics Committee.

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-lysine–coated 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 Gill’s 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 phosphatase–conjugated 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 anti–human factor VIII-related antigen antibody (1:10, Biomeda). Immunoreactivity was visualized by use of a Vector ABC–alkaline 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 red–conjugated 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

Top Abstract Introduction Methods Results Discussion References

 
Hsp60 and Hsp70 Are Detectable in Aortas of ApoE^-/- Mice From 3 Weeks of Age
Total plasma cholesterol levels of 3-week-old apoE^-/- mice were <12.6 mmol/L. For Western diet–fed 8-, 20-, and 40-week-old mice and chow diet–fed 69-week-old mice, the levels were hypercholesterolemic (20.74 to 61.68 mmol/L). The aortas of 3-week-old apoE^-/- mice displayed normal histology, with an orderly arrangement of medial SMCs and no signs of mononuclear infiltration. Focal lipid deposition detected with oil red O staining was confined to aortic valve commissures (Figure 1a). Hsp60 and hsp70 were homogenously expressed in aortic valve commissures, extending to the immediate free aortic wall in aortic root sections. They were not detected on the free aortic wall distant to the valves (Figure 1b through 1d) or on the valve commissures of normocholesterolemic wild-type apoE^+/+ mice (data not shown; see below).

View larger version (70K): [in this window] [in a new window]   Figure 1. Hsp60 and hsp70 are detectable in the aortas of 3-week-old apoE-/- mice and are highly expressed in the aortas of 8-week-old mice. a through d, Illustrated are serial sections from 3-week-old apoE-/- mice revealing focal lipid deposition (a, stained red with oil red O) and expression of hsp60 (b, stained brown with SPA-806 mAb) and hsp70 (c, stained brown with the SPA-810 mAb) at valve commissures in the aortic sinus region, extending to the immediate free aortic wall. Hsp60 is not expressed in the free aortic wall distant to valve commissures (d). e through k, In 8-week-old apoE-/- mice, hsp70 was strongly expressed at valve commissures of the aortic sinus (e), extending to the free aortic wall and including expression by endothelial (black arrowheads) and intimal (white arrowhead) cells (g). Panel f is a negative control serial section of panel e, in which primary anti-hsp70 mAb was omitted. Hsp60 was strongly expressed at the aortic valve commissures, extending to subendothelial regions of the free aortic wall (h). Panel i is a serial section of panel h, revealing the presence of lipid-filled foam cells (stained with oil red O) on valve commissures, extending to the intima. Panel j shows hsp60 expression in mononuclear cells adhering to nascent plaques in the proximal ascending aorta and on adjacent endothelium of uninvolved aorta. Hsp70 is intensely expressed by adjacent ECs (arrowheads) and by mononuclear cells (arrow on right), adhering to the proximal ascending aortic wall (k). l, The majority of hsp-positive cells adhering to aortic endothelium are monocytes/macrophages. MOMA-2–positive cells (stained red) can be seen adhering to hsp60-positive endothelium (stained brown) in aortic lesions of 8-week-old mice. Counterstain was Gill’s hematoxylin. Original magnifications x100 (a through c and i), x63 (j through l), and x40 (d through h).

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).

View larger version (129K): [in this window] [in a new window]   Figure 2. Hsp60 and hsp70 are heterogeneously expressed in lesions of 20-week-old mice and downregulated in aged mice. a, Hsp70 (stained brown) is strongly expressed in the intimal, necrotic core, and medial layer regions of an advanced plaque in the aortic sinus of 20-week-old mice. b, There was no staining when primary anti-hsp mAbs were omitted. c and d, Hsp60 (c) and hsp70 (d) were strongly expressed in advanced plaques of the abdominal aorta of 20-week-old mice, whereas medial layers lack expression. e, In contrast, hsp60 is absent from advanced plaque regions containing osteoclast-like cells (arrow). f and g, In 40-week-old mice, there is weak and patchy expression of hsp60 (f) and hsp70 (g) in the large expanding necrotic cores of chronic late-stage plaques of the aortic sinus. h through j, In 69-week-old mice, there is complete loss of hsp60 (h) and hsp70 (i) in advanced complicated collagen-rich plaques of the aortic sinus, whereas advanced plaques in the abdominal aorta show weak and patchy hsp70 expression (j). k, A representative aortic section from a 20-week-old normocholesterolemic apoE+/+ mouse displayed no hsp60 expression. Counterstain was Gill’s hematoxylin. Original magnifications x100 (a, g, h, and k), x40 (b through f), and x63 (i and j).

Downregulation of Hsp Expression in Advanced Plaques of 40- and 69-Week-Old ApoE^-/- Mice
The aortas of Western diet–fed 40-week-old and chow diet–fed 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).

View larger version (99K): [in this window] [in a new window]   Figure 3. Western blot analysis of aortic tissue homogenates. Homogenates of heat-shocked (1 hour at 42°C) EL-4 cells (lanes a), pooled lesion-prone and lesioned aortic segments from 3-, 8-, and 20-week-old apoE-/- mice (lanes b), pooled lesioned aortic segments from 40- and 69-week-old mice (lanes c), non–lesion-prone aortic segments from 3- and 8-week-old mice (lanes d), unstressed EL-4 cells (lanes e), and pooled aortic segments from 3-, 8-, and 20-week-old apoE+/+ mice (lanes f) were Western-blotted with SPA-806 (top) or SPA-810 (bottom) mAbs.

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 VIII–related 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 non–lesion-prone sites of the distal abdominal aorta lacked hsp expression (Figure 4d).

View larger version (82K): [in this window] [in a new window]   Figure 4. Colocalization of hsps with ECs and SMCs. Aortic sections from 3-week-old (a through d) and 20-week-old (e through h) apoE-/- mice were double-immunostained with mAbs against hsps and with markers for ECs (CD31 and factor VIII–related antigen [factor VIII RA], a through d) and SMCs (-SMC actin, e through h). a and b, Confocal images of an aortic arch section stained by double immunofluorescence for CD31 (a, green) and hsp70 (b, red). c and d, A lesion-prone site in the aortic arch (c) and a non–lesion-prone site in the distal abdominal aorta not expressing hsp60 (hsp60-ve) (d) double-stained for hsp60 (brown) and factor VIII RA (magenta red). Endothelial cells at the lesion-prone site were stained orange because of colocalization of hsp60 and factor VIII RA. e through h, Early (e and f) and late (g and h) lesions of aortic arch sections double-stained for hsps (brown) and -SMC actin (magenta red). SMCs expressing hsps are colored orange (compare with a single-arrowed orange cell in panel e). Panel f is near serial section of panel e, in which anti–-SMC actin mAb was omitted. Panel h is enlarged view of the rectangular portion of panel g. Counterstain was Gill’s hematoxylin. Magnifications x100 (a through f), x40 (g), and x80 (h).

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.

View larger version (96K): [in this window] [in a new window]   Figure 5. Colocalization of hsp60 and hsp70 with monocytes/macrophages and T cells. Shown are confocal images of an aortic arch section from 20-week-old apoE-/- mice, stained by double immunofluorescence for hsp60, hsp70, monocytes/macrophages, and T cells. a and b, Staining of monocytes/macrophages with MOMA-2 mAb (b, green) and anti-hsp70 mAb SPA-810 (a, red). c, Merging of images a and b. Abundant monocyte/macrophages expressing hsp70 were visualized as yellow-colored cells. d, e, g, and h, An aortic arch section from 20-week-old apoE-/- mice was double-stained for hsp60 (d, red), hsp70 (g, red), and T cells (e and h, green). f and i, Merging of images d and e (f ) and images g and h (i). Small numbers of T-cell clusters expressing hsp60 and hsp70 could be visualized as yellow-colored cells (arrowed). Original magnification x40. L indicates lumen of the aorta.

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

Top Abstract Introduction Methods Results Discussion References

 
The present study provides the first evidence that expression of hsp60 and hsp70 is strongly upregulated very early at lesion-prone sites in the aortas of young apoE^-/- knockout mice and then dramatically downregulated in the chronic lesions of aged mice. In contrast, the aortas of 3- to 69-week-old normocholesterolemic apoE^+/+ mice remained free of hsps. The apoE^+/+ mice were shelved next to apoE^-/- mice, indicating that increased hsp expression in apoE^-/- mice is a result of their hypercholesterolemia and is not due to an infectious agent. In accord, atherosclerosis in germ-free apoE^-/- mice is no different in animals raised with ambient levels of microbial challenge.¹⁷

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 O–detectable 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 autoantibodies^22,23 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.^34–36 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 cytokines^6,40 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

 
This work was supported in part by grants from the Heart Foundation of New Zealand, the Lottery Grants Board of New Zealand, the University of Auckland, and the Royal Society of New Zealand. G.W.K. was supported by a James Cook Research Fellowship funded by the Royal Society of New Zealand. The authors are grateful to Dr Annemarie Walsh and Susan Powell-Hayre of the Rockefeller University, New York, for the supply of apoE^+/- mouse embryos. We wish to thank the staff of the Biomedical Imaging Unit, Faculty of Medicine and Health Science, University of Auckland, and, in particular, Hilary Holloway for their support.

Received May 26, 2001; accepted September 6, 2001.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Libby P, Hansson GK. Involvement of the immune system in human atherogenesis: current knowledge and unanswered questions. Lab Invest. 1991; 64: 5–15.[Medline] [Order article via Infotrieve]
2. Wick G, Schett G, Amberger A, Kleindienst R, Xu Q. Is atherosclerosis an immunologically mediated disease? Immunol Today. 1995; 16: 27–33.[Medline] [Order article via Infotrieve]
3. Morimoto RI, Kline MP, Bimston DN, Cotto JJ. The heat shock response: regulation and function of heat shock proteins and molecular chaperones. Essays Biochem. 1997; 32: 17–29.[Medline] [Order article via Infotrieve]
4. Roma P, Catapano AL. Stress proteins and atherosclerosis. Atherosclerosis. 1996; 127: 147–154.[Medline] [Order article via Infotrieve]
5. Kleindienst R, Xu Q, Willeit J, Waldenberger FR, Weimann S, Wick G. Immunology of atherosclerosis: demonstration of heat shock protein 60 expression and T lymphocytes bearing /ß or / receptor in human atherosclerotic lesions. Am J Pathol. 1993; 142: 1927–1937.[Abstract]
6. Kol A, Sulhova GK, Licthman A, Libby P. Chlamydial heat shock protein 60 localizes in human atheroma and regulates macrophage tumor necrosis factor- and matrix metalloproteinase expression. Circulation. 1998; 98: 300–307.[Abstract/Free Full Text]
7. Berberian PA, Myers W, Tytell M, Challa V, Bond MG. Immunohistochemical localization of heat shock protein-70 in normal appearing and atherosclerotic specimens of human arteries. Am J Pathol. 1990; 136: 71–80.[Abstract]
8. Johnson AD, Berberian PA, Tytell M, Bond MG. Atherosclerosis alters the localization of HSP70 in human and macaque aortas. Exp Mol Pathol. 1993; 58: 155–168.[Medline] [Order article via Infotrieve]
9. Johnson AD, Berberian PA, Tytell M, Bond MG. Differential distribution of 70-kD heat shock protein in atherosclerosis: its potential role in arterial SMC survival. Arterioscler Thromb Vasc Biol. 1995; 15: 27–36.[Abstract/Free Full Text]
10. Zhu JH, Quyyumi AA, Rott D, Csako G, Wu HS, Halcox J, Epstein SE. Antibodies to human heat-shock protein 60 are associated with the presence and severity of coronary artery disease: evidence for an autoimmune component of atherogenesis. Circulation. 2001; 103: 1071–1075.[Abstract/Free Full Text]
11. Zugel U, Kaufmann SHE. Role of heat shock proteins in protection from and pathogenesis of infectious diseases. Clin Microbiol Rev. 1999; 12: 19–39.[Abstract/Free Full Text]
12. Smith JD, Breslow JL. The emergence of mouse models of atherosclerosis and their relevance to clinical research. J Intern Med. 1997; 242: 99–109.[Medline] [Order article via Infotrieve]
13. Plump AS, Smith JD, Hayek T, Alatosetala K, Walsh A, Verstuyft JG, Rubin EM, Breslow JL. Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells. Cell. 1992; 71: 343–353.[Medline] [Order article via Infotrieve]
14. Zhang SH, Reddick RL, Piedrahita JA, Maeda N. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science. 1992; 258: 468–471.[Abstract/Free Full Text]
15. Nakashima Y, Plump AS, Raines EW, Breslow JL, Ross R. ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree. Arterioscler Thromb. 1994; 14: 133–140.[Abstract/Free Full Text]
16. Ormrod DJ, Holmes CC, Miller TE. Dietary chitosan inhibits hypercholesterolaemia and atherogenesis in apolipoprotein E-deficient mouse model of atherosclerosis. Atherosclerosis. 1998; 138: 329–334.[Medline] [Order article via Infotrieve]
17. Wright SD, Burton C, Hernandez M, Hassing H, Montenegro J, Mundt S, Patel S, Card DJ, Hermanowski-Vosatka A, Bergstrom JD, et al. Infectious agents are not necessary for murine atherogenesis. J Exp Med. 2000; 191: 1437–1441.[Abstract/Free Full Text]
18. Hansson GK. T cells, specific immunity and atherosclerosis. Immunol News. 1999; 6: 33–35.
19. Walton KW, Williamson N, Johnson AG. The pathogenesis of atherosclerosis of the mitral and aortic valves. J Pathol. 1970; 101: 205–220.[Medline] [Order article via Infotrieve]
20. Peacock JA. An in vitro study of the onset of turbulence in the sinus of Valsalva. Circ Res. 1990; 67: 448–460.[Abstract/Free Full Text]
21. Crisby M, Kallin B, Thyberg J, Zhivotovsky B, Orrenius S, Kostulas V, Nilsson J. Cell death in human atherosclerotic plaques involves both oncosis and apoptosis. Atherosclerosis. 1997; 130: 17–27.[Medline] [Order article via Infotrieve]
22. Schett G, Metzler B, Mayr M, Amberger A, Niederwieser D, Gupta RS, Mizzen L, Xu Q, Wick G. Macrophage-lysis mediated by autoantibodies to heat shock protein 65/60. Atherosclerosis. 1997; 128: 27–38.[Medline] [Order article via Infotrieve]
23. Chan YC, Shukla N, Abdus-Samee M, Berwanger CS, Stanford J, Singh M, Mansfield AO, Stansby G. Anti-heat-shock protein 70 kDa antibodies in vascular patients. Eur J Vasc Endovasc Surg. 1999; 18: 381–385.[Medline] [Order article via Infotrieve]
24. Xu Q, Li DG, Holbrook NJ, Udelsmann R. Acute hypertension induces heat shock protein 70 gene expression in rat aorta. Circulation. 1995; 92: 1223–1229.[Abstract/Free Full Text]
25. Holbrook NJ, Udelsmann R. Heat shock protein gene expression in response to physiologic stress and aging.In: Morimoto RI, Tissieres A, Georgopoulous C, eds. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1994: 577–593.
26. Plump AS, Breslow JL. Apolipoprotein E and the apolipoprotein E-deficient mouse. Annu Rev Nutr. 1995; 15: 495–518.[Medline] [Order article via Infotrieve]
27. Walden CC, Hegele RA. Apolipoprotein E in hyperlipidemia. Ann Intern Med. 1994; 120: 1026–1036.[Abstract/Free Full Text]
28. Frostegard J, Nilsson J, Haegerstrand A, Hamsten A, Wigzell H, Gidlund M. Oxidized low-density lipoprotein induces differentiation and adhesion of human monocytes and the monocytic cell line U937. Proc Natl Acad Sci U S A. 1990; 87: 904–908.[Abstract/Free Full Text]
29. Frostegard J, Wu R, Giscombe R, Holm G, Lefvert AK, Nilsson J. Induction of T cell activation by oxidized low density lipoprotein. Arterioscler Thromb. 1992; 12: 461–467.[Abstract/Free Full Text]
30. Hessler JR, Morel DW, Lewis LJ, Chisholm GM. Lipoprotein oxidation and lipoprotein-induced cytotoxicity. Arteriosclerosis. 1983; 3: 215–222.[Abstract/Free Full Text]
31. Johnson AD. Heat shock proteins in atherosclerosis.In: Latchman DS, ed. Handbook of Experimental Pharmacology: Stress proteins, Volume 136: Heat Shock Proteins in Atherosclerosis. Berlin, Germany/New York, NY: Springer; 1999: 381–402.
32. Frostegard J, Kjellman B, Gidlung M, Andersson B, Jindal S, Kiessling R. Induction of heat shock protein in monocytic cells by oxidized low density lipoprotein. Atherosclerosis. 1996; 121: 93–103.[Medline] [Order article via Infotrieve]
33. Welch WJ. The mammalian stress response: cell physiology and biochemistry of stress proteins.In: Morimoto RI, Tissieres A, Geogopoulos C, eds. Stress Proteins in Biology and Medicine. Cold Spring Harbor, NY: Cold Spring Harbor Press; 1990: 223–278.
34. Xu Q, Dietrich H, Steiner HJ, Gown AM, Schoel B, Mikuz G, Kaufmann SHE, Wick G. Induction of atherosclerosis in normocholesterolemic rabbits by immunization with heat shock protein 65. Arterioscler Thromb. 1992; 12: 789–799.[Abstract/Free Full Text]
35. Afek A, George J, Gilburd B, Rauova L, Goldberg I, Kopolovic J, Harats D, Shoenfeld Y. Immunization of low-density lipoprotein receptor deficient (LDL-RD) mice with heat shock protein 65 (HSP-65) promotes early atherosclerosis. J Autoimmun. 2000; 14: 115–121.[Medline] [Order article via Infotrieve]
36. George J, Shoenfeld Y, Afek A, Gilburd B, Keren P, Shaish A, Kopolovic J, Wick G, Harats D. Enhanced fatty streak formation in C57BL/6J mice by immunization with heat shock protein-65. Arterioscler Thromb Vasc Biol. 1999; 19: 505–510.[Abstract/Free Full Text]
37. Pockley AG, Wu R, Lemne C, Kiessling R, de Faire U, Frostegard J. Circulating heat shock protein 60 is associated with early cardiovascular disease. Hypertension. 2000; 36: 303–307.[Abstract/Free Full Text]
38. Schett G, Amberger A, Van der Zee R, Recheis H, Willeit J, Wick G. Autoantibodies against heat shock protein 60 mediate endothelial cytotoxicity. J Clin Invest. 1995; 96: 2569–2577.[Medline] [Order article via Infotrieve]
39. Xu Q, Kiechl S, Mayr M, Metzler B, Egger G, Oberhollenzer F, Willeit J, Wick G. Association of serum antibodies to heat shock protein 65 with carotid atherosclerosis: clinical significance determined in a follow up study. Circulation. 1999; 100: 1169–1174.[Abstract/Free Full Text]
40. Chen W, Syldath U, Bellmann K, Burkat V, Kolb H. Human 60-kDa heat shock protein: a danger signal to the innate immune system. J Immunol. 1999; 162: 3212–3219.[Abstract/Free Full Text]
41. Kol A, Bourcier T, Licthman AH, Libby P. Chlamydial and human heat shock protein 60s activate human vascular endothelium, smooth muscle cells and macrophages. J Clin Invest. 1999; 103: 571–577.[Medline] [Order article via Infotrieve]
42. Asea A, Kraeft SK, Kurt-Jones EA, Stevenson MA, Chen LB, Finberg RW, Koo GC, Calderwood SK. HSP70 stimulates cytokine production through a CD14 dependent pathway, demonstrating its dual roles as a chaperone and cytokine. Nat Med. 2000; 6: 435–442.[Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				J. Herrmann, S. M. Soares, L. O. Lerman, and A. Lerman Potential Role of the Ubiquitin-Proteasome System in Atherosclerosis: Aspects of a Protein Quality Disease J. Am. Coll. Cardiol., May 27, 2008; 51(21): 2003 - 2010. [Abstract] [Full Text] [PDF]

				H. Nakashima, G. D. Frank, H. Shirai, A. Hinoki, S. Higuchi, H. Ohtsu, K. Eguchi, A. Sanjay, M. E. Reyland, P. J. Dempsey, et al. Novel Role of Protein Kinase C-{delta} Tyr311 Phosphorylation in Vascular Smooth Muscle Cell Hypertrophy by Angiotensin II Hypertension, February 1, 2008; 51(2): 232 - 238. [Abstract] [Full Text] [PDF]

				R. RIGANỎ, E. PROFUMO, B. BUTTARI, A. TAGLIANI, L. PETRONE, G. D'AMATI, F. IPPOLITI, R. CAPOANO, L. FUMAGALLI, B. SALVATI, et al. Heat Shock Proteins and Autoimmunity in Patients with Carotid Atherosclerosis Ann. N.Y. Acad. Sci., June 1, 2007; 1107(1): 1 - 10. [Abstract] [Full Text] [PDF]

				A. Shamaei-Tousi, J. P. Halcox, and B. Henderson Stressing the obvious? Cell stress and cell stress proteins in cardiovascular disease Cardiovasc Res, April 1, 2007; 74(1): 19 - 28. [Abstract] [Full Text] [PDF]

				M. Ghayour-Mobarhan, S. A New, D. J Lamb, B. J Starkey, C. Livingstone, T. Wang, N. Vaidya, and G. A Ferns Dietary antioxidants and fat are associated with plasma antibody titers to heat shock proteins 60, 65, and 70 in subjects with dyslipidemia Am. J. Clinical Nutrition, May 1, 2005; 81(5): 998 - 1004. [Abstract] [Full Text] [PDF]

				A.H. Schoneveld, M.M. Oude Nijhuis, B. van Middelaar, J.D. Laman, D.P.V. de Kleijn, and G. Pasterkamp Toll-like receptor 2 stimulation induces intimal hyperplasia and atherosclerotic lesion development Cardiovasc Res, April 1, 2005; 66(1): 162 - 169. [Abstract] [Full Text] [PDF]

				M. Ghayour-Mobarhan, D. J. Lamb, N. Vaidya, C. Livingstone, T. Wang, and G. A. A. Ferns Heat Shock Protein Antibody Titers Are Reduced by Statin Therapy in Dyslipidemic Subjects: A Pilot Study Angiology, January 1, 2005; 56(1): 61 - 68. [Abstract] [PDF]

				J. R. Kanwar, R. K. Kanwar, and G. W. Krissansen Simultaneous neuroprotection and blockade of inflammation reverses autoimmune encephalomyelitis Brain, June 1, 2004; 127(6): 1313 - 1331. [Abstract] [Full Text] [PDF]

				A. Khanna Concerted effect of transforming growth factor-{beta}, cyclin inhibitor p21, and c-myc on smooth muscle cell proliferation Am J Physiol Heart Circ Physiol, March 1, 2004; 286(3): H1133 - H1140. [Abstract] [Full Text] [PDF]

				B. Gao and M.-F. Tsan Recombinant Human Heat Shock Protein 60 Does Not Induce the Release of Tumor Necrosis Factor {alpha} from Murine Macrophages J. Biol. Chem., June 13, 2003; 278(25): 22523 - 22529. [Abstract] [Full Text] [PDF]

				B. Metzler, R. Abia, M. Ahmad, F. Wernig, O. Pachinger, Y. Hu, and Q. Xu Activation of Heat Shock Transcription Factor 1 in Atherosclerosis Am. J. Pathol., May 1, 2003; 162(5): 1669 - 1676. [Abstract] [Full Text] [PDF]

				Z. Han, Q. A. Truong, S. Park, and J. L. Breslow Two Hsp70 family members expressed in atherosclerotic lesions PNAS, February 4, 2003; 100(3): 1256 - 1261. [Abstract] [Full Text] [PDF]

				C. Patterson and D. Cyr Welcome to the Machine: A Cardiologist's Introduction to Protein Folding and Degradation Circulation, November 19, 2002; 106(21): 2741 - 2746. [Full Text] [PDF]

				Q. Xu Role of Heat Shock Proteins in Atherosclerosis Arterioscler. Thromb. Vasc. Biol., October 1, 2002; 22(10): 1547 - 1559. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Data Supplement Alert me when this article is cited Alert me if a correction is posted Citation Map 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 Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kanwar, R. K. Articles by Krissansen, G. W. Search for Related Content PubMed