© 1999 American Heart Association, Inc.
Original Contributions |
Enhanced Fatty Streak Formation in C57BL/6J Mice by Immunization With Heat Shock Protein-65
From the Research Unit of Autoimmune Diseases, Department of Medicine B (J.G., Y.S., B.G., P.K.), the Institute of Pathology (A.A., J.K.), and the Institute of Lipid and Atherosclerosis Research (P.K., A.S., D.H.), Sheba Medical Center, Tel Hashomer, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; and the Institute of Biomedical Aging Research, Austrian Academy of Sciences, and Institute for General and Experimental Pathology, University of Innsbruck Medical School, Innsbruck, Austria (G.W.).
Correspondence and reprint requests to Prof Yehuda Shoenfeld, Department of Medicine `B,' Sheba Medical Center, Tel-Hashomer 52621, Israel. E-mail shoenfel{at}post.tau.ac.il
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract—Recent data suggest that the immune system is involved in atherogenesis. Thus, interest has been raised as to the possible antigens that could serve as the initiators of the immune reaction. In the current work, we studied the effects of immunization with recombinant heat shock protein-65 (HSP-65) and HSP-65–rich Mycobacterium tuberculosis (MT) on early atherogenesis in C57BL/6J mice fed either a normal chow diet or a high-cholesterol diet (HCD). A rapid, cellular immune response to HSP-65 was evident in mice immunized with HSP-65 or with MT but not in the animals immunized with phosphate-buffered saline (PBS) alone. Early atherosclerosis was significantly enhanced in HCD-fed mice immunized with HSP-65 (n=10; mean aortic lesion size, 45 417±9258 µm2) or MT (n=15; 66 350±6850 µm2) compared with PBS-injected (n=10; 10 028±3599 µm2) or nonimmunized (n=10; 9500±2120 µm2) mice. No fatty streak lesions were observed in mice fed a chow diet regardless of the immunization protocol applied. Immunohistochemical analysis of atherosclerotic lesions from the HSP-65– and MT-immunized mice revealed infiltration of CD4 lymphocytes compared with the relatively lymphocyte-poor lesions in the PBS-treated or nonimmunized mice. Direct immunofluorescence analysis of lesions from HSP-65– and MT-immunized mice fed an HCD exhibited extensive deposits of immunoglobulins compared with the fatty streaks in the other study groups, consistent with the larger and more advanced lesions found in the former 2 groups. This model, which supports the involvement of HSP-65 in atherogenesis, furnishes a valuable tool to study the role of the immune system in atherogenesis.
Key Words: atherosclerosis • autoantibodies • heat shock protein-65 • oxidized LDL • Mycobacterium tuberculosis
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Atherosclerosis is a multifactorial process, the hallmark of which is an accumulation of lipids within the vessel wall, accompanied by mononuclear cell infiltration and proliferation of smooth muscle cells.1 In recent years, it has become apparent that the immune system plays an important role in the earliest and subsequent stages of atherosclerosis, as manifested by the finding of activated T cells and immunoglobulins within the lesions at different stages.2 3 4 5 These observations have prompted the search for possible (auto)antigenic candidates against which a local immune response is presumed to be triggered. The 2 major immunogens proposed by different groups are heat shock protein (HSP)-60/65 and oxidized LDL (oxLDL).5 6 7 8
The association of oxLDL with accelerated atherosclerosis has been suggested by direct evidence pointing to its existence within atherosclerotic plaques in humans and animals.9 10 Furthermore, circumstantial data suggest that T-lymphocyte clones extracted from human atherosclerotic lesions recognize oxLDL.11 However, experimental studies in animals indicate that immunization with homologous oxLDL elicits a protective, rather than a deleterious, effect on the development of atherosclerosis and neointimal formation.12 13 14 15
Probably the most illustrative evidence for the existence of an autoimmune reaction in atherosclerosis was provided in recent work by Wick and coworkers (Xu et al16 ), showing that normocholesterolemic rabbits immunized with recombinant HSP-65 or with Mycobacterium tuberculosis (MT; an HSP-65–rich bacterium) developed arteriosclerosis when fed a normal chow diet. These same authors have subsequently reinforced these findings by showing increased expression of HSP-60 within atherosclerotic lesions17 and documenting increased humoral response to the antigen in humans with carotid atherosclerosis and coronary heart disease.18 19
The aim of the present study was to extend these findings to a mouse model known to be genetically more resistant to atherosclerosis than are rabbits and to study the nature of the immune response toward HSP-65 and oxLDL. The advantage of having a mouse model with accelerated atherosclerosis induced by immunization would be the ability to conduct controlled studies with a large number of animals to further elucidate the role of the immune system in atherogenesis. We show herein that C57BL/6J mice fed a high-cholesterol diet (HCD) develop significantly enhanced fatty streak accumulation when immunized with recombinant HSP-65 or with MT containing the HSP-65.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Mice
Female C57BL/6J mice, 8 weeks old, were purchased from Tel Aviv University, Tel Aviv, Israel.
Diets
Mice were fed either an HCD (containing 1.25%
cholesterol and 0.5% sodium cholate; TD 88051, Harlan
Teklad) or a conventional mouse diet (chow). The diet and water
were provided ad libitum. During the study, mice remained healthy, as
evidenced by coat condition and body weight gain.
Antigens and Reagents: Recombinant HSP-65
Antigens and reagents for recombinant HSP-65 were purchased from
Dr M. Singh, Braunschweig, Germany.
Adjuvant
Incomplete Freund's adjuvant (IFA; HSP-65–free) was obtained
from DIFCO Laboratories and was fortified by adding 5 mg/mL MT, and the
mixture was sonicated on ice before emulsification.
Preparation of LDL and OxLDL
Blood for lipoprotein isolation was collected in EDTA from mice
after 12 hours of fasting. LDL (density=1.019 to 1.063 g/mL) was
isolated from plasma as previously described,20 after
density adjustment with added KBr, by preparative
ultracentrifugation at 50 000 rpm/min for 22 hours
with a type 50 rotor. LDL preparations were washed by
ultracentrifugation, dialyzed against 0.15 mol/L EDTA
(pH 7.4), passed through an Acrodisc filter (0.22-µm pore size) to
remove aggregates, and stored under N2 in the
dark.
LDL oxidation was performed by incubation of predialyzed LDL (1 mg protein/mL in EDTA-free PBS) with CuSO4 (10 µmol/L) for 24 hours at 37°C. Lipoprotein oxidation was confirmed by analysis of thiobarbituric acid–reactive substances, which measures malondialdehyde (MDA) equivalents by the lipid peroxidation test, and also by analysis of the conjugated-diene content of the lipoproteins.
Experimental Design
A pilot study performed before the main experiment indicated
that immunization of C57BL/6J mice with either HSP-65 or MT did not
lead to fatty streak formation when mice were fed a chow diet. Thus, in
the main experiment, most of the animals were fed the HCD. Mice were
immunized subcutaneously 3 times (every 3 weeks) with either HSP-65
(10 µm/dose), MT (5 mg/mL), or PBS, all emulsified in the
HSP-65–free IFA. The total volume injected into each mouse at each
immunization was 100 µL. An additional control group consisted of
nonimmunized, HCD-fed mice. The quantities used for immunization were
determined by following a preliminary experiment that had shown that
these were the minimum doses causing an effect on
atherosclerosis progression. A summary of the
experimental groups is given in Table 1
. The mice were fed either a chow
diet or an HCD, starting from the initial immunization, and all were
killed humanely 15 weeks later by cervical translocation. The
experimental protocol was approved by the Animal Subjects Committee of
our institute.
|
Cholesterol Levels
At the end of the experiment, 1 to 1.5 mL of blood was obtained
by cardiac puncture; 1000U/mL heparin was added to each sample. Total
plasma cholesterol levels were determined by using an
automated enzymatic technique (Boehringer Mannheim).
Proliferation Assays of Draining Lymph Node Lymphocytes From
Immunized Mice
Draining inguinal lymph nodes were collected from 4
HSP-65–, 4MT-, and 4PBS-immunized mice killed 11 days after the
primary immunization. The assays were performed as previously
described21 with minor modifications. In brief,
106 cells/mL were incubated in triplicate for 72
hours in 0.2 mL of culture medium in microtiter wells in the presence
of 2 or 10 µg/mL HSP-65. Cell proliferation was measured by the
incorporation of [3H]thymidine into DNA during
the final 12 hours of incubation. The results were computed as a
stimulation index (SI), as the ratio of the mean counts per minute
(cpm) of the antigen to the mean background cpm obtained in the absence
of the antigen. The SDs were always<10% of the mean cpm.
Detection of Anti–HSP-65 Antibodies
Recombinant HSP-65 (1 µg/mL) in PBS (pH 7.2) was coated onto
flat-bottom, 96-well ELISA plates (Nunc Maxisorp) by overnight
incubation at 4°C as previously described.16 After
washing with 0.02% PBS–Tween and blocking with 1% BSA in PBS, sera
were added in different dilutions (1:50, 1:100, and 1:200 in PBS) and
incubated for 1 hour at room temperature. Peroxidase-conjugated rabbit
anti-mouse IgG (Dako Ltd, High Wycombe, UK) was added, and incubation
was continued for 1 hour at room temperature, followed by 4 washes with
PBS/Tween. Finally, 100 µL of citrate phosphate buffer (0.1 mol/L, pH
4.2) containing 0.53 mg/mL
2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Sigma Chemical
Co) was added, and absorbance was measured after 30 minutes at 490 nm
in a Titertek ELISA reader.
Inhibition assays were performed to confirm the specificity of anti–HSP-65 antibodies and to check for a possible cross-reactivity with oxLDL, an important immunogen in atherosclerosis. The concentration of HSP-65–immunized mouse serum resulting in the half-maximal binding to HSP-65 was determined, and different inhibitors (ie, HSP-65, oxLDL, or BSA) were applied in increasing concentrations.
Detection of Anti-OxLDL Antibodies
Ninety-six-well polystyrene plates (Nunc) were coated with
either copper-oxidized LDL, native LDL (at a concentration of 5 µg/mL
in PBS), or PBS alone overnight at 4°C. After 4 washes with PBS
containing 0.05% Tween and 0.001% aprotinin (Sigma), the plates were
blocked with 2% BSA for 2 hours at room temperature. Serum fractions
were diluted to 1:50 in PBS–0.05% Tween–0.2% BSA and added to the
wells. After additional overnight incubation the plates were washed,
and alkaline phosphatase–conjugated goat anti-mouse IgG (Jackson
ImmunoResearch laboratories Inc), diluted 1:10 000 in PBS–0.05%
Tween–0.2% BSA, was added for 1 hour at room temperature. After
extensive washing, 1 mg/mL p-nitrophenyl phosphate (Sigma)
in 50 mmol/L carbonate buffer containing 1 mmol/L
MgCl2, pH 9.8, was added as a substrate. The
reaction was stopped after 30 minutes by adding 1 mol/L NaOH. The
optical density was read at a 405-nm wavelength in a Titertek ELISA
reader (SLT Laboratory Instruments). Levels of anti-oxLDL antibodies
were calculated as the level of binding to native LDL subtracted from
that for the binding to oxLDL.
Assessment of Atherosclerosis
Quantification of atherosclerotic fatty streak lesions was done
by calculation of lesions size in the aortic sinus as previously
described22 with a few modifications. In brief, the heart
and upper portion of the aorta were removed from the animals and the
peripheral fat was carefully removed. The upper section was
embedded in OCT medium and frozen. Every other section (10-µm thick)
throughout the aortic sinus (400 µm) was taken for
analysis. The distal portion of the aortic sinus is recognized
by the 3 valve cusps that form the junction of the aorta to the heart.
Sections were evaluated for fatty streak lesions after being stained
with oil red O. Lesion areas per section were counted on a grid by an
observer who was unfamiliar with the tested specimen.
Immunohistochemistry
Immunohistochemical staining (antibodies to mouse CD4, CD8a, and
macrophages; all from Serotek) of cryostat sections (5-µm
thick) of the aortic sinus was done as described
previously.22 For demonstration of oxLDL epitopes in the
lesions of the mice, the sections were fixed (with methanol and
subsequently with acetone) and blocked before incubation with mouse
monoclonal antibodies specific for MDA-modified lysines of LDL (MDA2;
obtained by immunization of BALB/c mice with MDA-LDL; a kind gift of Dr
S. Yla-Herttuala, Helsinki, Finland23 ). The assay
was performed using the Histomouse-SP bulk kit (Zymed Laboratories Inc)
for detection of mouse primary antibodies on mouse tissues. The results
were evaluated by light microscopy.
Direct Immunofluorescence to Detect Murine
Bound Antibodies
Mouse hearts were perfused with NaCl and quick-frozen in
LN2. Cryostat sections (5 µm) were stained
with FITC-conjugated, affinity-purified goat anti-mouse IgG and IgM
F(ab')2 (Sigma) and then examined under a
fluorescence microscope.
Statistical Analysis
Data are presented as mean±SEM. Statistical differences
were determined by 1-way ANOVA to determine interactions between
immunization groups. P<0.05 was accepted as statistically
significant.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Body Weights
Initial body weights for C57BL/6J mice were 22±2 g (mean±SEM). Final average body weights were 24 to 25 g. No statistically significant differences were found between weights in the various immunization groups at the end of the experiment.
Cholesterol Levels
Average cholesterol levels in the plasma of the
C57BL/6J mice fed an HCD were 2.549±0.226 mg/mL compared with their
littermates fed a chow diet (0.704±0.062 mg/mL). No differences in the
cholesterol levels were evident between the experimental
groups given the same diet.
Proliferative Responses of Draining Lymph Node Cells to
HSP-65
Significant SIs to 10 µg/mL HSP-65 were evident in both the
HSP-65– (7.9±0.55) and MT- (2.95±0.13) immunized mice but not in the
PBS-immunized mice (1.07±0.13; Figure 1). The results of the SI
measurements were also significant for lower concentrations of HSP-65
in the culture medium (2 µg/mL).
|
Antibodies to HSP-65
Anti–HSP-65 levels in the sera of chow-fed, MT-immunized and
HSP-65–immunized mice were higher than those in the chow-fed,
nonimmunized mice (Table 2). The
anti–HSP-65 levels in the HSP-65–immunized mice were considerably
higher than those in their MT-immunized littermates. Levels of HSP-65
antibodies were significantly lower in HCD-fed, MT-immunized mice
compared with their chow-fed, MT-immunized littermates.
|
BSA and oxLDL did not inhibit the binding of the antibodies (from
HSP-65–immunized mice) to HSP-65–coated plates, suggesting that the
response to HSP-65 may be specific and non–cross-reactive with other
potentially confounding antigens that may influence atherogenesis (ie,
oxLDL; Figure 2). A dose-dependent
inhibition of anti–HSP-65 binding to HSP-65–coated plates was
evident.
|
Antibodies to OxLDL
Levels of anti-oxLDL antibodies were significantly higher in the
chow-fed, MT-immunized mice; the HCD-fed, PBS-immunized mice; and the
nonimmunized mice fed an HCD compared with the HCD-fed mice immunized
with MT and the nonimmunized controls (Table 2
).
Early Development of Atherosclerosis in the
Aortic Sinuses
The mice that were fed a chow diet did not develop early fatty
streaks in their aortas regardless of the immunization protocol
applied. Mean aortic lesion size in the nonimmunized, HCD-fed mice was
9500±2120 µm2. Significantly enhanced
fatty streak formation, when compared with nonimmunized mice, was
evident in the HSP-65– (45 417±9258
µm2; P<0.05) and MT-
(66 350±6850 µm2; P<0.05)
immunized mice. Lesion development in the PBS-immunized mice
(10 028±3599 µm2 was similar to that in
their nonimmunized littermates (Figures 3 and 4).
|
|
Immunohistochemisty
Lesions from mice in all experimental groups were found to contain
macrophages. The lesions of HSP-65– and MT-immunized mice
appeared more mature and contained significantly more
macrophages. Fatty streaks in the aortic sinus sections from
the HCD-fed, MT- and HSP-65–immunized mice were found to possess
larger numbers of CD4+ lymphocytes (with minor amounts of CD8+
lymphocytes). Only low numbers of CD4+ and CD8+ lymphocytes were
evident in the lesions of the PBS-immunized or nonimmunized mice fed an
HCD.
More lesions from the HCD-fed, MT-immunized mice were found to exhibit MDA-LDL–specific epitopes, evident by staining with a monoclonal anti–MDA-LDL antibody. Direct immunofluorescence studies displayed significantly greater IgG deposition in the lesions from MT and HSP-65 immunization groups and in comparison with lesions taken from PBS-immunized or nonimmunized, HCD-fed mice. The larger quantities of lymphocytes and immunoglobulins found in the MT- and HSP-65–immunized mice were consistent with the more advanced lesions found in these groups.
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The principal aim of the current study was to establish an inbred small-animal model of atherosclerosis by immunization with either recombinant HSP-65 or the HSP-65–rich MT, a goal that has successfully been accomplished in normocholesterolemic rabbits.16 An additional purpose was to investigate the role of the immune response toward HSP-65 and oxLDL after immunization with heat-killed preparations of MT, which are known to contain HSP-65, or with the recombinant protein only. This finding is of particular interest, in view of the current controversy as to the principal autoantigen that triggers the immune response within atherosclerotic plaques.5 7 It has been suggested5 8 16 17 that the expression of HSP, probably due to various stressful insults, results in a respective autoimmune response that accelerates atherosclerosis. Further support for this hypothesis is the induction of arteriosclerosis by immunization of normocholesterolemic rabbits with HSP-65 and MT,16 or equivalently, by infections (Marek's disease in chickens; cytomegalovirus, herpes simplex virus, and Chlamydia pneumonia in humans; reviewed in Reference 2424 ). A leading notion7 9 holds that oxidation of LDL is an important factor in atherosclerosis, assuming that the subsequent cellular and immune response toward it plays a protective role.12 13 14 15
In the present study, we have shown that HCD-fed C57BL/6J mice immunized with MT or with HSP-65 developed accelerated fatty streak formation in their aortic sinuses compared with their PBS-immunized or nonimmunized littermates fed a similar diet. Both MT- and HSP-65–immunized mice exhibited a clear and specific cellular immune response toward HSP-65, evident by the proliferation of lymph node cells to the protein. Furthermore, it was apparent that the chow-fed mice immunized with MT, the HCD-fed mice, and particularly the HSP-65–immunized animals, developed higher levels of antibodies to HSP-65 compared with MT-immunized mice fed an HCD. Antibodies to oxLDL were also found to be lower in the HCD-fed, MT-immunized mice when compared with their chow-fed littermates. A possible explanation for the reduced oxLDL and HSP-65 levels in the MT-immunized mice became apparent during the immunohistochemistry studies. The lesions in the C57BL/6J mice immunized with MT or with HSP-65 and fed an HCD contained large deposits of immunoglobulins, which could not be detected in the lesions of their littermates (nonimmunized mice fed an HCD). We suggest that immunization with MT or HSP-65 induces humoral and cellular immune reactions against HSP-65, which then exerts a cross-reactive effect on arterial endothelial cells expressing HSP-60 due to feeding of the HCD. Indeed, HSP-65 and oxLDL antibodies can be detected in mice immunized with MT, as well as in those fed the HCD.
The unexpectedly low titers of anti-oxLDL and anti–HSP-65 antibodies in the HCD-fed, MT-immunized mice can be explained either by the occurrence of immune complexes or by the deposition of the antibodies in the fatty streaks. The levels of HSP-65 antibodies in the HSP-65–immunized mice were so high that deposition in the plaque did not influence the overall levels in the mouse sera. A very recent report17 supports this concept, showing that patients with acute myocardial events have significantly lower titers of anti–HSP-65 antibodies compared with subjects with coronary heart disease. This observation suggests that the expression of HSP-60 by the infarcted heart "consumes" the anti–HSP-65 previously present in the sera of these patients. These findings, combined with the in vitro assays demonstrating the potential of anti–HSP-60 antibodies to mediate endothelial cytotoxicity,25 lend support to the role of the humoral response in promoting the accelerated atherosclerotic process.
Recent studies12 13 14 15 have raised the issue of immunomodulatory roles of anti-oxLDL antibodies in the pathogenesis of atherosclerosis, suggesting that these antibodies, when induced by immunization with homologous oxLDL, are associated with the suppression of atherosclerosis in animals. These findings and the data from the current study suggest that antibodies against "injurious" oxLDL may lead to protection from atherosclerosis, whereas a humoral response to protective HSP-60 accelerates atherogenesis.
An interesting observation was that despite the lower anti–HSP-65 antibody levels in the MT-immunized mice in comparison with HSP-65–immunized animals, atherosclerosis was significantly enhanced in both groups. It is possible that antibodies of different specificity are induced by the 2 immunization protocols. Additionally, immunization with MT may have a more pronounced effect on the production of proinflammatory cytokines that may then exert an additive effect on atherogenesis in this mouse model.
The role of the cellular immune system in atherogenesis is also controversial. Several authors have shown that suppression of cellular immunity resulted in accelerated atherosclerosis in animals.26 27 Yet treatment with monoclonal antibodies to CD4 was found to suppress atherogenesis in mice,28 suggesting that the cellular immune response is involved in the progression of atherosclerosis. Two recent studies imply that apoE-deficient mice (an atherosclerosis-prone strain) develop similarly mature atherosclerotic lesions when deprived of their T- and B-cell functions by cross-breeding with recombinase-activating gene-1– and -2–deficient mice.29 30
How can these observations be reconciled with our current results pointing to the relative abundance of CD4 cells within lesions of MT- and HSP-65–immunized mice compared with the scarcity of these immunopotent cells in the nonimmunized mice? It appears that atherosclerosis is a multifactorial process involving several cellular components. Exogenous factors such as infections and encounters with additional antigens by various routes are likely to influence the progression of atherogenesis, in addition to the known major causes, such as hypercholesterolemia and genetic susceptibility. In our study, lymph node reactivity to HSP-65 was pronounced in MT- and HSP-65–immunized mice, and the lesions from these same animals were rich in CD4 cells. It may thus be proposed that the anti–HSP-65–specific T cells generated after immunization became localized in areas known to preferentially express HSP (ie, endothelial cells faced with the "burden" of hypercholesterolemia-induced formation of LDL), where they released mediators that propagated early atherosclerosis. It is noteworthy that in the current model, unlike in the rabbit model,16 normocholesterolemic mice immunized with HSP-65 did not develop fatty streaks. Thus, the primary initiating factor in the mouse is hypercholesterolemia, which exerts a "stressful insult" on the endothelial cells, leading to HSP expression. The immune response to HSP-65 thus acts to enhance the severity of the lesions induced by the HCD.
The results of the current study are in line with the several studies documenting infectious agents associated with accelerated atherosclerosis.24 Thus, Chlamydia pneumoniae31 and herpes simplex virus32 are examples of infectious agents that have been associated with accelerated atherosclerosis. In general, it has been suggested that infections elicit a nonspecific immune response that also acts at the local level (the atherosclerotic lesion), leading to an accumulation of inflammatory cells that attract macrophages, thus leading to increased uptake of lipids and accelerated atherosclerosis.
In conclusion, we have shown that immunization of mice with recombinant HSP-65 or with heat-killed, HSP-rich MT can lead to accelerated fatty streak formation. This small-animal model furnishes an improved tool for studying the contribution of the immune system to the development of atherosclerosis over the current atherosclerosis-prone murine models, which have been genetically manipulated. Moreover, this model could be applied as a means of studying immunomodulatory interventions in atherogenesis.
|
Acknowledgments |
|---|
This investigation received financial support from the Austrian Science Fund project No. 12213 (to G.W.), the Freda and Leon Schaller grant for research in autoimmunity, and the United States–Israel Binational Science Foundation No. 93-103 (to D.H.).
Received March 11, 1998; accepted July 29, 1998.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801–809.[Medline] [Order article via Infotrieve]
2. 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]
3. van der Wal AC. Atherosclerotic lesions in humans: in situ immunophenotypic analysis suggesting an immune mediated response. Lab Invest.. 1989;61:166–170.[Medline] [Order article via Infotrieve]
4.
Steinberg D, Witztum JL. Lipoproteins and
atherogenesis: current concepts. JAMA. 1990;264:3047–3052.
5. 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]
6. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol: modification of low density lipoprotein that increases its atherogenicity. N Engl J Med. 1989;320:915–924.[Medline] [Order article via Infotrieve]
7. Witztum JL. The oxidation hypothesis of atherosclerosis. Lancet. 1994;344:793–795.[Medline] [Order article via Infotrieve]
8. Xu Q, Wick G. The role of heat shock proteins in protection and pathophysiology of the arterial wall. Mol Med Today. 1996;2:372–379.[Medline] [Order article via Infotrieve]
9.
Palinski W, Rosenfeld ME, Yla-Herttuala S, Gurtner GC,
Socher SS, Butler SW, Parthasarathy S, Carew TE, Steinberg D, Witztum
JL. Low density lipoprotein undergoes oxidative modification in vivo.
Proc Natl Acad Sci U S A. 1989;86:1372–1376.
10.
Yla-Herttuala S, Palinski W, Butler SW, Picard W,
Steinberg W, Witztum JL. Rabbit and human atherosclerotic lesion
contain IgG that recognizes epitopes of oxidized LDL.
Arterioscler Thromb. 1994;14:32–40.
11.
Stemme S, Holm J, Wiklund O, Witztum JL, Hansson GK. T
lymphocytes from human atherosclerotic plaques recognize oxidized low
density lipoprotein. Proc Natl Acad Sci U S A. 1995;92:3893–3897.
12.
Palinski W, Miller E, Witztum JL. Immunization of low
density lipoprotein (LDL) deficient rabbits with homologous
malondialdehyde-modified LDL reduces atherogenesis. Proc Natl
Acad Sci U S A. 1995;92:821–825.
13.
Ameli SA, Hultgardh-Nilsson, Regnstrom J, Calara F,
Yana J, Cercek B, Shah PK, Nilsson J. Effect of immunization with
homologous LDL and oxidized LDL on early
atherosclerosis in hypercholesterolemic
rabbits. Arterioscler Thromb Vasc Biol. 1996;16:1074–1079.
14. Nilsson J, Calara F, Regnstrom J, Hultgardh-Nilsson A, Ameli S, Cercek B, Shah PK. Immunization with homologous oxidized low density lipoprotein reduces neointimal formation after balloon angioplasty in hypercholesterolemic rabbits. J Am Coll Cardiol. 1997;30:1886–1891.[Abstract]
15. George J, Afek A, Gilburd B, Levkovitz H, Shaish A, Goldberg I, Kopolovic J, Wick G, Shoenfeld Y, Harats D. Hyperimmunization of apoE deficient mice with homologous MDA-low density lipoprotein suppresses early atherogenesis. Atherosclerosis. 1998;138:147–152.[Medline] [Order article via Infotrieve]
16.
Xu Q, Dietrich H, Steiner HJ, Gown AM, Schoel B, Mikuz
G, Kaufman SHE, Wick G. Induction of
arteriosclerosis in
normocholesterolemic mice and rabbits by immunization
with heat shock protein 65. Arterioscler Thromb. 1992;12:789–799.
17. Xu Q, Kleindienst R, Waitz H, Dietrich H, Wick G. Increased expression of heat shock protein 65 coincides with a population of infiltrating T lymphocytes in atherosclerotic lesions of rabbits specifically responding to heat shock protein 65. J Clin Invest. 1993;91:2693–2702.
18. Xu Q, Willeit J, Marosi M, Kleindienst R, Oberhollenzer F, Kiechl S, Stulnig T, Luef G, Wick G. Association of serum antibodies to heat shock protein 65 with carotid atherosclerosis. Lancet. 1993;341:255–259.[Medline] [Order article via Infotrieve]
19. Hoppichler F, Lechleitner M, Traweger C, Schett G, Dzien A, Sturm W, Xu Q. Changes of serum antibodies to heat-shock protein 65 in coronary heart disease and acute myocardial infarction. Atherosclerosis. 1996;126:333–338.[Medline] [Order article via Infotrieve]
20. George J, Blank M, Hojnik M, Bar-Meir E, Koike T, Matsuura E, Lorber M, Aviram M, Shoenfeld Y. Oxidized low density lipoprotein (oxLDL) but not LDL aggravates the manifestations of antiphospholipid syndrome (APS). Clin Exp Immunol. 1997;108:227–233.[Medline] [Order article via Infotrieve]
21.
Elias D, Markovits D, Reshef T, van der Zee R, Cohen
IR. Induction and therapy in non-obese diabetic (NOD/Lt) mouse by a
65-kDa heat shock protein. Proc Natl Acad Sci U S A. 1990;87:1576–1580.
22.
Qiao JH, Xie PZ, Fishbein MC, Kreuzer J, Drake TA,
Demer LL, Lusis AJ. Pathology of atheromatous
lesions in inbred and genetically engineered mice: genetic
determinations of arterial calcification.
Arterioscler Thromb. 1994;14:1480–1497.
23.
Palinski W, Yla-Herttuala S, Rosenfeld ME, Butler S,
Socher SA, Parthasarathy S, Curtiss LK, Witztum JL. Antisera and
monoclonal antibodies specific for epitopes generated during oxidative
modification of low density lipoprotein.
Arteriosclerosis. 1990;10:325–335.
24.
Cook PJ, Lip GYH. Infectious agents and atherosclerotic
vascular disease. Q J Med. 1996;89:727–735.
25. Schett G, Xu Q, Amberger A, Van der Zee R, Recheis H, Willeit J, Wick G. Antibodies against heat shock protein 60 mediate endothelial cytotoxicity. J Clin Invest. 1995;96:2569–2577.
26. Emeson EE, Shen ML. Accelerated atherosclerosis in hyperlipidemic C57BL/6 mice treated with cyclosporine A. Am J Pathol. 1993;142:1906–1915.[Abstract]
27. Roselaar SE, Schonfeld G, Daugherty A. Enhanced development of atherosclerosis in cholesterol-fed rabbits by suppression of cell-mediated immunity. J Clin Invest. 1995;96:1389–1394.
28. Emeson EE, Shen ML, Bell CGH, Qureshi A. Inhibition of atherosclerosis in CD4 T-cell ablated and nude (nu/nu) C57BL/6 hyperlipidemic mice. Am J Pathol. 1996;149:675–685.[Abstract]
29.
Dansky HM, Charlton SA, Harper MM, Smith JD. T and B
lymphocytes play a minor role in atherosclerotic plaque formation in
the apolipoprotein E-deficient mouse. Proc Natl Acad Sci
U S A. 1997;94:4642–4646.
30. Daugherty A, Pure E, Delfel-Butteiger D, Chen S, Leferovich J, Roselaar SE, Rader DJ. The effects of total lymphocyte deficiency on the extent of atherosclerosis in apolipoprotein E-/- mice. J Clin Invest. 1997;100:1575–1580.[Medline] [Order article via Infotrieve]
31. Capron L. Chlamydia in coronary plaques: hidden culprits or harmless hobo? Nat Med.. 1996;8:856–857.
32. Hajjar DP, Pomerantz KB, Falcone DJ, Weksler BB, Grant AJ. Herpes simplex virus infection in human arterial cells: implications in atherosclerosis. J Clin Invest. 1987;80:1317–1321.
This article has been cited by other articles:
 |
|
 |
|
  I. Gotsman, A. H. Sharpe, and A. H. Lichtman T-Cell Costimulation and Coinhibition in Atherosclerosis Circ. Res., November 21, 2008; 103(11): 1220 - 1231. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Zal, C. Baboonian, and J. C. Kaski Letter to the Editor: Autoreactive CD4+CD28- T Cells and Acute Coronary Syndromes Arterioscler Thromb Vasc Biol, March 1, 2007; 27(3): E18 - E18. [Full Text] [PDF]
|
|
|
|
 |
|
 A. Mor, G. Luboshits, D. Planer, G. Keren, and J. George Altered status of CD4+CD25+ regulatory T cells in patients with acute coronary syndromes Eur. Heart J., November 1, 2006; 27(21): 2530 - 2537. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A.-K. L. Robertson and G. K Hansson T Cells in Atherogenesis: For Better or For Worse? Arterioscler Thromb Vasc Biol, November 1, 2006; 26(11): 2421 - 2432. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Tedgui and Z. Mallat Cytokines in Atherosclerosis: Pathogenic and Regulatory Pathways Physiol Rev, April 1, 2006; 86(2): 515 - 581. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Miyamoto, H. Yumoto, Y. Takahashi, M. Davey, F. C. Gibson III, and C. A. Genco Pathogen-Accelerated Atherosclerosis Occurs Early after Exposure and Can Be Prevented via Immunization Infect. Immun., February 1, 2006; 74(2): 1376 - 1380. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S G Baidya and Q-T Zeng Helper T cells and atherosclerosis: the cytokine web Postgrad. Med. J., December 1, 2005; 81(962): 746 - 752. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. C. Mehra, V. S. Ramgolam, and J. R. Bender Cytokines and cardiovascular disease J. Leukoc. Biol., October 1, 2005; 78(4): 805 - 818. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K Mandal, G Foteinos, M Jahangiri, and Q Xu Role of antiheat shock protein 60 autoantibodies in atherosclerosis Lupus, September 1, 2005; 14(9): 742 - 746. [Abstract] [PDF]
|
|
|
|
 |
|
 A G Pockley and J Frostegard Heat shock proteins in cardiovascular disease and the prognostic value of heat shock protein related measurements Heart, September 1, 2005; 91(9): 1124 - 1126. [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]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 Y. Shoenfeld, R. Wu, L. D. Dearing, and E. Matsuura Are Anti-Oxidized Low-Density Lipoprotein Antibodies Pathogenic or Protective? Circulation, October 26, 2004; 110(17): 2552 - 2558. [Full Text] [PDF]
|
|
|
|
|
|
B. Ludewig, P. Krebs, and E. Scandella Immunopathogenesis of atherosclerosis J. Leukoc. Biol., August 1, 2004; 76(2): 300 - 306. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. OSTERUD and E. BJORKLID Role of Monocytes in Atherogenesis Physiol Rev, October 1, 2003; 83(4): 1069 - 1112. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. N. Fredrikson, B. Hedblad, G. Berglund, R. Alm, M. Ares, B. Cercek, K.-Y. Chyu, P. K. Shah, and J. Nilsson Identification of Immune Responses Against Aldehyde-Modified Peptide Sequences in ApoB Associated With Cardiovascular Disease Arterioscler Thromb Vasc Biol, May 1, 2003; 23(5): 872 - 878. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Harats, N. Yacov, B. Gilburd, Y. Shoenfeld, and J. George Oral tolerance with heat shock protein 65 attenuates mycobacterium tuberculosis-inducedand high-fat-diet-driven atherosclerotic lesions J. Am. Coll. Cardiol., October 2, 2002; 40(7): 1333 - 1338. [Abstract] [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]
|
|
|
|
|
|
R. Maron, G. Sukhova, A.-M. Faria, E. Hoffmann, F. Mach, P. Libby, and H. L. Weiner Mucosal Administration of Heat Shock Protein-65 Decreases Atherosclerosis and Inflammation in Aortic Arch of Low-Density Lipoprotein Receptor-Deficient Mice Circulation, September 24, 2002; 106(13): 1708 - 1715. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. K. Hansson, P. Libby, U. Schonbeck, and Z.-Q. Yan Innate and Adaptive Immunity in the Pathogenesis of Atherosclerosis Circ. Res., August 23, 2002; 91(4): 281 - 291. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Dimayuga, B. Cercek, S. Oguchi, G. N. Fredrikson, J. Yano, P. K. Shah, S. Jovinge, and J. Nilsson Inhibitory Effect on Arterial Injury-Induced Neointimal Formation by Adoptive B-Cell Transfer in Rag-1 Knockout Mice Arterioscler Thromb Vasc Biol, April 1, 2002; 22(4): 644 - 649. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. A. Schreyer, C. M. Vick, and R. C. LeBoeuf Loss of Lymphotoxin-alpha but Not Tumor Necrosis Factor-alpha Reduces Atherosclerosis in Mice J. Biol. Chem., March 29, 2002; 277(14): 12364 - 12368. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Huittinen, M. Leinonen, L. Tenkanen, M. Manttari, H. Virkkunen, T. Pitkanen, E. Wahlstrom, T. Palosuo, V. Manninen, and P. Saikku Autoimmunity to Human Heat Shock Protein 60, Chlamydia pneumoniae Infection, and Inflammation in Predicting Coronary Risk Arterioscler Thromb Vasc Biol, March 1, 2002; 22(3): 431 - 437. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. G. Pockley Heat Shock Proteins, Inflammation, and Cardiovascular Disease Circulation, February 26, 2002; 105(8): 1012 - 1017. [Full Text] [PDF]
|
|
|
|
 |
|
 Y Sherer and Y Shoenfeld Atherosclerosis Ann Rheum Dis, February 1, 2002; 61(2): 97 - 99. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. K. Hansson Immune Mechanisms in Atherosclerosis Arterioscler Thromb Vasc Biol, December 1, 2001; 21(12): 1876 - 1890. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. K. Kanwar, J. R. Kanwar, D. Wang, D. J. Ormrod, and G. W. Krissansen Temporal Expression of Heat Shock Proteins 60 and 70 at Lesion-Prone Sites During Atherogenesis in ApoE-Deficient Mice Arterioscler Thromb Vasc Biol, December 1, 2001; 21(12): 1991 - 1997. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. George, S. Greenberg, I. Barshack, M. Singh, S. Pri-Chen, S. Laniado, and G. Keren Accelerated intimal thickening in carotid arteries of balloon-injured rats after immunization against heat shock protein 70 J. Am. Coll. Cardiol., November 1, 2001; 38(5): 1564 - 1569. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. George, A. Afek, B. Gilburd, Y. Shoenfeld, and D. Harats Cellular and humoral immune responses to heat shock protein 65 are both involved in promoting fatty-streak formation in LDL-receptor deficient mice J. Am. Coll. Cardiol., September 1, 2001; 38(3): 900 - 905. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P A Gordon, J George, M A Khamashta, D Harats, G Hughes, and Y Shoenfeld Atherosclerosis and autoimmunity Lupus, April 1, 2001; 10(4): 249 - 252. [Abstract] [PDF]
|
|
|
|
 |
|
 B. Ludewig, S. Freigang, M. Jäggi, M. O. Kurrer, Y.-C. Pei, L. Vlk, B. Odermatt, R. M. Zinkernagel, and H. Hengartner Linking immune-mediated arterial inflammation and cholesterol-induced atherosclerosis in a transgenic mouse model PNAS, October 23, 2000; (2000) 220427097. [Abstract] [Full Text]
|
|
|
|
|
|
J. George, Y. Shoenfeld, B. Gilburd, A. Afek, A. Shaish, and D. Harats Requisite Role for Interleukin-4 in the Acceleration of Fatty Streaks Induced by Heat Shock Protein 65 or Mycobacterium tuberculosis Circ. Res., June 23, 2000; 86(12): 1203 - 1210. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J George, A Afek, B Gilburd, D Harats, and Y Shoenfeld Autoimmunity in atherosclerosis: lessons from experimental models Lupus, March 1, 2000; 9(3): 223 - 227. [Abstract] [PDF]
|
|
|
|
|
|
S. E. Epstein, Y. F. Zhou, and J. Zhu Infection and Atherosclerosis : Emerging Mechanistic Paradigms Circulation, July 27, 1999; 100 (4): e20 - e28. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Ludewig, S. Freigang, M. Jaggi, M. O. Kurrer, Y.-C. Pei, L. Vlk, B. Odermatt, R. M. Zinkernagel, and H. Hengartner Linking immune-mediated arterial inflammation and cholesterol-induced atherosclerosis in a transgenic mouse model PNAS, November 7, 2000; 97(23): 12752 - 12757. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Dimayuga, B. Cercek, S. Oguchi, G. N. Fredrikson, J. Yano, P. K. Shah, S. Jovinge, and J. Nilsson Inhibitory Effect on Arterial Injury-Induced Neointimal Formation by Adoptive B-Cell Transfer in Rag-1 Knockout Mice Arterioscler Thromb Vasc Biol, April 1, 2002; 22(4): 644 - 649. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||