SHAPE Society

1. Editorial Slides
VP Watch, December 19, 2001, Volume 1, Issue 38
Atherosclerosis, An Autoimmune Disease?
What could be the culprit antigen(s)? Ox-LDL, HSP, or what??

2.  In1856 Virchow described atherosclerosis as
“endarteritis”. A century later Russel Ross
named atherosclerosis “an inflammatory
disease”. Ross likened atherosclerosis to other
chronic inflammatory diseases such as
rheumatoid arthritis and glomerulonephritis. 1
 The central role of immune system in
atherosclerosis and it’s clinical complications is
now widely accepted. Many investigators are
searching to find out what antigens attract
immune cells into arterial wall and possibly later
on into atherosclerotic plaques. 2,3,4
 Autoantibodies against oxidized low-density
lipoprotein (oxLDL), cardiolipin, beta2-
glycoprotein-I and heat-shock protein 60/65
have been suggested. 2

3.  Georg Wick, Qingbo Xu, and colleagues have
hypothesized that an autoimmune reaction
against heat shock protein 60s, expressed by
endothelial cells in areas that are subject to
increased hemodynamic stress, is the initiating
event in atherogenesis. 5,6
 The hypothesis indicates that Because a high
degree of antigenic homology exists between
microbial (bacterial and parasitic) and human
HSP60, the 'cost' of immunity to microbes
might be the danger of cross-reactivity with
human HSP60 expressed by the endothelial
cells of stressed arteries subjected to classical
risk factors.7

4.  Two major families of HSPs (60s and 70s) have
been related to atherosclerosis. Unlike HSP60s,
HSP70s are not reported as strong triggers of
autoimmune reactions, however, Bond, Johnson
and colleagues have suggested certain role for
HSP70s in atherosclerosis. 8,9
 Chen et al described autologous hsp60 as a danger
signal to the innate immune system.10
 Xu et al showed induction of arteriosclerosis in
normocholesterolemic rabbits by immunization with
heat shock protein 65. 5
 George, Afek, and colleagues reported induction of
arteriosclerosis in normocholesterolemic rabbits by
immunization with heat shock protein 65. 11,12

5.  A number of other experimental and observational
studies have shown a significant relationship between
heat shock proteins and atherosclerosis. 9,11,13,14
 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. 15
 Bocharov et al reported that heat shock protein 60 is a
high-affinity high-density lipoprotein binding protein
suggesting a potential mechanism to explain the
known association between immunity developed
against hsp60 and the development of atherosclerosis.
16

6.  Comparing the similarities between atherosclerosis
and other autoimmune disorders such as
rheumatoid arthritis (as indicated by Ross in the
following slide) can also hint about the potential role
of autoimmune mechanisms in atherosclerosis and
it’s complications. 1
 Interestingly, recent studies have uncovered an
important role for heat shock proteins in
pathogenesis of rheumatoid arthritis. 17,18
 Like in rheumatoid arthritis, the suggested role of
HSPs in atherosclerosis may also in part explain
the missing link between infectious agents and
atherosclerosis where a high degree of antigenic
homology between human and microbial HSPs can
cause cross-reaction. 17,7

7. Disease Monocytes &
Macrophage
Lymphocyte Granulocyte Connective-
Tissue Cells
Extracellular
Matrix
Pathogenetic Mechanisms
Atherosclerosis
+ + -
SMCs Collagen type
I, III, IV,
elastin,
fibronectin,
proteoglycan
Endothelial-cell injury and
dysfunction; fibrous cap;
new matrix formation &
degeneration; necrotic core
Cirrhosis
+ + -
Fibroblasts Collagen type
I, III
Parenchymal cell injury, new
matrix and scarring
replacing necrotic
parenchyma
Rheumatoid
arthritis + + +/-
Synovial
fibroblasts
Collagen type
I, III,
fibronectin,
proteoglycan
Synovial-cell injury; erosion
of cartilage; new matrix
scarring (pannus)
Glomeruloscle-
rosis + + -
Mesangial
cells
Collagen type
I, IV,
fibronectin
Epithelial- and endothelial-
cell injury and dysfunction;
decrease in glomerular
filtration; new matrix
formation;
Pulmonary
fibrosis + + +/-
SMCs,
Fibroblasts
Collagen type
III, IV,
fibronectin
Inflammatory exudate in
alveoli & bronchi; organized
by extensive matrix
deposition and scarring
Chronic
pancreatitis + + -
Fibroblasts Collagen,
fibronectin,
proteoglycan
Epithelial injury; periductal
inflammation; interstitial fat
necrosis; new matrix
formation
Ross R. Atherosclerosis--an inflammatory disease. N Engl J Med. 1999 Jan 14;340(2):115-26. Review

8.  As highlighted in this week of VP Watch,
Kanwar, Krissansen, et al. found that
expression of hsp60 and hsp70 was 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. 20
 They showed that Hsp60 and hsp70 were
detectable in the aortas of 3-week-old apoE-/-
mice and were highly expressed in the aortas
of 8-week-old mice. 20

9.  Kanwar et al. indicated that in 8-week-old
apoE-/- mice, hsp60 and 70 was strongly
expressed at valve commissures of the aortic
sinus, extending to the free aortic wall and
including expression by endothelial and intimal
cells. 20
 In this study they resulted that Hsp60 and hsp70
were heterogeneously expressed in lesions of
20-week-old mice. Hsp60 and hsp70 were
strongly expressed in advanced plaques of the
abdominal aorta of 20-week-old mice, whereas
medial layers lack expression. 20

10.  In 69-week-old mice, there was complete loss
of hsp60 and hsp70 in advanced complicated
collagen-rich plaques of the aortic sinus.
(down regulated in aged mice) 20
 As a result of this study, lesion-prone sites
displayed strong endothelial hsp60
expression, whereas non–lesion-prone sites
of the distal abdominal aorta lacked hsp
expression. 20
 Monocytes/macrophages expressing hsp70
and hsp60 (data not shown) were the most
prominent cell type in lesions. 20

11. 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.
Rupinder K. Kanwar, Jagat R. Kanwar, Dongmao Wang, Douglas J. Ormrod, and Geoffrey 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 2001 21: 1991-1997.

12. 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).
Rupinder K. Kanwar, Jagat R. Kanwar, Dongmao Wang, Douglas J. Ormrod, and Geoffrey 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 2001 21: 1991-1997.

13. Conclusion:
1- Autoimmune reactions (cellular and humoral) against
HSPs particularly HSP60s may play an important role
in early stage development of atherosclerosis.
2- HSP60s and HSP70s released from necrotic cells in
the core area of advanced plaques may stimulate the
innate immune response to promote inflammation and
attract new inflammatory cells thereby may link to
complications of plaque such as rupture and or
thrombosis.

14. Questions:
I. According to our current body of knowledge, the
development of atherosclerosis seems to have two
major preceding components, metabolic disorder
(lipid abnormality etc.) and inflammatory disorder
(enhanced immune or autoimmune response). The
question is which one comes first?
II. Since the complication of atherosclerosis (vulnerable
plaque) is more important than it’s development
(stable plaque), the question is which one of the two
(1-metabolic, 2-Immune) components of
atherosclerosis plays a more important role?

15. Questions:
III. Do you find the idea of vaccination against HSPs or
oxidized-LDL or other suggested antigens, clinically
feasible?
IV. If yes, which one seems more feasible to you,
eradication of atherosclerosis by vaccination against
triggers of plaque development, or, eradication of
vulnerable plaque by vaccination against triggers of
plaque vulnerability?

16. Suggestion:
I. More studies are needed to characterize the weakest
points in the chain of atherosclerosis, any
suggestion?
II. Please email your thoughts to:
Discussion-Group@VP.org or DG@VP.org

17. 1. Ross R. Atherosclerosis--an inflammatory disease.
N Engl J Med. 1999 Jan 14;340(2):115-26. Review
2. Shoenfeld Y, Sherer Y, George J, Harats D. ;Autoantibodies
associated with atherosclerosis. Ann Med. 2000 Dec;32 Suppl 1:37-40.
Review.
3. Hansson, G.; Immunological markers of atherosclerosis.
Lancet. 1993 Jan 30;341(8840):278.
4. Witztum JL, Palinski W. ; Are immunological mechanisms relevant for
the development of atherosclerosis? Clin Immunol. 1999
Feb;90(2):153-6. Review.
5. Xu Q, Dietrich H, Steiner HJ, Gown AM, Schoel B, Mikuz G, Kaufmann
SH, Wick G. ; Induction of arteriosclerosis in normocholesterolemic
rabbits by immunization with heat shock protein 65. Arterioscler
Thromb. 1992 Jul;12(7):789-99.
6. Wick G, Schett G, Amberger A, Kleindienst R, Xu Q.; Is
atherosclerosis an immunologically mediated disease?; Immunol
Today. 1995 Jan;16(1):27-33. Review.
References

18. 7. Wick G, Perschinka H, Millonig G. ; Atherosclerosis as an autoimmune disease:
an update.; Trends Immunol. 2001 Dec 1;22(12):665-669.
8. 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 Jan;15(1):27-36.
9. 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 Jan;136(1):71-80.
10. Chen W, Syldath U, Bellmann K, Burkart V, Kolb H.; Human 60-kDa heat-shock
protein: a danger signal to the innate immune system.; J Immunol. 1999 Mar
15;162(6):3212-9.
11. 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
Mar;19(3):505-10.
References

19. 12. 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 Mar;14(2):115-21.
13. Hansen PR, Chew M, Zhou J, Daugherty A, Heegaard N, Jensen P, Mouritsen
S, Falk E.; Freunds adjuvant alone is antiatherogenic in apoE-deficient mice and
specific immunization against TNFalpha confers no additional benefit.
Atherosclerosis. 2001 Sep;158(1):87-94.
14. George J, Afek A, Gilburd B, Shoenfeld Y, Harats D.; 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. 2001 Sep;38(3):900-5.
15. 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 alpha/beta or gamma/delta receptor in
human atherosclerotic lesions.; Am J Pathol. 1993 Jun;142(6):1927-37.
References

20. 16. Bocharov AV, Vishnyakova TG, Baranova IN, Remaley AT, Patterson AP,
Eggerman TL.; Heat shock protein 60 is a high-affinity high-density lipoprotein
binding protein.; Biochem Biophys Res Commun. 2000 Oct 14;277(1):228-35.
17. Gaston, JS.; Heat shock proteins and arthritis--new readers start here.
Autoimmunity. 1997;26(1):33-42. Review.
18. Schett G, Tohidast-Akrad M, Steiner G, Smolen J.; The stressed synovium.;
Arthritis Res. 2001;3(2):80-6. Review.
19. Gaston, JS. ; Heat shock proteins and arthritis--new readers start here.;
Autoimmunity. 1997;26(1):33-42. Review.
20. Rupinder K. Kanwar, Jagat R. Kanwar, Dongmao Wang, Douglas J. Ormrod,
and Geoffrey 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 2001 21: 1991-1997.
References