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??

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

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

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

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

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

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

fibroblastsCollagen type

I, III, fibronectin,

proteoglycan

Synovial-cell injury; erosion of cartilage; new matrix

scarring (pannus)

Glomeruloscle-rosis + + - Mesangial

cellsCollagen type

I, IV, fibronectin

Epithelial- and endothelial-cell injury and dysfunction;

decrease in glomerular filtration; new matrix

formation;

Pulmonary fibrosis + + +/- SMCs,

FibroblastsCollagen 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

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

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

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

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.

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.

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.

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?

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?

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

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

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

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

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