- Email: [email protected]
Potential Role of the Ubiquitin-Proteasome System in Atherosclerosis
Journal of the American College of Cardiology © 2008 by the American College of Cardiology Foundation Published by Elsevier Inc.
Vol. 51, No. 21, 2008 ISSN 0735-1097/08/$34.00 doi:10.1016/j.jacc.2008.02.047
STATE-OF-THE-ART PAPER
Potential Role of the Ubiquitin-Proteasome System in Atherosclerosis Aspects of a Protein Quality Disease Joerg Herrmann, MD, Sandra M. Soares, MD, Lilach O. Lerman, MD, PHD, Amir Lerman, MD Rochester, Minnesota Misfolded or damaged proteins are recognized intracellularly by protein quality mechanisms. These include chaperones and the ubiquitin-proteasome system, which aim at restoration of protein function and protein removal, respectively. A number of studies have outlined the functional significance of the ubiquitin-proteasome system for the heart and, as of recently, for the vascular system. This review summarizes these recent findings with a focus on atherosclerosis. In particular, this paper reflects on the viewpoint of atherosclerosis as a protein quality disease. (J Am Coll Cardiol 2008;51:2003–10) © 2008 by the American College of Cardiology Foundation
The structure and function of cells and related organs, including the cardiovascular system, is determined to a significant degree by the structure and function of their proteins. The synthesis of new proteins is balanced by the degradation of those that have become disposable for various reasons. Recent research findings have highlighted the significance of these biological processes for cardiovascular diseases. The present review will summarize these advances, introducing the viewpoint of atherosclerosis as a protein quality disease. Protein Quality Mechanisms The quality of a protein and its function relies on its 3-dimensional structure and folding dynamics. The loss of proper 3-dimensional arrangement, referred to as unfolding, impairs not only protein function but eventually also cellular function and viability. To prevent these detrimental consequences, each cell is equipped with 2 operational systems: chaperones and proteases. Chaperones recognize protein substrates based on hydrophobic protein regions, which are usually buried within the tertiary and quarternary structures but surface as the protein structure unfolds after translational or post-translational damage. Variations in the protein sequences that flank these hydrophobic sequences determine the large number and various types of chaperones across the different cellular compartments (1). Their main function is to hold misfolded From the Department of Internal Medicine, Mayo Clinic, Rochester, Minnesota. Supported by the National Institutes of Health (R01 HL63911-04, K24 HL6984001), the Mayo Stiftung, and the Mayo Foundation. Manuscript received November 7, 2007; revised manuscript received January 28, 2008, accepted February 12, 2008.
proteins, to fold them back into their native structure, and to promote the degradation of proteins resistant to this process by making them more accessible to proteases. This latter group of enzymes cleaves proteins based on amino acid sequence patterns. Up to 90% of all intracellular proteins are degraded, both in a chaperone-dependent and in a chaperone-independent manner, by a specialized protease system, called the ubiquitin-proteasome system (UPS) (2). This system operates mainly in the cytoplasm and recognizes protein substrates, like chaperones, on the basis of bulky hydrophobic residues but also based on basic residues at the NH2-terminus (so-called N-end rule). Thus, numerous chaperones and the UPS work independently and in synergy to assure the quality of intracellular proteins. The kinetics of the interactions between chaperones, the UPS, and damaged proteins were summarized in a triage model (Fig. 1) (3). One particular subtype of protein quality control mechanisms is the unfolded protein response (4). This response is triggered by the accumulation of unfolded or misfolded proteins in the lumen of the endoplasmic reticulum (ER), which is also referred to as ER stress and leads to the modification of transducer proteins in the ER membrane and the activation of their related pathways. These pathways include the inositol-requiring protein 1, the activating transcription factor-6, and the protein kinase ribonucleic acid-like kinase signaling pathways. Collectively, these pathways orchestrate the down-regulation of the synthesis and the translocation of proteins into the ER, which leads to a reduction in protein load. They also mediate the expression of chaperones and proteinmodifying enzymes, which facilitates protein refolding. Furthermore, they engage the endoplasmic reticulumassociated protein degradation pathway, which entails the
2004
Herrmann et al. Protein Quality and Atherosclerosis
retrotranslocation of unfolded proteins into the cytosol and their proteolysis by the proteaAbeta ⴝ amyloid beta some. In case these mechanisms protein fail to resolve the mismatch beAPP ⴝ amyloid precursor tween protein load and handling protein capacity over a period of time, ER ⴝ endoplasmic the unfolded protein response inreticulum duces autophagy as an alternative ERAD ⴝ endoplasmic reticulum-associated coping mechanism. This process protein degradation involves the sequestration of the HSP ⴝ heat shock protein ER into membrane-bounded compartments (autophagosomes), LDL ⴝ low-density lipoprotein its fusion with lysosomes, and the ROS ⴝ reactive oxygen degradation of its content. The species ER-specific autophagy is seemTIA ⴝ transient ischemic ingly essential for cells to survive attack severe ER stress (5). However, UPS ⴝ ubiquitinwith overwhelming ER stress cell proteasome system death pathways are nevertheless activated. In summary, cells are equipped with a robust apparatus to repair or remove damaged proteins. This includes chaperones, proteases, and the ER-related unfolded protein response along the general concept of protein triaging. The competitive balance between the activity of protein quality control mechAbbreviations and Acronyms
Figure 1
JACC Vol. 51, No. 21, 2008 May 27, 2008:2003–10
anisms, on the one hand, and the generation of low-quality proteins, on the other hand, is the hallmark of this concept.
Protein Quality Diseases Protein quality diseases, also called protein misfolding, precipitation, or conformational diseases, are a group of disorders that are associated with and occasionally caused by the conformational change, aggregation, and deposition of 1 or more proteins (6). Neurodegenerative diseases were the first prominent example for this entity. Mutation analyses in familial counterparts to sporadic neurologic disorders and experimental studies highlighted the pathogenetic role of the accumulation of misfolded proteins in these disease processes (7,8). Research efforts also showed that the accumulation of these proteins is due to their increased production, abnormal processing, and/or decreased elimination via chaperones, the UPS, or the unfolded protein response (8,9). The “amyloid hypothesis” of neurodegenerative diseases links the aggregation of misfolded proteins into ordered protease-resistant structures, called amyloid fibrils, to aberrant protein interactions that culminate in neuronal dysfunction and ultimately neurodegeneration (10). Indeed, the pathological momentum seems to be with the very process of amyloid formation.
Protein Triage System
Because of translational or post-translational damage, proteins can become misfolded, leading to surface exposure of hydrophobic regions. These regions are recognition sites for chaperones, which aim at restoring the native protein structure or triage to the ubiquitin-proteasome system (UPS). This system can also recognize damaged proteins directly and mediates the attachment of at least 4 ubiquitin molecules, which directs proteins to degradation by the proteasome. Deubiquitinating enzymes can reverse the ubiquitin-chain degradation signal, and damaged proteins receive a new chance for refolding. Some proteins, such as oxidatively damaged proteins, can be recognized directly by the proteasome complex. A protein’s propensity to fold to a state with particular binding characteristics, including the affinity for chaperones versus proteases, will determine its fate. This includes restoration of protein structure and function, protein degradation, and protein cross-linking and aggregation.
Herrmann et al. Protein Quality and Atherosclerosis
JACC Vol. 51, No. 21, 2008 May 27, 2008:2003–10
Amyloid formation is an ordered process and begins when misfolded proteins start to assume a beta-pleated sheet structure and self-associate to form soluble preamyloid oligomers. As these soluble preamyloid oligomers start to form protofibrils and coalesce, classic amyloid fibrils, plaques, and tangles are formed, which then exhibit Congo red-positive staining. Classic amyloid plaques are therefore a late and final reflection of a process that remains undetected in its peak toxicity. Consistent with this view, neurologic disease precedes the histologic appearance of classic amyloid plaques. As another implication, the focus of attention has shifted from the large aggregates and microscopic hallmarks to the small aggregates and molecular processes of the pre-amyloid stage both extracellularly and intracellularly (11). Moreover, there has been the realization that the process of aggregation is not inherent to only a few proteins but can be observed with various proteins once misfolding remains unresolved (12). In summary, unfolded/misfolded proteins can accumulate in cells and tissues when they are produced in large amounts and/or not cleared sufficiently. These proteins then form aggregates, which can remove amino acids from the recycling pool, tie up chaperones and proteases, serve as foci for the aggregation of other unrelated proteins, and finally disrupt
Aortic sinus 3-week-old mice
2005
cellular and tissue functions. Over time, the accumulation of low-quality proteins can therefore result in disease, a so-called protein quality disease. Precipitates such as amyloid are characteristic late-stage histologic fingerprints. Protein Quality Control in Atherosclerosis Besides inflammation, excess generation of reactive oxygen species (ROS), so-called oxidative stress, is considered to be an important element in the pathophysiology of atherosclerosis (13). Among the various effects, oxidative stress has been shown to increase the expression of chaperones, including those of the family of heat shock proteins (HSPs). The HSPs most closely associated with atherosclerosis include HSP60 and HSP70. The expression of HSP60 in the vascular wall correlates with the severity of atherosclerosis and is particularly prominent in the shoulder regions and around necrotic cores of atherosclerotic plaques (14). The HSP70 is mainly expressed in more advanced atheromas around sites of necrosis and lipid accumulation (15). Importantly, these findings can be reproduced experimentally in hypercholesterolemic apolipoprotein E⫺/⫺ mice, in which the expression of both HSPs increases in the early and progressive disease stages (Fig. 2) (16).
Aorta commissure 8-week-old mice
Abdominal aorta 20-week-old mice
Aortic sinus 69-week-old mice
A
B
C
D
E
F
G
H
HSP 60
HSP 70
Aortic non-lesions 3-8-weeks old mice
Aortic lesions 3-20-weeks old mice
Aortic lesions 40-69-weeks old mice
HSP 60
HSP 70
Figure 2
Chaperone Expression in Early and Advanced Experimental Atherosclerosis
Aortic samples from apolipoprotein E⫺/⫺ mice fed a Western diet for up to 40 weeks and a chow diet for 69 weeks. As presented in the upper panels, there is an increase in the expression of heat shock protein (HSP) 60 and HSP70 by immunohistochemistry from 3 to 20 weeks. Immunoblotting, as presented in the lower panels, confirms these findings and shows that this increase in expression is specific for lesion sites and decreases in chronic lesions of aged mice. Images used with permission of the American Heart Association (16).
2006
Figure 3
Herrmann et al. Protein Quality and Atherosclerosis
JACC Vol. 51, No. 21, 2008 May 27, 2008:2003–10
Ubiquitin Expression and Proteasome Activity in Advanced Atherosclerosis
Increase in ubiquitin immunoreactivity in complicated plaques of fatal acute myocardial infarction-related coronary arteries compared with advanced plaques in the noninfarction-related coronary arteries of the same patient, relating to differences in the shoulder and fibrous cap areas (left). Images on the left and data for the bottom left graph adapted, with permission, from Herrmann et al. (17). In carotid artery plaques of patients with symptoms of transient ischemic attack (TIA), stroke, or amaurosis (Am.) fugax, the level of ubiquitin-protein conjugates, but not of free ubiquitin, is higher and proteasome function is lower than in carotid plaques from asymptomatic patients (right). Graphs on the right used with permission of the American Heart Association (19).
There is also evidence for stimulation of the UPS in atherosclerosis. Coronary artery plaques associated with fatal acute myocardial infarction are characterized by increased expression of ubiquitin/ubiquitinated proteins, especially in the shoulder and cap regions (Fig. 3) (17). Similarly, carotid artery plaques from patients with symptoms of focal cerebral ischemia display a higher level of ubiquitinated proteins than those obtained from asymptomatic patients (Fig. 3) (18,19). In experimental models of atherosclerosis, we and others demonstrated that levels of ubiquitinated proteins start to increase at early disease stages (20,21). Furthermore, there is evidence of a concomitant increase in proteasome activity in experimental hypercholesterolemia and hyperglycemia, probably to compensate for the increased amount of substrates for the system, including misfolded proteins, under those circumstances (21–23).
Along these lines, activation of the unfolded protein response has been reported as a very early phenomenon in atherosclerosis (24). Cholesterol accumulation and oxidative stress products such as peroxynitrite seem to be the most potent inducers for the unfolded protein response in macrophages and endothelial cells, respectively (25–27). In these cells, activation of the unfolded protein response has been linked to cytotoxicity but might not be sufficient to trigger cell death by itself (24,28). How the unfolded protein response directs to life or death of a cell is currently incompletely understood. Nevertheless, there is consistency in the view that cell death pathways are activated under circumstances of unmitigated or overwhelming ER stress. Taken together, there is up-regulation of the expression and activity of chaperones, the UPS, and the unfolded protein response with overlapping features in atherosclerosis. These findings point to an increased demand of protein quality
Herrmann et al. Protein Quality and Atherosclerosis
JACC Vol. 51, No. 21, 2008 May 27, 2008:2003–10
2007
mechanisms to prevent the vascular accumulation of misfolded proteins increasingly generated by oxidative stress and other factors. The compensatory efforts might be successful in the beginning but might not suffice over a long period of time. Protein Quality Disease Aspects of Atherosclerosis Autopsy-based studies highlighted lower-level expression of HSPs in complicated, acellular, and collagenous plaques in both human disease and experimental models (15,29). Furthermore, in hypercholesterolemic apolipoprotein E-deficient mice, HSP expression decreases in the more advanced plaques before the development of complications (Fig. 2) (16). These findings indicate that chaperone function may become relatively insufficient in the aged and advanced disease stages. Although the activity of the ubiquitin system remains seemingly unimpaired even in advanced atherosclerotic plaques, increase as well as decrease in proteasome activity has been observed in symptomatic carotid artery plaques (18,19). In a large animal model, we have recently demonstrated that chronic inhibition of the proteasome contributes to endogenous oxidative stress and atherogenesis (22). In contrast, other groups have noted that in vivo or ex vivo proteasome inhibition for a relatively short period of time yields beneficial vascular effects (23,30,31). The complexity and diversity of the data is recaptured in the in vitro finding that high concentrations of oxidized low-density lipoprotein (LDL) lead to an initial transient activation of proteasome function followed by a sustained decay of proteasome activity and that proteasome inhibition potentiates the cytotoxic effects of oxidized LDL (32). Intensity and duration of the modulation of proteasome function and its specific environment are seemingly key variables for a number of biological processes, and their interaction determines the ultimate consequences for cells and tissues. If protein quality mechanisms decreased in their activity over time, one would expect accumulation and aggregation of misfolded proteins in “aged” arteries. If causally related to atherogenesis, these changes might also be particularly prominent in atherosclerotic lesions. As recently shown in experimental hypercholesterolemia, oxidatively modified and ubiquitinated proteins accumulate in the vascular wall, especially with inhibition of the compensatory increase in proteasome activity (22). Under those circumstances, prominent intimal thickening can be observed as well (22). With progression of disease, deposition of amyloid can be found in the intima adjacent to atherosclerotic plaques in up to one-half of the cases and to a similar extent within the plaque area, mainly in the necrotic core regions (Fig. 4) (33,34). Accumulation of intracellular proteins, modified by oxidation, nitration, phosphorylation, or (as particularly prominent in diabetes) glycylation, may represent part of the amyloidogenic burden. Apolipoproteins constitute the most important extracellular source for amyloid fibrils in atherosclerotic plaques. This relates to mutations as well as their limited conformational stability in the absence of
Figure 4
Amyloid Staining in Advanced Human Atherosclerosis
Advanced human atherosclerotic plaques in the aorta reacting with antiserum to apolipoprotein A-1 (A and C) (bar ⫽ 50 m) in colocalization with amyloid, which was visualized as green birefringence by Congo red staining (B and D). Images used with permission of John Wiley & Sons (33). Hematoxylin-eosin staining of an advanced carotid atherosclerotic plaque (E), exhibiting green birefringence by Congo red staining as an indicator of amyloid deposition in areas near the shoulder (F) and the lipid core (G) in the absence of primary amyloidosis (original magnifications: E: ⫻2; F, G: ⫻5).
lipids, which can result from enzymatic or oxidative particle modification, dissociation, or displacement (35,36). Furthermore, members of the cathepsin family of proteases may cleave apolipoproteins to generate amyloid-prone fragments (37). Apolipoprotein amyloidosis may therefore be simply a reflection of lipoprotein metabolism. However, fibrillar amyloid-like proteins have been shown to stimulate CD36 signaling in atherosclerotic plaques, thereby assuming a more active role in tissue inflammation and ROS production (38). Finally, the load of amyloidogenic particles in atherosclerotic plaques might be increased by platelet-derived and locally expressed amyloid precursor protein (APP), which has been linked to a number of vascular changes. The APP processing leads to formation of amyloid beta protein (Abeta), causally related to Alzheimer’s disease. Intriguingly, overexpression of a mutant form of APP in
2008
Figure 5
Herrmann et al. Protein Quality and Atherosclerosis
JACC Vol. 51, No. 21, 2008 May 27, 2008:2003–10
Vascular Changes Due to Overexpression of Mutant Amyloid Precursor Protein
Compared with normal cerebral vascular anatomy and flow dynamics in 10-month-old wild mice (A), magnetic resonance angiography shows minor flow voids (arrow) in 6-monthold mice overexpressing a mutant form of amyloid precursor protein (APP23) with fairly preserved structures on corrosion casts (B). Twenty-month-old APP23 mice display significant flow abnormalities, including partial and complete flow voids (C). The corresponding corrosion cast demonstrates constriction, inclusions, and vessel elimination (D). Also noteworthy is the presence of a small artery connecting the left and the right sides of the circle of Willis (# in panels C and D) likely to maintain a dimunitive level of blood flow under the circumstance of vessel substitution at the posterior cerebral artery level. Images used with permission of the Society of Neuroscience (39).
mice (Tg-APP23) leads to progressive flow abnormalities within the cerebral circulation on magnetic resonance angiography, relating to vessel constriction, deformation, and closure (Fig. 5) (39). Moreover, impairment of endotheliumdependent vasorelaxation of carotid arteries and the aorta can be found in related mice (Tg2576) and is attenuated by endothelin-receptor antagonism (40). Likewise in a mouse model, systemic infusion of soluble Abeta leads to an increase in vascular resistance and a decrease in cerebral perfusion (41). These findings correspond to the provasoconstricting/ antivasorelaxing properties of Abeta, which were documented in in vitro studies and ultimately attributed to Abeta-induced inhibitory signaling on endothelial nitric oxide synthase activity (42). Of further note, histologic studies confirm the capacity of Abeta to cause structural damage of endothelial cells and to induce tissue inflammation (43,44). Intracellular accumulation of Abeta can also lead to mitochondrial dysfunction, with release of free radicals and its important implications (11). It can also impair proteasome function, resulting in further Abeta accumulation (11). As amyloid deposition progresses, so do the degenerative changes of the vascular wall. These include aneurysmal dilation, vascular rupture, and hemorrhage on the one end and narrowing and occlusion of vessels on the
other end of the spectrum. In its entirety, this vascular process has become known as cerebral amyloid angiopathy and is frequently seen in patients with Alzheimer’s disease (45). In fact, the neuropsychologic deficits of Alzheimer’s disease seem to correlate better with the degree of amyloid deposition in the cerebral vessels than in the brain. Last but not least, learning disabilities and cerebral Abeta deposits are greater in atherosclerosis-susceptible APPoverexpressing Tg2576 mice fed an atherogenic diet, and the extent of aortic atherosclerosis is enhanced by the APPtransgenic trait and correlates with the cerebral Abeta burden in these mice (46). In summary, nonfibrillar and fibrillar forms of the classic amyloidogenic protein Abeta have been shown to cause endothelial injury and dysfunction as well as progressive structural changes of the entire vascular wall in the context of cerebral amyloid angiopathy over time. Similar degenerative changes can be observed in atherosclerosis, and synergism has been suggested in the pathophysiology of Alzheimer’s disease and atherosclerosis. Within the framework of protein quality diseases, the accumulation of damaged intracellular proteins and the deposition of amyloidogenic extracellular proteins may be of significance for the atherosclerotic disease process (Fig. 6).
Herrmann et al. Protein Quality and Atherosclerosis
JACC Vol. 51, No. 21, 2008 May 27, 2008:2003–10
Figure 6
2009
Illustration of Atherosclerosis as a Protein Quality Disease
In the initial phase of cardiovascular risk factor exposure, compensatory up-regulation of chaperones and the ubiquitin-proteasome system (UPS) prevents the overwhelming intracellular accumulation of damaged and dysfunctional proteins. The UPS activity also contributes to the classical activation pathway of nuclear factor kappa-B and thereby to inflammation and cell proliferation. With the formation and growth of a metabolically active atherosclerotic plaque, there is further production of misfolded and damaged proteins in the progression phase. Once the classical protein quality mechanisms are overwhelmed and fail, these dysfunctional proteins accumulate (and aggregate) and autophagy remains the final clearance pathway. The accumulating proteins can undergo further oxidation, ubiquitination, and cross-linking. As yet another unique characteristic, beta-pleated sheets can be formed and hence amyloid fibrils via the intermediate steps of pre-amyloid oligomers and protofibrils. In addition to intracellular proteins, proteins in the extracellular matrix can undergo conformational changes. For instance, oxidation and phospholipid hydrolysis of lowdensity lipoprotein (LDL) produces oxidatively modified and electronegative particles with unfolding of the apolipoprotein components (electron microscopic images used with permission of Elsevier Science [35]). The generation of amyloid-like structures in this process serves as a potent “key” to the uptake of these modified LDLs by macrophages via scavenger receptors (37). Recognition of amyloid-like fibrils by CD36 (and conceivably the receptor for advanced glycation end-products) leads to the production of reactive oxygen species, chemokines, and cytokines, which contributes further to the atherosclerotic disease process, including its complication phase.
Future Cardiovascular Perspective Certainly, one of the central remaining questions is whether the accumulation of dysfunctional proteins is a causal factor, a contributing factor, or simply an epiphenomenon in atherosclerosis, similar to the current discussion in heart failure (47,48). Mice expressing an anchorless prion protein that is insoluble and resistant to proteolysis develop cardiomyopathy, which constitutes the strongest experimental evidence so far for the detrimental consequences of protein precipitation on cardiac function (49). Intriguingly, in this model, amyloid deposition can be seen primarily within and around endothelial cells (49,50). Further studies on the vasofunctional and vasostructural consequences of these findings would be very helpful in attesting that protein precipitation by itself exerts a pathophysiological and notably atherogenic momentum. Positive results would prompt further studies on how proteins can escape protein quality control mechanisms and perturb vascular homeostasis in the human vasculature. These studies should be complemented by gain-of-function and loss-of-function studies
on central components of the 2 arms of the protein quality control system. Clinical studies evaluating the effect of selective positive or negative modulation of the systems in relation to the stage of disease will give final answers from a human disease and therapy standpoint. With this perspective, the viewpoint of atherosclerosis as a protein quality disease may contribute to current theory and future treatment of atherosclerotic cardiovascular disease. Reprint requests and correspondence: Dr. Joerg Herrmann, Department of Internal Medicine, Mayo Clinic Rochester, 200 First Street Southwest, Rochester, Minnesota 55905. E-mail: herrmann. [email protected].
REFERENCES
1. Bukau B, Weissman J, Horwich A. Molecular chaperones and protein quality control. Cell 2006;125:443–51. 2. Herrmann J, Lerman LO, Lerman A. Ubiquitin and ubiquitin-like proteins in protein regulation. Circ Res 2007;100:1276 –91. 3. Wickner S, Maurizi MR, Gottesman S. Posttranslational quality control: folding, refolding, and degrading proteins. Science 1999;286: 1888 –93.
2010
Herrmann et al. Protein Quality and Atherosclerosis
4. Ron D, Walter P. Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol 2007;8:519 –29. 5. Bernales S, McDonald KL, Walter P. Autophagy counterbalances endoplasmic reticulum expansion during the unfolded protein response. PLoS Biol 2006;4:e423. 6. Carrell RW, Lomas DA. Conformational disease. Lancet 1997;350: 134 – 8. 7. Taylor JP, Hardy J, Fischbeck KH. Toxic proteins in neurodegenerative disease. Science 2002;296:1991–5. 8. Ross CA, Poirier MA. Protein aggregation and neurodegenerative disease. Nat Med 2004;10 Suppl:S10 –7. 9. Forman MS, Trojanowski JQ, Lee VM. Neurodegenerative diseases: a decade of discoveries paves the way for therapeutic breakthroughs. Nat Med 2004;10:1055– 63. 10. Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 2002;297: 353– 6. 11. LaFerla FM, Green KN, Oddo S. Intracellular amyloid-beta in Alzheimer’s disease. Nat Rev Neurosci 2007;8:499 –509. 12. Bucciantini M, Giannoni E, Chiti F, et al. Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature 2002;416:507–11. 13. Stocker R, Keaney JF Jr. Role of oxidative modifications in atherosclerosis. Physiol Rev 2004;84:1381– 478. 14. 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;142:1927–37. 15. Berberian PA, Myers W, Tytell M, Challa V, Bond MG. Immunohistochemical localization of heat shock protein-70 in normalappearing and atherosclerotic specimens of human arteries. Am J Pathol 1990;136:71– 80. 16. Kanwar RK, Kanwar JR, Wang D, Ormrod DJ, Krissansen GW. Temporal expression of heat shock proteins 60 and 70 at lesion-prone sites during atherogenesis in ApoE-deficient mice. Arterioscler Thromb Vasc Biol 2001;21:1991–7. 17. Herrmann J, Edwards WD, Holmes DR Jr., et al. Increased ubiquitin immunoreactivity in unstable atherosclerotic plaques associated with acute coronary syndromes. J Am Coll Cardiol 2002;40:1919 –27. 18. Marfella R, D’Amico M, Di Filippo C, et al. Increased activity of the ubiquitin-proteasome system in patients with symptomatic carotid disease is associated with enhanced inflammation and may destabilize the atherosclerotic plaque: effects of rosiglitazone treatment. J Am Coll Cardiol 2006;47:2444 –55. 19. Versari D, Herrmann J, Gossl M, et al. Dysregulation of the ubiquitin-proteasome system in human carotid atherosclerosis. Arterioscler Thromb Vasc Biol 2006;26:2132–9. 20. Herrmann J, Gulati R, Napoli C, et al. Oxidative stress-related increase in ubiquitination in early coronary atherogenesis. FASEB J 2003;17:1730 –2. 21. Tan C, Li Y, Tan X, Pan H, Huang W. Inhibition of the ubiquitinproteasome system: a new avenue for atherosclerosis. Clin Chem Lab Med 2006;44:1218 –25. 22. Herrmann J, Saguner AM, Versari D, et al. Chronic proteasome inhibition contributes to coronary atherosclerosis. Circ Res 2007;101: 865–74. 23. Xu J, Wu Y, Song P, Zhang M, Wang S, Zou MH. Proteasomedependent degradation of guanosine 5=-triphosphate cyclohydrolase I causes tetrahydrobiopterin deficiency in diabetes mellitus. Circulation 2007;116:944 –53. 24. Zhou J, Lhotak S, Hilditch BA, Austin RC. Activation of the unfolded protein response occurs at all stages of atherosclerotic lesion development in apolipoprotein E-deficient mice. Circulation 2005; 111:1814 –21. 25. Feng B, Yao PM, Li Y, et al. The endoplasmic reticulum is the site of cholesterol-induced cytotoxicity in macrophages. Nat Cell Biol 2003; 5:781–92. 26. Dickhout JG, Hossain GS, Pozza LM, Zhou J, Lhotak S, Austin RC. Peroxynitrite causes endoplasmic reticulum stress and apoptosis in human vascular endothelium: implications in atherogenesis. Arterioscler Thromb Vasc Biol 2005;25:2623–9.
JACC Vol. 51, No. 21, 2008 May 27, 2008:2003–10 27. Gargalovic PS, Gharavi NM, Clark MJ, et al. The unfolded protein response is an important regulator of inflammatory genes in endothelial cells. Arterioscler Thromb Vasc Biol 2006;26:2490 – 6. 28. Horke S, Witte I, Wilgenbus P, Kruger M, Strand D, Forstermann U. Paraoxonase-2 reduces oxidative stress in vascular cells and decreases endoplasmic reticulum stress-induced caspase activation. Circulation 2007;115:2055– 64. 29. 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. 30. Meiners S, Laule M, Rother W, et al. Ubiquitin-proteasome pathway as a new target for the prevention of restenosis. Circulation 2002;105: 483–9. 31. Stangl V, Lorenz M, Meiners S, et al. Long-term up-regulation of eNOS and improvement of endothelial function by inhibition of the ubiquitin-proteasome pathway. Faseb J 2004;18:272–9. 32. Vieira O, Escargueil-Blanc I, Jurgens G, et al. Oxidized LDLs alter the activity of the ubiquitin-proteasome pathway: potential role in oxidized LDL-induced apoptosis. FASEB J 2000;14:532– 42. 33. Mucchiano GI, Haggqvist B, Sletten K, Westermark P. Apolipoprotein A-1– derived amyloid in atherosclerotic plaques of the human aorta. J Pathol 2001;193:270 –5. 34. Rocken C, Tautenhahn J, Buhling F, et al. Prevalence and pathology of amyloid in atherosclerotic arteries. Arterioscler Thromb Vasc Biol 2006;26:676 –7. 35. Ursini F, Davies KJ, Maiorino M, Parasassi T, Sevanian A. Atherosclerosis: another protein misfolding disease? Trends Mol Med 2002; 8:370 – 4. 36. Stewart CR, Tseng AA, Mok YF, et al. Oxidation of low-density lipoproteins induces amyloid-like structures that are recognized by macrophages. Biochemistry 2005;44:9108 –16. 37. Howlett GJ, Moore KJ. Untangling the role of amyloid in atherosclerosis. Curr Opin Lipidol 2006;17:541–7. 38. Medeiros LA, Khan T, El Khoury JB, et al. Fibrillar amyloid protein present in atheroma activates CD36 signal transduction. J Biol Chem 2004;279:10643– 8. 39. Beckmann N, Schuler A, Mueggler T, et al. Age-dependent cerebrovascular abnormalities and blood flow disturbances in APP23 mice modeling Alzheimer’s disease. J Neurosci 2003;23:8453–9. 40. Elesber AA, Bonetti PO, Woodrum JE, et al. Bosentan preserves endothelial function in mice overexpressing APP. Neurobiol Aging 2006;27:446 –50. 41. Suo Z, Humphrey J, Kundtz A, et al. Soluble Alzheimers betaamyloid constricts the cerebral vasculature in vivo. Neurosci Lett 1998;257:77– 80. 42. Gentile MT, Vecchione C, Maffei A, et al. Mechanisms of soluble beta-amyloid impairment of endothelial function. J Biol Chem 2004; 279:48135– 42. 43. Thomas T, McLendon C, Sutton ET, Thomas G. Cerebrovascular endothelial dysfunction mediated by beta-amyloid. Neuroreport 1997; 8:1387–91. 44. Thomas T, Sutton ET, Bryant MW, Rhodin JA. In vivo vascular damage, leukocyte activation and inflammatory response induced by beta-amyloid. J Submicrosc Cytol Pathol 1997;29:293–304. 45. Revesz T, Ghiso J, Lashley T, et al. Cerebral amyloid angiopathies: a pathologic, biochemical, and genetic view. J Neuropathol Exp Neurol 2003;62:885–98. 46. Li L, Cao D, Garber DW, Kim H, Fukuchi K. Association of aortic atherosclerosis with cerebral beta-amyloidosis and learning deficits in a mouse model of Alzheimer’s disease. Am J Pathol 2003;163:2155– 64. 47. Wang X, Robbins J. Heart failure and protein quality control. Circ Res 2006;99:1315–28. 48. Patterson C, Ike C, Willis PWt, Stouffer GA, Willis MS. The bitter end: the ubiquitin-proteasome system and cardiac dysfunction. Circulation 2007;115:1456 – 63. 49. Trifilo MJ, Yajima T, Gu Y, et al. Prion-induced amyloid heart disease with high blood infectivity in transgenic mice. Science 2006;313:94 –7. 50. Chesebro B, Trifilo M, Race R, et al. Anchorless prion protein results in infectious amyloid disease without clinical scrapie. Science 2005;308:1435–9.
Vol. 51, No. 21, 2008 ISSN 0735-1097/08/$34.00 doi:10.1016/j.jacc.2008.02.047
STATE-OF-THE-ART PAPER
Potential Role of the Ubiquitin-Proteasome System in Atherosclerosis Aspects of a Protein Quality Disease Joerg Herrmann, MD, Sandra M. Soares, MD, Lilach O. Lerman, MD, PHD, Amir Lerman, MD Rochester, Minnesota Misfolded or damaged proteins are recognized intracellularly by protein quality mechanisms. These include chaperones and the ubiquitin-proteasome system, which aim at restoration of protein function and protein removal, respectively. A number of studies have outlined the functional significance of the ubiquitin-proteasome system for the heart and, as of recently, for the vascular system. This review summarizes these recent findings with a focus on atherosclerosis. In particular, this paper reflects on the viewpoint of atherosclerosis as a protein quality disease. (J Am Coll Cardiol 2008;51:2003–10) © 2008 by the American College of Cardiology Foundation
The structure and function of cells and related organs, including the cardiovascular system, is determined to a significant degree by the structure and function of their proteins. The synthesis of new proteins is balanced by the degradation of those that have become disposable for various reasons. Recent research findings have highlighted the significance of these biological processes for cardiovascular diseases. The present review will summarize these advances, introducing the viewpoint of atherosclerosis as a protein quality disease. Protein Quality Mechanisms The quality of a protein and its function relies on its 3-dimensional structure and folding dynamics. The loss of proper 3-dimensional arrangement, referred to as unfolding, impairs not only protein function but eventually also cellular function and viability. To prevent these detrimental consequences, each cell is equipped with 2 operational systems: chaperones and proteases. Chaperones recognize protein substrates based on hydrophobic protein regions, which are usually buried within the tertiary and quarternary structures but surface as the protein structure unfolds after translational or post-translational damage. Variations in the protein sequences that flank these hydrophobic sequences determine the large number and various types of chaperones across the different cellular compartments (1). Their main function is to hold misfolded From the Department of Internal Medicine, Mayo Clinic, Rochester, Minnesota. Supported by the National Institutes of Health (R01 HL63911-04, K24 HL6984001), the Mayo Stiftung, and the Mayo Foundation. Manuscript received November 7, 2007; revised manuscript received January 28, 2008, accepted February 12, 2008.
proteins, to fold them back into their native structure, and to promote the degradation of proteins resistant to this process by making them more accessible to proteases. This latter group of enzymes cleaves proteins based on amino acid sequence patterns. Up to 90% of all intracellular proteins are degraded, both in a chaperone-dependent and in a chaperone-independent manner, by a specialized protease system, called the ubiquitin-proteasome system (UPS) (2). This system operates mainly in the cytoplasm and recognizes protein substrates, like chaperones, on the basis of bulky hydrophobic residues but also based on basic residues at the NH2-terminus (so-called N-end rule). Thus, numerous chaperones and the UPS work independently and in synergy to assure the quality of intracellular proteins. The kinetics of the interactions between chaperones, the UPS, and damaged proteins were summarized in a triage model (Fig. 1) (3). One particular subtype of protein quality control mechanisms is the unfolded protein response (4). This response is triggered by the accumulation of unfolded or misfolded proteins in the lumen of the endoplasmic reticulum (ER), which is also referred to as ER stress and leads to the modification of transducer proteins in the ER membrane and the activation of their related pathways. These pathways include the inositol-requiring protein 1, the activating transcription factor-6, and the protein kinase ribonucleic acid-like kinase signaling pathways. Collectively, these pathways orchestrate the down-regulation of the synthesis and the translocation of proteins into the ER, which leads to a reduction in protein load. They also mediate the expression of chaperones and proteinmodifying enzymes, which facilitates protein refolding. Furthermore, they engage the endoplasmic reticulumassociated protein degradation pathway, which entails the
2004
Herrmann et al. Protein Quality and Atherosclerosis
retrotranslocation of unfolded proteins into the cytosol and their proteolysis by the proteaAbeta ⴝ amyloid beta some. In case these mechanisms protein fail to resolve the mismatch beAPP ⴝ amyloid precursor tween protein load and handling protein capacity over a period of time, ER ⴝ endoplasmic the unfolded protein response inreticulum duces autophagy as an alternative ERAD ⴝ endoplasmic reticulum-associated coping mechanism. This process protein degradation involves the sequestration of the HSP ⴝ heat shock protein ER into membrane-bounded compartments (autophagosomes), LDL ⴝ low-density lipoprotein its fusion with lysosomes, and the ROS ⴝ reactive oxygen degradation of its content. The species ER-specific autophagy is seemTIA ⴝ transient ischemic ingly essential for cells to survive attack severe ER stress (5). However, UPS ⴝ ubiquitinwith overwhelming ER stress cell proteasome system death pathways are nevertheless activated. In summary, cells are equipped with a robust apparatus to repair or remove damaged proteins. This includes chaperones, proteases, and the ER-related unfolded protein response along the general concept of protein triaging. The competitive balance between the activity of protein quality control mechAbbreviations and Acronyms
Figure 1
JACC Vol. 51, No. 21, 2008 May 27, 2008:2003–10
anisms, on the one hand, and the generation of low-quality proteins, on the other hand, is the hallmark of this concept.
Protein Quality Diseases Protein quality diseases, also called protein misfolding, precipitation, or conformational diseases, are a group of disorders that are associated with and occasionally caused by the conformational change, aggregation, and deposition of 1 or more proteins (6). Neurodegenerative diseases were the first prominent example for this entity. Mutation analyses in familial counterparts to sporadic neurologic disorders and experimental studies highlighted the pathogenetic role of the accumulation of misfolded proteins in these disease processes (7,8). Research efforts also showed that the accumulation of these proteins is due to their increased production, abnormal processing, and/or decreased elimination via chaperones, the UPS, or the unfolded protein response (8,9). The “amyloid hypothesis” of neurodegenerative diseases links the aggregation of misfolded proteins into ordered protease-resistant structures, called amyloid fibrils, to aberrant protein interactions that culminate in neuronal dysfunction and ultimately neurodegeneration (10). Indeed, the pathological momentum seems to be with the very process of amyloid formation.
Protein Triage System
Because of translational or post-translational damage, proteins can become misfolded, leading to surface exposure of hydrophobic regions. These regions are recognition sites for chaperones, which aim at restoring the native protein structure or triage to the ubiquitin-proteasome system (UPS). This system can also recognize damaged proteins directly and mediates the attachment of at least 4 ubiquitin molecules, which directs proteins to degradation by the proteasome. Deubiquitinating enzymes can reverse the ubiquitin-chain degradation signal, and damaged proteins receive a new chance for refolding. Some proteins, such as oxidatively damaged proteins, can be recognized directly by the proteasome complex. A protein’s propensity to fold to a state with particular binding characteristics, including the affinity for chaperones versus proteases, will determine its fate. This includes restoration of protein structure and function, protein degradation, and protein cross-linking and aggregation.
Herrmann et al. Protein Quality and Atherosclerosis
JACC Vol. 51, No. 21, 2008 May 27, 2008:2003–10
Amyloid formation is an ordered process and begins when misfolded proteins start to assume a beta-pleated sheet structure and self-associate to form soluble preamyloid oligomers. As these soluble preamyloid oligomers start to form protofibrils and coalesce, classic amyloid fibrils, plaques, and tangles are formed, which then exhibit Congo red-positive staining. Classic amyloid plaques are therefore a late and final reflection of a process that remains undetected in its peak toxicity. Consistent with this view, neurologic disease precedes the histologic appearance of classic amyloid plaques. As another implication, the focus of attention has shifted from the large aggregates and microscopic hallmarks to the small aggregates and molecular processes of the pre-amyloid stage both extracellularly and intracellularly (11). Moreover, there has been the realization that the process of aggregation is not inherent to only a few proteins but can be observed with various proteins once misfolding remains unresolved (12). In summary, unfolded/misfolded proteins can accumulate in cells and tissues when they are produced in large amounts and/or not cleared sufficiently. These proteins then form aggregates, which can remove amino acids from the recycling pool, tie up chaperones and proteases, serve as foci for the aggregation of other unrelated proteins, and finally disrupt
Aortic sinus 3-week-old mice
2005
cellular and tissue functions. Over time, the accumulation of low-quality proteins can therefore result in disease, a so-called protein quality disease. Precipitates such as amyloid are characteristic late-stage histologic fingerprints. Protein Quality Control in Atherosclerosis Besides inflammation, excess generation of reactive oxygen species (ROS), so-called oxidative stress, is considered to be an important element in the pathophysiology of atherosclerosis (13). Among the various effects, oxidative stress has been shown to increase the expression of chaperones, including those of the family of heat shock proteins (HSPs). The HSPs most closely associated with atherosclerosis include HSP60 and HSP70. The expression of HSP60 in the vascular wall correlates with the severity of atherosclerosis and is particularly prominent in the shoulder regions and around necrotic cores of atherosclerotic plaques (14). The HSP70 is mainly expressed in more advanced atheromas around sites of necrosis and lipid accumulation (15). Importantly, these findings can be reproduced experimentally in hypercholesterolemic apolipoprotein E⫺/⫺ mice, in which the expression of both HSPs increases in the early and progressive disease stages (Fig. 2) (16).
Aorta commissure 8-week-old mice
Abdominal aorta 20-week-old mice
Aortic sinus 69-week-old mice
A
B
C
D
E
F
G
H
HSP 60
HSP 70
Aortic non-lesions 3-8-weeks old mice
Aortic lesions 3-20-weeks old mice
Aortic lesions 40-69-weeks old mice
HSP 60
HSP 70
Figure 2
Chaperone Expression in Early and Advanced Experimental Atherosclerosis
Aortic samples from apolipoprotein E⫺/⫺ mice fed a Western diet for up to 40 weeks and a chow diet for 69 weeks. As presented in the upper panels, there is an increase in the expression of heat shock protein (HSP) 60 and HSP70 by immunohistochemistry from 3 to 20 weeks. Immunoblotting, as presented in the lower panels, confirms these findings and shows that this increase in expression is specific for lesion sites and decreases in chronic lesions of aged mice. Images used with permission of the American Heart Association (16).
2006
Figure 3
Herrmann et al. Protein Quality and Atherosclerosis
JACC Vol. 51, No. 21, 2008 May 27, 2008:2003–10
Ubiquitin Expression and Proteasome Activity in Advanced Atherosclerosis
Increase in ubiquitin immunoreactivity in complicated plaques of fatal acute myocardial infarction-related coronary arteries compared with advanced plaques in the noninfarction-related coronary arteries of the same patient, relating to differences in the shoulder and fibrous cap areas (left). Images on the left and data for the bottom left graph adapted, with permission, from Herrmann et al. (17). In carotid artery plaques of patients with symptoms of transient ischemic attack (TIA), stroke, or amaurosis (Am.) fugax, the level of ubiquitin-protein conjugates, but not of free ubiquitin, is higher and proteasome function is lower than in carotid plaques from asymptomatic patients (right). Graphs on the right used with permission of the American Heart Association (19).
There is also evidence for stimulation of the UPS in atherosclerosis. Coronary artery plaques associated with fatal acute myocardial infarction are characterized by increased expression of ubiquitin/ubiquitinated proteins, especially in the shoulder and cap regions (Fig. 3) (17). Similarly, carotid artery plaques from patients with symptoms of focal cerebral ischemia display a higher level of ubiquitinated proteins than those obtained from asymptomatic patients (Fig. 3) (18,19). In experimental models of atherosclerosis, we and others demonstrated that levels of ubiquitinated proteins start to increase at early disease stages (20,21). Furthermore, there is evidence of a concomitant increase in proteasome activity in experimental hypercholesterolemia and hyperglycemia, probably to compensate for the increased amount of substrates for the system, including misfolded proteins, under those circumstances (21–23).
Along these lines, activation of the unfolded protein response has been reported as a very early phenomenon in atherosclerosis (24). Cholesterol accumulation and oxidative stress products such as peroxynitrite seem to be the most potent inducers for the unfolded protein response in macrophages and endothelial cells, respectively (25–27). In these cells, activation of the unfolded protein response has been linked to cytotoxicity but might not be sufficient to trigger cell death by itself (24,28). How the unfolded protein response directs to life or death of a cell is currently incompletely understood. Nevertheless, there is consistency in the view that cell death pathways are activated under circumstances of unmitigated or overwhelming ER stress. Taken together, there is up-regulation of the expression and activity of chaperones, the UPS, and the unfolded protein response with overlapping features in atherosclerosis. These findings point to an increased demand of protein quality
Herrmann et al. Protein Quality and Atherosclerosis
JACC Vol. 51, No. 21, 2008 May 27, 2008:2003–10
2007
mechanisms to prevent the vascular accumulation of misfolded proteins increasingly generated by oxidative stress and other factors. The compensatory efforts might be successful in the beginning but might not suffice over a long period of time. Protein Quality Disease Aspects of Atherosclerosis Autopsy-based studies highlighted lower-level expression of HSPs in complicated, acellular, and collagenous plaques in both human disease and experimental models (15,29). Furthermore, in hypercholesterolemic apolipoprotein E-deficient mice, HSP expression decreases in the more advanced plaques before the development of complications (Fig. 2) (16). These findings indicate that chaperone function may become relatively insufficient in the aged and advanced disease stages. Although the activity of the ubiquitin system remains seemingly unimpaired even in advanced atherosclerotic plaques, increase as well as decrease in proteasome activity has been observed in symptomatic carotid artery plaques (18,19). In a large animal model, we have recently demonstrated that chronic inhibition of the proteasome contributes to endogenous oxidative stress and atherogenesis (22). In contrast, other groups have noted that in vivo or ex vivo proteasome inhibition for a relatively short period of time yields beneficial vascular effects (23,30,31). The complexity and diversity of the data is recaptured in the in vitro finding that high concentrations of oxidized low-density lipoprotein (LDL) lead to an initial transient activation of proteasome function followed by a sustained decay of proteasome activity and that proteasome inhibition potentiates the cytotoxic effects of oxidized LDL (32). Intensity and duration of the modulation of proteasome function and its specific environment are seemingly key variables for a number of biological processes, and their interaction determines the ultimate consequences for cells and tissues. If protein quality mechanisms decreased in their activity over time, one would expect accumulation and aggregation of misfolded proteins in “aged” arteries. If causally related to atherogenesis, these changes might also be particularly prominent in atherosclerotic lesions. As recently shown in experimental hypercholesterolemia, oxidatively modified and ubiquitinated proteins accumulate in the vascular wall, especially with inhibition of the compensatory increase in proteasome activity (22). Under those circumstances, prominent intimal thickening can be observed as well (22). With progression of disease, deposition of amyloid can be found in the intima adjacent to atherosclerotic plaques in up to one-half of the cases and to a similar extent within the plaque area, mainly in the necrotic core regions (Fig. 4) (33,34). Accumulation of intracellular proteins, modified by oxidation, nitration, phosphorylation, or (as particularly prominent in diabetes) glycylation, may represent part of the amyloidogenic burden. Apolipoproteins constitute the most important extracellular source for amyloid fibrils in atherosclerotic plaques. This relates to mutations as well as their limited conformational stability in the absence of
Figure 4
Amyloid Staining in Advanced Human Atherosclerosis
Advanced human atherosclerotic plaques in the aorta reacting with antiserum to apolipoprotein A-1 (A and C) (bar ⫽ 50 m) in colocalization with amyloid, which was visualized as green birefringence by Congo red staining (B and D). Images used with permission of John Wiley & Sons (33). Hematoxylin-eosin staining of an advanced carotid atherosclerotic plaque (E), exhibiting green birefringence by Congo red staining as an indicator of amyloid deposition in areas near the shoulder (F) and the lipid core (G) in the absence of primary amyloidosis (original magnifications: E: ⫻2; F, G: ⫻5).
lipids, which can result from enzymatic or oxidative particle modification, dissociation, or displacement (35,36). Furthermore, members of the cathepsin family of proteases may cleave apolipoproteins to generate amyloid-prone fragments (37). Apolipoprotein amyloidosis may therefore be simply a reflection of lipoprotein metabolism. However, fibrillar amyloid-like proteins have been shown to stimulate CD36 signaling in atherosclerotic plaques, thereby assuming a more active role in tissue inflammation and ROS production (38). Finally, the load of amyloidogenic particles in atherosclerotic plaques might be increased by platelet-derived and locally expressed amyloid precursor protein (APP), which has been linked to a number of vascular changes. The APP processing leads to formation of amyloid beta protein (Abeta), causally related to Alzheimer’s disease. Intriguingly, overexpression of a mutant form of APP in
2008
Figure 5
Herrmann et al. Protein Quality and Atherosclerosis
JACC Vol. 51, No. 21, 2008 May 27, 2008:2003–10
Vascular Changes Due to Overexpression of Mutant Amyloid Precursor Protein
Compared with normal cerebral vascular anatomy and flow dynamics in 10-month-old wild mice (A), magnetic resonance angiography shows minor flow voids (arrow) in 6-monthold mice overexpressing a mutant form of amyloid precursor protein (APP23) with fairly preserved structures on corrosion casts (B). Twenty-month-old APP23 mice display significant flow abnormalities, including partial and complete flow voids (C). The corresponding corrosion cast demonstrates constriction, inclusions, and vessel elimination (D). Also noteworthy is the presence of a small artery connecting the left and the right sides of the circle of Willis (# in panels C and D) likely to maintain a dimunitive level of blood flow under the circumstance of vessel substitution at the posterior cerebral artery level. Images used with permission of the Society of Neuroscience (39).
mice (Tg-APP23) leads to progressive flow abnormalities within the cerebral circulation on magnetic resonance angiography, relating to vessel constriction, deformation, and closure (Fig. 5) (39). Moreover, impairment of endotheliumdependent vasorelaxation of carotid arteries and the aorta can be found in related mice (Tg2576) and is attenuated by endothelin-receptor antagonism (40). Likewise in a mouse model, systemic infusion of soluble Abeta leads to an increase in vascular resistance and a decrease in cerebral perfusion (41). These findings correspond to the provasoconstricting/ antivasorelaxing properties of Abeta, which were documented in in vitro studies and ultimately attributed to Abeta-induced inhibitory signaling on endothelial nitric oxide synthase activity (42). Of further note, histologic studies confirm the capacity of Abeta to cause structural damage of endothelial cells and to induce tissue inflammation (43,44). Intracellular accumulation of Abeta can also lead to mitochondrial dysfunction, with release of free radicals and its important implications (11). It can also impair proteasome function, resulting in further Abeta accumulation (11). As amyloid deposition progresses, so do the degenerative changes of the vascular wall. These include aneurysmal dilation, vascular rupture, and hemorrhage on the one end and narrowing and occlusion of vessels on the
other end of the spectrum. In its entirety, this vascular process has become known as cerebral amyloid angiopathy and is frequently seen in patients with Alzheimer’s disease (45). In fact, the neuropsychologic deficits of Alzheimer’s disease seem to correlate better with the degree of amyloid deposition in the cerebral vessels than in the brain. Last but not least, learning disabilities and cerebral Abeta deposits are greater in atherosclerosis-susceptible APPoverexpressing Tg2576 mice fed an atherogenic diet, and the extent of aortic atherosclerosis is enhanced by the APPtransgenic trait and correlates with the cerebral Abeta burden in these mice (46). In summary, nonfibrillar and fibrillar forms of the classic amyloidogenic protein Abeta have been shown to cause endothelial injury and dysfunction as well as progressive structural changes of the entire vascular wall in the context of cerebral amyloid angiopathy over time. Similar degenerative changes can be observed in atherosclerosis, and synergism has been suggested in the pathophysiology of Alzheimer’s disease and atherosclerosis. Within the framework of protein quality diseases, the accumulation of damaged intracellular proteins and the deposition of amyloidogenic extracellular proteins may be of significance for the atherosclerotic disease process (Fig. 6).
Herrmann et al. Protein Quality and Atherosclerosis
JACC Vol. 51, No. 21, 2008 May 27, 2008:2003–10
Figure 6
2009
Illustration of Atherosclerosis as a Protein Quality Disease
In the initial phase of cardiovascular risk factor exposure, compensatory up-regulation of chaperones and the ubiquitin-proteasome system (UPS) prevents the overwhelming intracellular accumulation of damaged and dysfunctional proteins. The UPS activity also contributes to the classical activation pathway of nuclear factor kappa-B and thereby to inflammation and cell proliferation. With the formation and growth of a metabolically active atherosclerotic plaque, there is further production of misfolded and damaged proteins in the progression phase. Once the classical protein quality mechanisms are overwhelmed and fail, these dysfunctional proteins accumulate (and aggregate) and autophagy remains the final clearance pathway. The accumulating proteins can undergo further oxidation, ubiquitination, and cross-linking. As yet another unique characteristic, beta-pleated sheets can be formed and hence amyloid fibrils via the intermediate steps of pre-amyloid oligomers and protofibrils. In addition to intracellular proteins, proteins in the extracellular matrix can undergo conformational changes. For instance, oxidation and phospholipid hydrolysis of lowdensity lipoprotein (LDL) produces oxidatively modified and electronegative particles with unfolding of the apolipoprotein components (electron microscopic images used with permission of Elsevier Science [35]). The generation of amyloid-like structures in this process serves as a potent “key” to the uptake of these modified LDLs by macrophages via scavenger receptors (37). Recognition of amyloid-like fibrils by CD36 (and conceivably the receptor for advanced glycation end-products) leads to the production of reactive oxygen species, chemokines, and cytokines, which contributes further to the atherosclerotic disease process, including its complication phase.
Future Cardiovascular Perspective Certainly, one of the central remaining questions is whether the accumulation of dysfunctional proteins is a causal factor, a contributing factor, or simply an epiphenomenon in atherosclerosis, similar to the current discussion in heart failure (47,48). Mice expressing an anchorless prion protein that is insoluble and resistant to proteolysis develop cardiomyopathy, which constitutes the strongest experimental evidence so far for the detrimental consequences of protein precipitation on cardiac function (49). Intriguingly, in this model, amyloid deposition can be seen primarily within and around endothelial cells (49,50). Further studies on the vasofunctional and vasostructural consequences of these findings would be very helpful in attesting that protein precipitation by itself exerts a pathophysiological and notably atherogenic momentum. Positive results would prompt further studies on how proteins can escape protein quality control mechanisms and perturb vascular homeostasis in the human vasculature. These studies should be complemented by gain-of-function and loss-of-function studies
on central components of the 2 arms of the protein quality control system. Clinical studies evaluating the effect of selective positive or negative modulation of the systems in relation to the stage of disease will give final answers from a human disease and therapy standpoint. With this perspective, the viewpoint of atherosclerosis as a protein quality disease may contribute to current theory and future treatment of atherosclerotic cardiovascular disease. Reprint requests and correspondence: Dr. Joerg Herrmann, Department of Internal Medicine, Mayo Clinic Rochester, 200 First Street Southwest, Rochester, Minnesota 55905. E-mail: herrmann. [email protected].
REFERENCES
1. Bukau B, Weissman J, Horwich A. Molecular chaperones and protein quality control. Cell 2006;125:443–51. 2. Herrmann J, Lerman LO, Lerman A. Ubiquitin and ubiquitin-like proteins in protein regulation. Circ Res 2007;100:1276 –91. 3. Wickner S, Maurizi MR, Gottesman S. Posttranslational quality control: folding, refolding, and degrading proteins. Science 1999;286: 1888 –93.
2010
Herrmann et al. Protein Quality and Atherosclerosis
4. Ron D, Walter P. Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol 2007;8:519 –29. 5. Bernales S, McDonald KL, Walter P. Autophagy counterbalances endoplasmic reticulum expansion during the unfolded protein response. PLoS Biol 2006;4:e423. 6. Carrell RW, Lomas DA. Conformational disease. Lancet 1997;350: 134 – 8. 7. Taylor JP, Hardy J, Fischbeck KH. Toxic proteins in neurodegenerative disease. Science 2002;296:1991–5. 8. Ross CA, Poirier MA. Protein aggregation and neurodegenerative disease. Nat Med 2004;10 Suppl:S10 –7. 9. Forman MS, Trojanowski JQ, Lee VM. Neurodegenerative diseases: a decade of discoveries paves the way for therapeutic breakthroughs. Nat Med 2004;10:1055– 63. 10. Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 2002;297: 353– 6. 11. LaFerla FM, Green KN, Oddo S. Intracellular amyloid-beta in Alzheimer’s disease. Nat Rev Neurosci 2007;8:499 –509. 12. Bucciantini M, Giannoni E, Chiti F, et al. Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature 2002;416:507–11. 13. Stocker R, Keaney JF Jr. Role of oxidative modifications in atherosclerosis. Physiol Rev 2004;84:1381– 478. 14. 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;142:1927–37. 15. Berberian PA, Myers W, Tytell M, Challa V, Bond MG. Immunohistochemical localization of heat shock protein-70 in normalappearing and atherosclerotic specimens of human arteries. Am J Pathol 1990;136:71– 80. 16. Kanwar RK, Kanwar JR, Wang D, Ormrod DJ, Krissansen GW. Temporal expression of heat shock proteins 60 and 70 at lesion-prone sites during atherogenesis in ApoE-deficient mice. Arterioscler Thromb Vasc Biol 2001;21:1991–7. 17. Herrmann J, Edwards WD, Holmes DR Jr., et al. Increased ubiquitin immunoreactivity in unstable atherosclerotic plaques associated with acute coronary syndromes. J Am Coll Cardiol 2002;40:1919 –27. 18. Marfella R, D’Amico M, Di Filippo C, et al. Increased activity of the ubiquitin-proteasome system in patients with symptomatic carotid disease is associated with enhanced inflammation and may destabilize the atherosclerotic plaque: effects of rosiglitazone treatment. J Am Coll Cardiol 2006;47:2444 –55. 19. Versari D, Herrmann J, Gossl M, et al. Dysregulation of the ubiquitin-proteasome system in human carotid atherosclerosis. Arterioscler Thromb Vasc Biol 2006;26:2132–9. 20. Herrmann J, Gulati R, Napoli C, et al. Oxidative stress-related increase in ubiquitination in early coronary atherogenesis. FASEB J 2003;17:1730 –2. 21. Tan C, Li Y, Tan X, Pan H, Huang W. Inhibition of the ubiquitinproteasome system: a new avenue for atherosclerosis. Clin Chem Lab Med 2006;44:1218 –25. 22. Herrmann J, Saguner AM, Versari D, et al. Chronic proteasome inhibition contributes to coronary atherosclerosis. Circ Res 2007;101: 865–74. 23. Xu J, Wu Y, Song P, Zhang M, Wang S, Zou MH. Proteasomedependent degradation of guanosine 5=-triphosphate cyclohydrolase I causes tetrahydrobiopterin deficiency in diabetes mellitus. Circulation 2007;116:944 –53. 24. Zhou J, Lhotak S, Hilditch BA, Austin RC. Activation of the unfolded protein response occurs at all stages of atherosclerotic lesion development in apolipoprotein E-deficient mice. Circulation 2005; 111:1814 –21. 25. Feng B, Yao PM, Li Y, et al. The endoplasmic reticulum is the site of cholesterol-induced cytotoxicity in macrophages. Nat Cell Biol 2003; 5:781–92. 26. Dickhout JG, Hossain GS, Pozza LM, Zhou J, Lhotak S, Austin RC. Peroxynitrite causes endoplasmic reticulum stress and apoptosis in human vascular endothelium: implications in atherogenesis. Arterioscler Thromb Vasc Biol 2005;25:2623–9.
JACC Vol. 51, No. 21, 2008 May 27, 2008:2003–10 27. Gargalovic PS, Gharavi NM, Clark MJ, et al. The unfolded protein response is an important regulator of inflammatory genes in endothelial cells. Arterioscler Thromb Vasc Biol 2006;26:2490 – 6. 28. Horke S, Witte I, Wilgenbus P, Kruger M, Strand D, Forstermann U. Paraoxonase-2 reduces oxidative stress in vascular cells and decreases endoplasmic reticulum stress-induced caspase activation. Circulation 2007;115:2055– 64. 29. 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. 30. Meiners S, Laule M, Rother W, et al. Ubiquitin-proteasome pathway as a new target for the prevention of restenosis. Circulation 2002;105: 483–9. 31. Stangl V, Lorenz M, Meiners S, et al. Long-term up-regulation of eNOS and improvement of endothelial function by inhibition of the ubiquitin-proteasome pathway. Faseb J 2004;18:272–9. 32. Vieira O, Escargueil-Blanc I, Jurgens G, et al. Oxidized LDLs alter the activity of the ubiquitin-proteasome pathway: potential role in oxidized LDL-induced apoptosis. FASEB J 2000;14:532– 42. 33. Mucchiano GI, Haggqvist B, Sletten K, Westermark P. Apolipoprotein A-1– derived amyloid in atherosclerotic plaques of the human aorta. J Pathol 2001;193:270 –5. 34. Rocken C, Tautenhahn J, Buhling F, et al. Prevalence and pathology of amyloid in atherosclerotic arteries. Arterioscler Thromb Vasc Biol 2006;26:676 –7. 35. Ursini F, Davies KJ, Maiorino M, Parasassi T, Sevanian A. Atherosclerosis: another protein misfolding disease? Trends Mol Med 2002; 8:370 – 4. 36. Stewart CR, Tseng AA, Mok YF, et al. Oxidation of low-density lipoproteins induces amyloid-like structures that are recognized by macrophages. Biochemistry 2005;44:9108 –16. 37. Howlett GJ, Moore KJ. Untangling the role of amyloid in atherosclerosis. Curr Opin Lipidol 2006;17:541–7. 38. Medeiros LA, Khan T, El Khoury JB, et al. Fibrillar amyloid protein present in atheroma activates CD36 signal transduction. J Biol Chem 2004;279:10643– 8. 39. Beckmann N, Schuler A, Mueggler T, et al. Age-dependent cerebrovascular abnormalities and blood flow disturbances in APP23 mice modeling Alzheimer’s disease. J Neurosci 2003;23:8453–9. 40. Elesber AA, Bonetti PO, Woodrum JE, et al. Bosentan preserves endothelial function in mice overexpressing APP. Neurobiol Aging 2006;27:446 –50. 41. Suo Z, Humphrey J, Kundtz A, et al. Soluble Alzheimers betaamyloid constricts the cerebral vasculature in vivo. Neurosci Lett 1998;257:77– 80. 42. Gentile MT, Vecchione C, Maffei A, et al. Mechanisms of soluble beta-amyloid impairment of endothelial function. J Biol Chem 2004; 279:48135– 42. 43. Thomas T, McLendon C, Sutton ET, Thomas G. Cerebrovascular endothelial dysfunction mediated by beta-amyloid. Neuroreport 1997; 8:1387–91. 44. Thomas T, Sutton ET, Bryant MW, Rhodin JA. In vivo vascular damage, leukocyte activation and inflammatory response induced by beta-amyloid. J Submicrosc Cytol Pathol 1997;29:293–304. 45. Revesz T, Ghiso J, Lashley T, et al. Cerebral amyloid angiopathies: a pathologic, biochemical, and genetic view. J Neuropathol Exp Neurol 2003;62:885–98. 46. Li L, Cao D, Garber DW, Kim H, Fukuchi K. Association of aortic atherosclerosis with cerebral beta-amyloidosis and learning deficits in a mouse model of Alzheimer’s disease. Am J Pathol 2003;163:2155– 64. 47. Wang X, Robbins J. Heart failure and protein quality control. Circ Res 2006;99:1315–28. 48. Patterson C, Ike C, Willis PWt, Stouffer GA, Willis MS. The bitter end: the ubiquitin-proteasome system and cardiac dysfunction. Circulation 2007;115:1456 – 63. 49. Trifilo MJ, Yajima T, Gu Y, et al. Prion-induced amyloid heart disease with high blood infectivity in transgenic mice. Science 2006;313:94 –7. 50. Chesebro B, Trifilo M, Race R, et al. Anchorless prion protein results in infectious amyloid disease without clinical scrapie. Science 2005;308:1435–9.