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.

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).

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).

Advanced human atherosclerotic plaques in the aorta reacting with antiserum to apolipoprotein A-1 (A and C) (bar = 50 µm) 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: x2; F, G: x5).

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-month-old 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).

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 low-density 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.