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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:676.)
© 2006 American Heart Association, Inc.

Letters to the Editor

Prevalence and Pathology of Amyloid in Atherosclerotic Arteries

Christoph Röcken; Jörg Tautenhahn; Frank Bühling; Daniela Sachwitz; Steffi Vöckler; Andreas Goette; Thomas Bürger

From the Departments of Pathology (C.R., D.S., S.V.), General Surgery (J.T.), and Immunology (F.B.), Otto-von-Guericke-University Magdeburg, Germany; the Department of Clinical and Laboratory Medicine (F.B.), Cottbus, Germany; the Division of Cardiology (A.G.), of the Otto-von-Guericke-University Magdeburg, Germany; and the Diakonissen Krankenhaus (T.B.), Kassel, Germany.

Correspondence to Prof Dr med Christoph Röcken, Department of Pathology, Charité University Hospital, Schumannstr. 20/21, D-10117 Berlin, Germany. E-mail rockenc{at}gmx.de

To the Editor:

Apolipoprotein AI (AApoAI)–associated amyloidosis is characterized by the deposition of apolipoprotein AI (apoAI) and occurs as a hereditary and a nonhereditary form. Hereditary AApoAI amyloidosis is a systemic disease leading to the deposition of amyloid in various organs and tissues and is caused by germline mutations in the APOA1 gene. Nonhereditary AApoAI amyloid is far more prevalent and characterized by deposits of nonvariant protein in atherosclerotic arteries.^1–3 Despite being linked to the most common cause of morbidity and mortality in Western societies, nonhereditary AApoAI amyloid has achieved only little attention.^1–4 It shares several striking similarities with secondary or reactive AA amyloidosis. Nonhereditary AApoAI amyloid occurs in the background of a local chronic inflammatory reaction, it originates from an apolipoprotein that largely associates with high-density lipoproteins (HDLs), and apoAI-containing HDL is endocytosed and retroendocytosed by macrophages, which, in themselves, are able to form amyloid in vitro. Finally, AApoAI amyloidosis is characterized by the deposition of proteolytic fragments of the precursor protein, leading us to speculate that proteolysis is involved in the pathogenesis of AApoAI amyloid.^1,3 The aim of this study was to gain further insights into the pathology of nonhereditary AApoAI amyloid.

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The prevalence and spatial distribution of amyloid, macrophages, cathepsin B (CathB), cathepsin K (CathK), cathepsin L (CathL), and carboxy methyl lysine (CML) was studied using carotid artery specimens obtained from a consecutive series of all 225 patients undergoing carotid endarterectomy with polyester patch angioplasty (Table I, available online at http://atvb.ahajournals.org) during the period from 1997 to 2003. All patients were scheduled for elective therapeutic endarterectomy and gave written informed consent in the surgical procedure. Patient characteristics were retrieved from hospital records. This study was in accordance with the guidelines of the ethics committee of the University of Magdeburg. Tissue samples were formalin fixed and paraffin embedded. Deparaffinized serial sections were stained with hematoxylin and eosin, Elastic van Gieson stain, and Congo Red. Immunostaining was performed with specific antibodies directed against apoAI, CathB, CathK, CathL, CD68, CML, serum amyloid A (SAA), and transthyretin as described previously.^5–7 In vitro degradation experiments with apoAI-enriched HDL apolipoproteins (purchased from Calbiochem) were performed using recombinant human CathB (1.5 µmol/L final concentration), Cath K (3.0 µmol/L), and CathL (0.15 µmol/L and 30 nmol/L).^5–7 Degradation was performed at 37°C for 10, 30, 120, or 240 minutes at pH 5.5. Proteins were resolved in polyacrylamide gels and visualized by Coomassie blue staining.⁵ Enzymatic activity was studied in 6 unfixed carotid artery specimens (2 with and 4 without intimal amyloid).

Table I summarizes the patients’ characteristics. Amyloid was found in the intima and in atherosclerotic plaques in 122 (54%) patients (Figure; Table I).

View larger version (109K): [in this window] [in a new window]   Carotid artery specimens from a patient with symptomatic carotid artery stenosis. Green birefringent amyloid deposits were found in Congo red–stained specimens (Congo red), which were immunoreactive for apoAI (arrow). Note abundant apoAI immunostaining in the surrounding nonamyloidotic plaque area. CathL was found in all arteries, being the most abundant cysteine protease in macrophages of atherosclerotic arteries, and was also found extracellularly. Congo red staining in polarized light; immunostaining with anti-apoAI and anti-CathL antibodies; hematoxylin counterstain. Original magnifications x200. In vitro degradation experiments using native apoAI-enriched HDL apolipoproteins were performed with CathL (30 nmol/L) for 10 minutes, 30 minutes, 2 hours, and 4 hours. Incubation for 4 hours at 37°C in the presence of proteases and E64 served as a control. NP denotes no protease added. SDS-PAGE and Coomassie blue staining.

Patients with amyloid were significantly older than patients without amyloid (P<0.001). The presence of amyloid correlated only with triglyceride levels. Fifty-one amyloid-containing specimens were subjected to immunohistochemical staining with anti–apoAI– and anti–SAA-antibodies. Extracellular apoAI immunoreactivity was most prominent as diffuse staining in atherosclerotic plaques and rarely in the arterial media. In addition, macrophages and foam cells commonly stained for apoAI. In 45 (88%) arteries, amyloid deposits clearly stained with the anti-apoAI antibody (Figure). In six (12%) specimens, amyloid could no longer be discerned in the anti–apoAI-immunostained sections. SAA was not detected in any specimen. The presence and distribution of CML was studied in 20 specimens with amyloid and was detected in every specimen. CML was not found within amyloid. However, it was interesting to note that amyloid deposits were always surrounded by CML immunoreactivity. Twenty-four resection specimens were studied for CD68-immunoreactive macrophages and the spatial distribution of cysteine proteases, including 16 specimens with and 8 without amyloid. CD68-immunoreactive macrophages and CathB were found in every specimen. CathB was present in macrophages (83% of the patients), multinucleated histiocytic giant cells (MGCs; 13%), endothelial cells (4%), and myocytes (4%). Extracellular immunostaining was also commonly observed. CathK was found in 11 (69%) amyloidotic and 6 (63%) nonamyloidotic arteries and was the least commonly found cysteine protease. CathK was also present in macrophages (46%), MGCs (13%), and myocytes (54%). CathL was expressed in all arteries, being present in macrophages (100%), MGCs (17%), and extracellularly (Figure). Almost all macrophages stained for CathL, whereas only a small fraction was immunoreactive for CathB and CathK. CathL was the most abundant cysteine protease in macrophages of atherosclerotic arteries. The pattern of immunostaining for cysteine proteases did not show any differences between amyloidotic and nonamyloidotic arteries. Next, we studied enzymatic activity by fluorospectroscopy (excitation 345 nm, emission 440 nm; Spectramax Gemini Dual-Scanning Microplate Spectrofluorometer, Molecular Devices Cooperation) using a Z-R-R-AMC (CathB), Z-G-P-R-AMC (CathK), or Z-F-R-AMC (CathL; all Bachem) fluorogenic substrate. CathB (V_max/mg=6.83±4.99) and CathL (V_max/mg=15.70±10.47) activity was found in every carotid artery specimen. CathK activity (V_max/mg=6.73±3.79) was detectable in 3 carotid artery specimens, including 1 with intimal amyloid deposits. We then examined whether native human apoAI obtained commercially is susceptible to degradation by cysteine proteases. All 3 proteases were found to be potentially able to degrade apoAI at a concentrations of 30 nmol/L (CathL), 0.15 µmol/L (CathL), 1.5 µmol/L (CathB), and 3 µmol/L (CathK) generating differently sized protein and peptide fragments. No degradation was observed in the absence of active protease or in the presence of a cysteine protease inhibitor (E64).

After investigating a large unselected series of resection specimens, we show here that amyloid is a common pathological change in atherosclerotic carotid arteries. The occurrence of amyloid correlated significantly with patient age. Because atherosclerosis was more prevalent than amyloid leads to the conjecture that atherosclerosis precedes or promotes the formation of amyloid. The majority of our patients tested had apoAI-immunoreactive amyloid deposits, consistent with the known origin of intimal amyloid. Despite being common, little is known about the pathogenesis and significance of nonhereditary AApoAI amyloid in atherosclerotic arteries. Serum levels of apoAI correlate inversely to the risk and severity of coronary artery disease, and therefore, it is unlikely that high apoAI–serum levels precede the development of amyloid. However, apoAI is present in atherosclerotic arteries already at an early stage,^8,9 and the amount of apoAI correlates with patient age and the severity of atherosclerosis.⁸ These findings indicate that apoAI is enriched in atherosclerotic arteries leading to a high local concentration, which, on its own, is known to increase the risk of amyloid formation. In support of this notion, we found abundant apoAI immunoreactivity outside the amyloid deposits.

A hallmark of atherosclerosis is the post-translational modification of proteins and lipids by advanced-glycation end products (AGEs). The biological effect of AGEs is mediated, at least partly, by the receptor of AGEs (RAGE). Yan et al¹⁰ have shown that canceling out the activation of cellular RAGE delayed the onset of reactive amyloidosis in mice, thus describing a putative pathophysiological pathway by which AGEs may influence amyloid formation. In this study, we show that amyloid-containing arteries are rich in CML, a distinct, chemically characterized type of AGE. Thus, AGEs may also be involved in the pathology of nonhereditary AApoAI amyloid.

Apart from the primary structure, local or systemic protein concentrations, and the presence of AGEs, other factors contribute to the pathology of amyloid and amyloidoses, including proteolysis of the precursor protein and amyloid deposits, as well as macrophages. We believe that we are the first to show the presence of proteolytically active CathB, CathK, and CathL in atherosclerotic arteries, which have been shown previously to also be potentially involved in the pathology and pathogenesis of AA- and immunoglobulin-derived AL amyloid.^5,7 Interestingly, and sharing another similarity with other forms of amyloid,⁶ we also found CathK-immunoreactive MGCs in amyloidotic arteries. CathK belongs to the most active human elastases described until now and probably represents an enhanced specific proteolytic capability of histiocytic cells.¹¹ Furthermore, we show that all 3 proteases are able to degrade apoAI, generating intermediate-sized fragments, some having a molecular weight similar to AApoAI amyloid proteins.

With an increasing knowledge about conformational diseases, it has become evident that protein misfoldings and aggregates can be pathogenic.¹² Amyloid in atherosclerotic plaques might be just the tip of an iceberg. Large amounts of aging proteins in the plaque are prone to a multitude of conformational changes and formation of supramolecular structures, not all of which necessarily have to form amyloid to gain a pathologic function. In this respect, atherosclerosis may share similarities with Alzheimer disease. Further studies into this topic are warranted.

Acknowledgments

This work was supported by grants from the Deutsche Forschungsgemeinschaft (grant RO1173/3-3), Bonn Bad-Godesberg, Germany. The authors thank Dr Rosemarie Kientsch-Engel (Roche Diagnostics GmbH; Penzberg, Germany) for kindly providing the anti-CML antibody and Dr Robert Menard (Biotechnology Research Institute, NRCC; Montreal, Canada) for kindly providing recombinant human cathepsin B and L.

References

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