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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

Raffaele Marfella, MD, PhD^_*,,_*, Michele D’Amico, PhD^,, Clara Di Filippo, PhD^,, Alfonso Baldi, MD, Mario Siniscalchi, MD, PhD^_*, Ferndinando Carlo Sasso, MD, PhD^_*, Michele Portoghese, MD^||, Ornella Carbonara, MD^_*, Basilio Crescenzi, MD^¶, Paolo Sangiuolo, MD^¶, Giovanni Francesco Nicoletti, MD^#, Raffaele Rossiello, MD^_**, Franca Ferraraccio, MD, Federico Cacciapuoti, MD^_*, Mario Verza, MD^_*, Ludovico Coppola, MD^_*, Francesco Rossi, MD^, and Giuseppe Paolisso, MD, PhD^_*,

^_* Department of Geriatrics and Metabolic Diseases, Section of Pathology, Second University of Naples, Naples, Italy
"Centro di Eccellenza Cardiovascolare,", Section of Pathology, Second University of Naples, Naples, Italy
Department of Experimental Medicine, Section of Pathology, Second University of Naples, Naples, Italy
Department of Biochemistry, Section of Pathology, Second University of Naples, Naples, Italy
^|| Cardiovascular Surgery Unit, Sassari Hospital, Naples, Italy
^¶ Cardiovascular Surgery Unit, Hospital V. Monaldi, Naples, Italy
^# Department of Surgery, Second University of Naples, Naples, Italy
^_** Department of Biochemistry and Biophysics "F. Cedrangolo," Section of Anatomic Pathology, Second University of Naples, Naples, Italy

View larger version (92K): [in a new window]   Figure 1 Representative example of plaques studied. This section is representative of the plaque region analyzed. The section was stained with hematoxylin-eosin. Plaque section at low magnification. (A) A carotid plaque from a patient affected by ipsilateral major stroke who underwent carotid endarterectomy within two months of symptom onset. Fibrous cap plaque rupture with intraluminal thrombus is evident (magnification x2). (B) Carotid plaque from an asymptomatic patient, characterized by a large lipid core and a thin fibrous cap, without the presence of acute thrombus within the lumen (magnification x2). (C) Micrograph showing immunohistochemical staining of carotid plaque from patient affected by ipsilateral major stroke. Fibrous cap adjacent to rupture site containing numerous macrophage-foam cells positive to CD68 antibody (brown chromogen) (magnification x100).

View larger version (64K): [in a new window]   Figure 2 (A) Immunochemistry for matrix metalloproteinase (MMP)-9 and sirius red staining (x630; box, x400) for collagen content (x100) in asymptomatic, placebo-treated, and rosiglitazone-treated symptomatic plaques. Similar regions of plaque are shown. These results are typical of asymptomatic, placebo-treated and rosiglitazone-treated symptomatic plaques. (B) Enzyme-linked immunosorbent assay for MMP-9 and sirius red staining for collagen content in asymptomatic, placebo-treated, and rosiglitazone-treated symptomatic plaques. (The central line represents the median, the boxes span from the 25th to 75th percentiles, and the error bars extend from the 10th to 90th percentiles.) *p < 0.05 compared with placebo group. p < 0.05 compared with rosiglitazone group.

View larger version (64K): [in a new window]   Figure 3 (A) Proteasome 20S (x1,000) and ubiquitin (x630) by immunohistochemistry in asymptomatic, placebo-treated, and rosiglitazone-treated symptomatic plaques. Similar regions of the plaque are shown (boxes, x200). These results are typical of asymptomatic, placebo-treated, and rosiglitazone-treated symptomatic plaques. (B) Levels of proteasome 20S by specific SDS activation kit, ubiquitin levels by enzyme-linked immunosorbent assay kit *p < 0.05 compared with placebo group. p < 0.05 compared with rosiglitazone group.

View larger version (53K): [in a new window]   Figure 4 Consecutive immunostaining (x200) using serial sections of symptomatic plaques demonstrated that cells positive for CD68 were also positive for proteasome 20S and ubiquitin. These results are typical of 12 placebo symptomatic plaques.

View larger version (50K): [in a new window]   Figure 5 (A) Levels of activated nuclear factor kappa B (specific Trans-AM p50 and p65 subunit assay kit) and inhibitory kappa B (IkB)-beta asymptomatic, placebo-treated, and rosiglitazone-treated symptomatic plaques; (B) immunohistochemistry for activated p65 (x630) and p50 (x630) and IkB-beta (x800). Similar regions of the plaque are shown. These results are typical of asymptomatic, placebo-treated, and rosiglitazone-treated symptomatic plaques. *p < 0.05 compared with placebo group. p < 0.05 compared with rosiglitazone group.

View larger version (25K): [in a new window]   Figure 6 Ubiquitin, proteasome 20S, activated NFkB, IkB-beta, and O2– production in symptomatic and asymptomatic monocytes. Purified symptomatic monocytes were cultured in presence or absence (48 h) of rosiglitazone (7.0 µM). *p < 0.05 compared with symptomatic monocytes. p < 0.05 compared with rosiglitazone-treated symptomatic monocytes. Abbreviations as in Figure 5.