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Oxidative stress-related increase in ubiquitination in early coronary atherogenesis¹

JOERG HERMANN^*, RAJIV GULATI^*, CLAUDIO NAPOLI, JULIE E. WOODRUM^*, LILACH O. LERMAN, MARTIN RODRIGUEZ-PORCEL^*, VINCENZO SICA, ROBERT D. SIMARI^*, AARON CIECHANOVER and AMIR LERMAN^*,2

Divisions of
^* Cardiovascular Diseases and
Hypertension, Mayo Clinic, Rochester, Minnesota, USA;
Department of Medicine and Clinical Pathology, University of Naples, Naples, Italy; and
Rapport Institute for Research in Medical Sciences, Technion-Israel Institue of Technology, Haifa, Israel

²Correspondence: Division of Cardiovascular Diseases, Mayo Clinic Rochester, 200 First Street S.W., Rochester, MN 55905, USA. E-mail: lerman.amir{at}mayo.edu

SPECIFIC AIMS

The ubiquitin proteasome system (UPS) may be impaired, hence of questionable pathophysiologic significance under conditions of increased oxidative stress, including atherosclerosis. Thus, this study was performed to test the hypothesis that the coronary artery UPS is functionally active in an experimental model of early atherogenesis.

PRINCIPAL FINDINGS

1. Experimental hypercholesterolemia induces an increase in ubiquitin-protein conjugates in the coronary arterial wall
To examine whether exposure to the cardiovascular risk factor hypercholesterolemia is associated with enhanced ubiquitination in the coronary arterial wall, we subjected female domestic pigs to a normal or a high-cholesterol diet for 12 weeks and analyzed the extent of ubiquitin-protein conjugates in coronary arteries by immunoblotting and immunostaining using an antibody that detects ubiquitin/ubiquitin-substrate conjugates (Babco, Richmond, CA). Compared with coronary artery samples from animals on a normal diet, the amount of ubiquitinated proteins was higher (increase in densitometric value by 35%) in coronary artery samples from animals on a high-cholesterol diet (Fig. 1 ). As demonstrated by immunohistochemistry, ubiquitin/ubiquitin-substrate conjugates were detected more extensively in coronary artery samples from pigs on a high-cholesterol diet, predominantly in vascular smooth muscle cells (VSMCs) of the media.

View larger version (67K): [in this window] [in a new window]   Figure 1. Immunoblot for ubiquitin/ubiquitin-protein conjugates in tissue homogenates of coronary arteries from pigs on a normal diet (N) or on a hypercholesterolemic diet without (HC) or with (HC+VIT) vitamin C and E supplementation for 12 weeks (upper panel). An increase in the extent of ubiquitin-protein conjugates is found in HC but not in HC+VIT, as illustrated by the bar graphs of the densitometric results (lower panel; mean±SD; *, P<0.05, for HC compared with N and HC+VIT; n=5 for all groups).

2. Increase in ubiquitin-protein conjugates in the coronary arterial wall in experimental hypercholesterolemia is related to oxidative stress
To further examine whether the increase in ubiquitination in the coronary artery wall in hypercholesterolemia was related to oxidative stress, we determined systemic parameters of oxidative stress and local radical scavenger activities and exposed pigs to vitamin C (1000 mg/day) and vitamin E (100 IU/kg/day) in addition to a high-cholesterol diet for 12 weeks. Compared with animals on a normal diet, low-density lipoprotein (LDL)–malondialdehyde and LDL–relative electrophoretic mobility were higher in animals on a high-cholesterol diet (8.5±0.3 nmol/mg protein and 12.2±0.4 mm from baseline vs. 6.8±0.7 nmol/mg protein and 10.7±0.5 mm from baseline, respectively; P<0.05 for both). Catalase and manganese superoxide dismutase activities of the coronary artery wall were lower in animals on a high-cholesterol diet compared with animals on a normal diet (11.3±1.2 and 2.0±0.3 IU/mg protein vs. 19.3±2.1 and 2.5±0.1 IU/mg protein, respectively; P<0.05 for both). Dietary vitamin treatment normalized oxidative stress parameters in the presence of elevated plasma concentrations of cholesterol (329±102 vs. 76±21 mg/dL in animals on normal diet, P<0.05, and 400±148 in animals on high-cholesterol diet alone, P=NS). Antioxidant vitamin supplementation fully prevented the increase of the extent of ubiquitinated conjugates in the coronary arterial wall otherwise observed in hypercholesterolemia (Fig. 1) .

3. Increase in coronary artery ubiquitin-protein conjugates in experimental hypercholesterolemia is not a result of impairment in proteasome- proteolytic activity
To examine if the increase in ubiquitinated products was the consequence of a decrease in their degradation as a result of an impairment of the proteolytic activity of the proteasome, we analyzed the functional activity of the proteasome on the level of the vascular wall. Coronary artery homogenates were incubated with 7-amino, 4-methyl-coumarin (AMC)-labeled substrates for the chymotrypsin-like activity [Leu-Leu-Val-Tyr (LLVY)-AMC] and the peptidylglutamyl hydrolase (PDGH) activity [Leu-Leu-Gln (LLE)-AMC] of the proteasome for 30 min at 37°C, and the increase in the concentration of AMC in the assay probe was measured by use of a spectrofluorometer. The proteasome-specific proteolytic activity was derived from the difference in AMC fluorescence in the absence and presence of MG-132, a peptide aldehyde, which blocks proteasome chymotrypsin-like and PDGH activities. As demonstrated in Figure 2 , no intergroup differences in proteasome-proteolytic activities were observed.

View larger version (18K): [in this window] [in a new window]   Figure 2. Bar graphs illustrating proteasome-proteolytic activity in tissue homogenates of coronary arteries from pigs on a normal diet (N) or on a hypercholesterolemic diet without (HC) or with (HC+VIT) vitamin C and E supplementation for 12 weeks (mean±SD; n=5 for each group). Proteasome-proteolytic activity was calculated for each coronary artery sample from the difference in proteolytic activity of the tissue homogenate without and with incubation of the proteasome inhibitor MG-132.

4. Increase in ubiquitin-protein conjugates can be seen in porcine VSMCs exposed to oxidative stress in vitro
To further assess if the increase in ubiquitination can be seen in vitro in the cell type in which it was predominantly seen in the coronary arterial wall in experimental hypercholesterolemia, we harvested porcine VSMCs from the media of the aorta of normal, female domestic pigs. We then cultured them, exposed them to increasing doses of hydrogen peroxide (H₂O₂; 100–500 µmol/L) for 12 h—the highest dose without or with pretreatment with vitamin C and E (150 µmol/L)—and analyzed the extent of ubiquitin-protein conjugates in these cells using the aforementioned antibody. Compared with untreated cells, there was an increase in ubiquitin-protein conjugates, particularly in cells treated with 500 µmol/L H₂O₂, which was attenuated by antioxidant vitamin pretreatment.

CONCLUSIONS

The UPS has been identified as the major route of nonlysosomal protein degradation and as a central element in the regulation of signaling pathways. Previous studies, however, indicated that the UPS could be functionally impaired under conditions of increased endogenous oxidative stress. Hence, these studies questioned a pathophysiological role of the UPS in diseases associated with enhanced endogenous oxidative stress, such as coronary artery disease. The current study now demonstrates an increase in the extent of ubiquitinated substrates in the coronary artery, primarily in media VSMCs in experimental hypercholesterolemia, an established model of early atherogenesis. Addition of antioxidant vitamins to the high-cholesterol diet normalized oxidative stress parameters and prevented the increase in ubiquitinated substrates in the absence of any lipid-lowering effect. Of further note, increase in ubiquitination was also seen in VSMCs exposed to H₂O₂ in vitro, which was attenuated by antioxidant vitamin treatment, similar to the findings in vivo.

These findings underscore the pathophysiologic role of oxidative stress for the increase in ubiquitinated products in experimental hypercholesterolemia. Furthermore, they indicate unimpaired ubiquitination under these experimental conditions of early atherogenesis. In addition, the current finding of normal proteasome-proteolytic activity in experimental hypercholesterolemia indicates that the increase in the extent of ubiquitinated substrates in the coronary artery does not reflect accumulation of ubiquitinated substrates as a consequence of oxidative stress-mediated, functional impairment of the proteasome-proteolytic core. Thus, the increased extent of ubiquitinated substrates in experimental hypercholesterolemia most likely reflects increase in substrate availability and/or increase in ubiquitination capacity (Fig. 3 ).

View larger version (89K): [in this window] [in a new window]   Figure 3. Schematic illustration. In association with an increase in plasma and tissue cholesterol concentrations, increase in plasma and tissue oxidative stress can be noted, which leads to the modulation of signaling pathways and the modification of proteins. Modified signaling molecules and proteins can subsequently be recognized by the ubiquitin system to undergo ubiquitin-dependent proteolysis. Oxidized proteins were, furthermore, reported to be direct substrates for the 20S proteasome, the proteolytic core of the 26S proteasome. When the generation of modified proteins exceeds proteasome-proteolytic capacity, oxidized proteins can form hydrophobic aggregates, which are excellent substrates for the ubiquitin system but become progressively resistant to proteolysis. This can lead to an increase in the level of ubiquitin-protein conjugates in the coronary arterial wall under conditions of chronically enhanced oxidative stress, for instance, in experimental hypercholesterolemia, a model of early atherosclerosis.

Among the substrates to be taken into consideration are oxidized proteins, which may be generated to an extent that exceeds the proteolytic capacity of the proteasome, even in the absence of any functional impairment. Under such conditions, oxidized proteins can form hydrophobic aggregates, which would become progressively, covalently cross-linked and eventually, excellent substrates for the ubiquitin system, yet also increasingly resistant to proteolysis. These aggregates of oxidized proteins may, therefore, be the basis for the increased levels of ubiquitin-protein conjugates. Other substrates, which have to be considered as well, are signaling molecules, which are increasingly modified under conditions of enhanced oxidative stress. Intriguing examples in this regard are the inhibitory molecules of the IB family. These molecules noncovalently attach to the dimeric transcription factor nuclear factor-B (NF-B), hence keeping it in an inactive state in the cytosol. In response to various cellular stimuli, such as exaggerated oxidative stress, IBs are, however, rapidly phosphorylated and ubiquitinated to allow nuclear translocation and transcription activity of NF-B. Thus, the current findings may be at least in part a reflection of the involvement of the UPS in oxidative stress-related protein modification and oxidative stress-related activation of signaling pathways in the coronary artery in early atherogenesis. Future studies are needed to define the predominant pathophysiologic role of the UPS and hence, the potential therapeutic value of its inhibition in atherosclerosis, including coronary artery disease.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0841fje; doi: 10.1096/fj.02-0841fje

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