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vol. 5

Local and systemic 20S proteasome release in patients with stenting of stenotic saphenous vein bypass grafts – a pilot study

Stephan Urs Sixt
Kirsten Leineweber
Dirk Böse
Thomas Konorza
Michael Haude
Stephan Hogrebe
Petra Kleinbongard
Raimund Erbel
Jürgen Peters
Gerd Heusch

Arch Med Sci 2009; 5, 4: 559-563
Online publish date: 2009/12/30
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The ubiquitin-proteasome system is the major pathway of non-lysosomal degradation of intracellular proteins and is central to processes such as apoptosis, inflammation and proliferation, which are all decisive elements of atherosclerosis [1]. Apart from the more established 26S proteasome, there is also a 20S proteasome which is functional in the extracellular space; its plasma concentration is increased in a variety of notably inflammatory states [2]. Increased activity of the ubiquitin system is associated with unstable coronary atherosclerotic plaques and acute coronary syndromes [3]. Of note, decreased vascular 20S proteasome activity increases ubiquitin levels and contributes to atherosclerosis progression in human carotid plaques [4]. Diabetic patients have greater 20S proteasome activity in their carotid plaques, and rosiglitazone attenuates the inflammation, including 20S proteasome activity, in the atherosclerotic lesion [5].
In the present study we therefore investigated systemic 20S proteasome concentrations in the plasma of patients with a severe stenosis in their saphenous vein aortocoronary bypass graft (SVG) and related them to disease progression. To obtain information on the proteasome concentration in coronary atherosclerotic plaques, 20S proteasome concentrations were also determined in the aspirate sampled during stent implantation under protection of an occlusion/aspiration device [6].

Material and methods

Thirty-nine symptomatic male patients (68 ±1 years, 82 ±2 kg body weight, Canadian Cardiova-scular Society (CCS) class [7] 2.4 ±0.1; NYHA class 1.9 ±0.1) with a flow-limiting stenosis in their SVG were studied; 4 patients were treated for 2 stenoses each such that a total of 43 stenoses were studied. Clinical and laboratory data, current medication, graft age, target vessel and stent type are presented in Table I. Twenty-eight apparently healthy subjects (44 ±19 years) served as controls. Full informed consent was obtained from all subjects and patients before participating in the study following approval by the local ethics committee of the Medical Faculty, University of Duisburg-Essen, and conforming with the principles outlined in the revised Declaration of Helsinki.

Intervention and follow-up
The interventional procedure, using a distal balloon occlusion device (TriAktiv®, Kensey Nash, Exton, PA, USA) during stenting, and quantitative angiography prior to and after stent implantation were performed as described before [6-8]. Six months after stent implantation quantitative coronary angiography was performed to determine the degree of restenosis.

Blood and aspirate samples
Venous blood (VB; 10 ml; potassium-EDTA S-Monovette; Sarstedt, Germany) was obtained via the cubital vein in controls and via the femoral vein in patients prior to intervention. Twenty ml of coronary arterial blood was aspirated distal to the lesion via the flushing catheter prior to (AB) and after stent implantation (ASP); the latter was diluted approximately two-fold with saline and filtered through a 40 mm mesh filter. In each instance, visible particulate plaque-derived debris was retained on the filter. VB, AB and filtered ASP were immediately centrifuged (800 γ for 10 min at 4°C), and the plasma was removed, quickly frozen in liquid nitrogen and stored at –80°C until further analysis.
Filters were washed with 100 µl cell lysis buffer (PromoCell GmbH, Heidelberg, DE) and controlled microscopically to ensure complete removal of the plaque-derived particulate debris. The particulate debris was dissolved by sonification (3 ´ 30 s with 1 min intervals on ice). The samples were incubated on ice for a further 15 min and then centrifuged for 10 min at 13 000 γ and 4°C. The supernatants were removed and stored at –80°C until further analysis.
The concentration of 20S proteasome present within VB, AB and ASP plasma and the plaque-derived particulate debris extracts was measured using an enzyme-linked immunosorbent assay as described recently [9, 10].

Statistic analysis
Data are presented as means (± SEM) and compared between groups with the unpaired two-sided Student t-test; linear regression was performed; statistics were done using dedicated software (Graph-Pad Software, San Diego, CA); p values < 0.05 were considered significant. The 20S proteasome data were normally distributed in the observed ranges.

Prior to stent implantation, the stenosis amounted to 63 ±1% diameter reduction, and mini-mal lumen diameter averaged 1.4 ±0.1 mm. Immediately after stent implantation, the diameter stenosis was reduced to 6 ±1%, and minimal lumen diameter was increased to 3.6 ±0.1 mm. Following stent implantation diameter re-stenosis was 28 ±5% after 6 ±0.3 months.

20S proteasome in plasma and aspirate
The 20S proteasome concentration in venous plasma was significantly lower in controls (447 ±29 ng/ml) than in patients undergoing SVG stenting (1327 ±102 ng/ml, Figure 1). In patients, similar 20S proteasome concentrations were obtained in the soluble aspirate fraction before (AB, 1001 ±128 ng/ml) and after stenting (ASP, 934 ±171 ng/ml).
The 20S proteasome concentration in the particulate debris-derived extracts averaged 7010 ±890 ng/ml and was thus 5- to 7-fold higher than in venous or coronary arterial plasma, respectively. The 20S proteasome venous plasma concentration was related to diameter stenosis at baseline (r = 0.3596, p = 0.0179, n = 43 stenoses, Figure 2).
When grouping patients according to their median venous 20S proteasome concentration of 1335 ng/ml (794 ±65 ng/ml, n = 22 stenoses vs. 1836 ±106 ng/ml, n = 21 stenoses, Figure 2), restenosis was more pronounced (40 ±9% within 6.0 ±0.4 months, n = 20 stenoses) in those with higher 20S proteasome concentration than in those with a lower concentration (17 ±3% within 6.4 ±0.5 months, n = 21 stenoses, p = 0.0059, Figure 2). In those with higher venous 20S proteasome concentration, the 20S proteasome concentration in the plaque-derived particular debris extract was lower (5846 ±993 ng/ml, n = 21stenoses) than in those with lower venous 20S proteasome concentration (8538 ±1548 ng/ml, n = 22 stenoses, p = 0.0308, Figure 2).

To date, almost no information exists about the origin or biological role of the extracellular 20S proteasome in health and disease. In human coronary artery plaques high amounts of 20S proteasome have been found. They most likely represent clots of inactivated 20S proteasomes [11] which have become insufficient to clear the cell from damaged proteins, leading to an accumulation of oxidized and ubiquitinated protein aggregates [3] and possibly contributing to apoptosis, plaque instability and acute complications [12, 13]. We have previously shown that plaque rupture during stent implantation induces local release of serotonin and thromboxane A2 [6] and TNF-α [8] and is associated with peri-interventional coronary microembolization [14-16] with consequent microinfarction [17-20] and reduced coronary reserve [17, 19]. We can only speculate that the containment of 20S proteasome in the particulate plaque debris in our present study represents a more innocuous storage compartment, whereas the circulating systemic 20S proteasome reflects release from the unstable plaque and is therefore more closely related to complications such as restenosis. Such release from the unstable plaque would suggest the use of systemic 20S proteasome as a potential biomarker of disease progression.
The current study is a pilot study with several limitations: the number of patients is small and without age-, sex-, or BMI-matched controls, and the results must be confirmed in future, prospective, larger scale studies. Of note, the present study was done in saphenous vein bypass grafts, and arterial grafts may have a different profile. Aspirin was present in 92% of our study population, and aspirin reduces 20S proteasome activity in atherosclerotic rabbits [21]; it will be difficult to recruit aspirin-naI¨ve patients undergoing PCI. Also, the use of statins in 85% of our study population may have impacted on our results, since statins largely reduce periprocedural coronary microembolization and its inflammatory consequences [22]; again, statin-naI¨ve patients are increasingly rare.

1. Herrmann J, Ciechanover A, Lerman LO, Lerman A. The ubiquitin-proteasome system in cardiovascular diseases – a hypothesis extended. Cardiovasc Res 2004; 61: 11-21.
2. Sixt SU, Dahlmann B. Extracellular, circulating proteasomes and ubiquitin – incidence and relevance. Biochim Biophys Acta 2008; 1782: 817-23.
3. 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.
4. Marfella R, Di Filippo C, Laieta MT, et al. Effects of ubiquitin-proteasome system deregulation on the vascular senescence and atherosclerosis process in elderly patients. J Gerontol A Biol Sci Med Sci 2008; 63:
5. Marfella R, D’Amico M, Esposito K, et al. The ubiquitin-proteasome system and inflammatory activity in diabetic atherosclerotic plaques: effects of rosiglitazone treatment. Diabetes 2006; 55: 622-32.
6. Leineweber K, Böse D, Vogelsang M, Haude M, Erbel R, Heusch G. Intense vasoconstriction in response to aspirate from stented saphenous vein aortocoronary bypass grafts. J Am Coll Cardiol 2006; 47: 981-6.
7. Campeau L. The Canadian Cardiovascular Society grading of angina pectoris revisited 30 years later. Can J Cardiol 2002; 18: 371-9.
8. Böse D, Leineweber K, Konorza T, et al. Release of TNF-αlpha during stent implantation into saphenous vein aortocoronary bypass grafts and its relation to plaque extrusion and restenosis. Am J Physiol Heart Circ Physiol 2007; 292: H2295-9.
9. Sixt SU, Beiderlinden M, Jennissen HP, Peters J. Extracellular proteasome in the human alveolar space:
a new housekeeping enzyme? Am J Physiol Lung Cell Mol Physiol 2007; 292: L1280-8.
10. Sixt SU, Adamzik M, Spyrka D, et al. Alveolar extracellular 20S proteasome in patients with acute respiratory distress syndrome. Am J Respir Crit Care Med 2009; 179: 1098-106.
11. Grune T, Jung T, Merker K, Davies KJ. Decreased proteolysis caused by protein aggregates, inclusion bodies, plaques, lipofuscin, ceroid, and 'aggresomes' during oxidative stress, aging, and disease. Int J Biochem Cell Biol 2004; 36: 2519-30.
12. Stoneman VE, Bennett MR. Role of apoptosis in atherosclerosis and its therapeutic implications. Clin Sci (Lond) 2004; 107: 343-54.
13. Versari D, Herrmann J, Gössl M, et al. Dysregulation of the ubiquitin-proteasome system in human carotid atherosclerosis. Arterioscler Thromb Vasc Biol 2006; 26: 2132-9.
14. Erbel R, Heusch G. Brief review: coronary micro-embolization. J Am Coll Cardiol 2000; 36: 22-4.
15. Skyschally A, Leineweber K, Gres P, Haude M, Erbel R, Heusch G. Coronary microembolization. Basic Res Cardiol 2006; 101: 373-82.
16. Heusch P, Skyschally A, Leineweber K, Haude M, Erbel R, Heusch G. The interaction of coronary microembolization and ischemic preconditioning: a third window of cardioprotection through TNF-αlpha. Arch Med Sci 2007; 2: 83-92.
17. Herrmann J, Haude M, Lerman A, et al. Abnormal coronary flow velocity reserve after coronary intervention is associated with cardiac marker elevation. Circulation 2001; 103: 2339-45.
18. Herrmann J, Lerman A, Baumgart D, et al. Preprocedural statin medication reduces the extent of periprocedural non-Q-wave myocardial infarction. Circulation 2002; 106: 2180-3.
19. Bahrmann P, Werner GS, Heusch G, et al. Detection of coronary microembolization by Doppler ultrasound in patients with stable angina pectoris undergoing elective percutaneous coronary interventions. Circulation 2007; 115: 600-8.
20. Böse D, von Birgelen C, Zhou XY, et al. Impact of atherosclerotic plaque composition on coronary microembolization during percutaneous coronary interventions. Basic Res Cardiol 2008; 103: 587-97.
21. Tan C, Li Y, Tan X, Pan H, Huang W. Inhibition of the ubiquitin-proteasome system: a new avenue for atherosclerosis. Clin Chem Lab Med 2006; 44: 1218-25.
22. Herrmann J, Lerman A, Baumgart D, et al. Pre-procedural statin medication reduces the extent of peri-procedural non-Q-wave myocardial infarction. Circulation 2002; 106: 2180-3.
Copyright: © 2009 Termedia & Banach. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0) License (http://creativecommons.org/licenses/by-nc-sa/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material, provided the original work is properly cited and states its license.
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