Elsevier

Atherosclerosis

Volume 212, Issue 1, September 2010, Pages 333-338
Atherosclerosis

Increased levels of thioredoxin in patients with abdominal aortic aneurysms (AAAs). A potential link of oxidative stress with AAA evolution

https://doi.org/10.1016/j.atherosclerosis.2010.05.031Get rights and content

Abstract

Objective

Oxidative stress is a main mechanism involved in vascular pathologies. Increased thioredoxin (TRX) levels have been observed in several oxidative stress-associated cardiovascular diseases. We aim to test the potential role of TRX as a biomarker of oxidative stress in abdominal aortic aneurysm (AAA).

Methods

TRX levels were analysed in both AAA intraluminal thrombus (ILT) tissue and in tissue-conditioned media by immunohistochemistry, Western blot and ELISA. Moreover, serum TRX levels were assessed in AAA Caucasian patients by ELISA.

Results

TRX was mainly localized in the luminal part of ILT in AAA. Compared with the abluminal layer, TRX release was increased in the luminal layer of the ILT of AAA (31 ± 9 ng/ml vs. 9 ± 3 ng/ml, p < 0.05). The interest of this approach is that we can identify proteins potentially released into the blood compartment, which could serve as biomarkers of the pathology. In a training population, serum TRX levels were significantly increased in patients with AAA relative to healthy subjects (50 ± 6 ng/ml vs. 26 ± 3 ng/ml, p < 0.05). These results were validated in a second independent group of patients. Moreover, a positive correlation between TRX and AAA size (rho = 0.5, p < 0.001) was observed. Finally, in AAA samples with follow-up, TRX was positively associated to aneurismal growth rate (rho = 0.25, p = 0.027).

Conclusions

TRX release is increased in the luminal part of AAA and TRX serum levels are increased in AAA patients compared with healthy subjects. TRX levels correlates with AAA size and expansion, suggesting its potential role as a biomarker of AAA evolution.

Introduction

Abdominal aortic aneurysm (AAA) is an important health problem in elderly. In cross-sectional studies the prevalence varies from 3% to 8%. In elderly men AAA may cause as much as 2–3% of all deaths. The incidence of asymptomatic and ruptured AAA has increased during the last decades [1].

Efforts to limit the mortality rate from AAA rupture depend on early detection and elective AAA repair. The indication for elective repair preventing rupture is mainly based upon the maximal diameter of AAA above 5 or 5.5 cm. The benefit of early detection of AAAs by ultrasound screening is limited because most AAAs are too small to operate at the time of diagnosis, early repair of small AAA has been demonstrated to be inefficient and there is currently no established treatment for small AAAs [2], [3]. Most AAAs shows discontinuous growth patterns and alternate periods of stability and non-growth with periods of acute expansion and occasionally rupture. In this respect, the diameter of the AAA is a surrogate marker of the growth rate, which reflects the magnitude of the degenerative process of the wall, and thus becomes a surrogate marker of rupture. Consequently, biomarkers of size, growth and rupture could give us a more nuanced indication for surgery. Moreover, identification of such biomarkers could also afford novel pathogenic pathways and may thus open possibilities for pharmacological inhibition of growth, providing tools for monitoring this inhibition [4].

AAA are characterized by the presence of a mural hemo-thrombus containing non-cellular and cellular blood components including platelets, red blood cells (RBCs) and polymorphonuclear neutrophils (PMN) which represent the major class of leukocytes, particularly abundant within the luminal layer of the thrombus. Upon activation, PMN release proteases but also myeloperoxidase (MPO) causing oxidative damages. The combination of NADPH oxidase, NO synthase and MPO leads to the production of a variety of reactive oxygen species such as superoxide anion, hydroxyl radicals or peroxynitrite. In addition, the trapping of RBCs within the thrombus may lead to hemolysis and subsequent release of the pro-oxidant hemoglobin, in particular able to generate OH° from H2O2 by the Fenton reaction. This biological activity of the luminal layer of the thrombus is therefore a source of potential biomarkers coming from activated or dying PMNs and RBCs. Thioredoxin (TRX) is an important intracellular antioxidant enzyme that is elevated in plasma of patients with coronary atherosclerosis [5], [6] and associated with the presence of intraplaque hemorrhage [7]. In the present work, we sought for the presence of TRX in the AAA thrombus, which could be released into the blood compartment. We thus assessed TRX in conditioned media from the different layers of AAA thrombus, and then in the serum of patients with AAA.

Section snippets

AAA tissue and tissue-conditioned media

Ten AAA thrombus samples were collected during surgical repair and dissected into luminal and abluminal parts (respectively at the interface with circulating blood and with the remaining media). AAA samples were obtained from patients undergoing surgery, enrolled in the RESAA protocol (REflet Sanguin de l’évolutivité des Anévrysmes de l’Aorte abdominale, CCPPRB Paris-Cochin n° 2095, n° 1930 and n° 1931) [8]. All patients gave their informed written consent, and the protocol was approved by a

TRX is mainly localized and released by the luminal part of AAA thrombus

We first analysed the presence and localization of TRX in human AAA thrombus. As shown in Fig. 1A, the luminal part of the thrombus shows an important staining associated with poly-lobed nuclei cells, likely to be neutrophils. In some areas, corresponding to most recently formed thrombus, fibrin and erythrocytes show an intense positivity, which may also participate in TRX release by the luminal part of the thrombus. Since TRX is released from cells in response to oxidative stress, we analysed

Discussion

AAA result from atherothrombotic complications in an infrarenal segment of the aorta, leading to a permanent arterial dilatation associated with proteolytic injury of the media. In contrast to stenosing evolution of atherothrombosis such as in carotid, femoral or coronary arteries, AAA presents an outward progression due to the weakening of the media as a consequence of extracellular matrix proteolysis and smooth muscle cell rarefaction. The formation of a luminal thrombus may be considered as

Acknowledgements

The paper have been supported by the European Community, FAD project (FP-7, HEALTH F2-2008-200647), the Spanish Ministerio de Ciencia y Tecnología (SAF2007/63648), (SAF2010-21852, SAF 2007-60896) HF2007-0120, Fundacion Ramon Areces, CAM (S2006/GEN-0247), Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III, Redes RECAVA (RD06/0014/0035) (Programa Miguel Servet to L.M.B.-C.) and EUS2008-03565. TJ was supported by grants from the Nouvelle Société Française d’Athérosclérose and Fondation

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