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Aims: Abdominal aortic aneurysm (AAA) is a particular form of atherothrombotic disease characterized by the dilation of the aortic wall and the presence of an intraluminal thrombus (ILT). The objective of the present study was to evaluate the pro-oxidant properties of the ILT and to characterize the anti-oxidant capacity of high-density lipoproteins (HDLs)., Methods and Results: Our results show that ILT, adventitia, and plasma from AAA patients contained high concentrations of lipid and protein oxidation products. Mediators produced within or released by the thrombus and the adventitia were shown to induce reactive oxygen species (ROS) production by cultured aortic smooth muscle cells (AoSMCs) and to trigger the onset of apoptosis (an increase in mitochondrial membrane potential). Iron chelation limited these effects. Both concentration and functionality of HDLs were altered in AAA patients. Plasma levels of Apo A-I were lower, and small HDL subclasses were decreased in AAA patients. Circulating HDLs in AAA patients displayed an impaired capacity to inhibit copper-induced low-density lipoprotein oxidation and AoSMC ROS production. Western blot analyses of HDLs demonstrated that myeloperoxidase is associated with HDL particles in AAA patients., Conclusion: ILT and adventitia are a source of pro-oxidant products, in particular haemoglobin, which may impact on the wall stability/rupture in AAA. In addition, HDLs from AAA patients exhibit an impaired anti-oxidant activity. In this context, restoring HDL functionality may represent a new therapeutic option in AAA.

Objective: In addition to its role in the clearance of haptoglobin-hemoglobin (Hp-Hb) complexes, CD163 is a macrophage scavenger receptor for tumor necrosis factor-like weak inducer of apoptosis (TWEAK). We recently reported that the CD163/TWEAK plasma ratio could be a potential biomarker of atherothrombosis in asymptomatic subjects. In this study, we assessed soluble TWEAK (sTWEAK) and soluble CD163 (sCD163) plasma levels in white males with peripheral artery disease (PAD) and in atherothrombotic femoral plaques to evaluate their relationship with disease. We also analyzed whether Hp-Hb complexes could compete for CD163-mediated TWEAK uptake., Methods and Results: Patients with PAD (n=155) showed a trend toward lower sTWEAK (median [interquartile range]: 134 [110-204] versus 147 [119-205] pg/mL; P=0.067) and higher sCD163 (median [interquartile range]: 367 [269-506] versus 288 [234-369] ng/mL; P<0.001) plasma concentrations than age-matched controls (n=251). sCD163 and sTWEAK plasma levels were negatively correlated in both patients and controls. After stratification according to the severity of disease, sCD163/sTWEAK ratio was significantly increased in patients with more severe disease relative to the other groups (P=0.049). Analysis of conditioned medium obtained from cultured human atherothrombotic femoral plaque samples (n=36) and healthy aortas (n=14) revealed that high amounts of sCD163 were released by the atherothrombotic tissue, whereas sTWEAK presented the opposite trend (P<0.05). Finally, we report a potential association between CD163 shedding and oxidative stress., Conclusions: Our results suggest that the sCD163/sTWEAK plasma ratio may be associated with atherothrombosis burden in PAD. We hypothesize that an imbalance between TWEAK and CD163 could reflect the progression of atherothrombosis.

Fibrinolysis and pericellular proteolysis depend on molecular coassembly of plasminogen and its activator on cell, fibrin, or matrix surfaces. We report here the existence of a fibrinolytic cross-talk mechanism bypassing the requirement for their molecular coassembly on the same surface. First, we demonstrate that, despite impaired binding of Glu-plasminogen to the cell membrane by epsilon-aminocaproic acid (epsilon-ACA) or by a lysine-binding site-specific mAb, plasmin is unexpectedly formed by cell-associated urokinase (uPA). Second, we show that Glu-plasminogen bound to carboxy-terminal lysine residues in platelets, fibrin, or extracellular matrix components (fibronectin, laminin) is transformed into plasmin by uPA expressed on monocytes or endothelial cell-derived microparticles but not by tissue-type plasminogen activator (tPA) expressed on neurons. A 2-fold increase in plasmin formation was observed over activation on the same surface. Altogether, these data indicate that cellular uPA but not tPA expressed by distinct cells is specifically involved in the recognition of conformational changes and activation of Glu-plasminogen bound to other biologic surfaces via a lysine-dependent mechanism. This uPA-driven cross-talk mechanism generates plasmin in situ with a high efficiency, thus highlighting its potential physiologic relevance in fibrinolysis and matrix proteolysis induced by inflammatory cells or cell-derived microparticles.

Objective: In human abdominal aortic aneurysm, the accumulation of blood-derived cells and proteases within the mural thrombus plays a pivotal role in the evolution toward vessel wall rupture. We sought to identify peptides released from abdominal aortic aneurysm specimens, characterized by an intraluminal thrombus., Methods and Results: Intraluminal thrombus samples were analyzed by differential proteomics, using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. A 1309-Da peptide was detected in larger amounts in the newly formed luminal thrombus layer relative to older layers. It was identified as being LVVYPWTQRF (known as LVV-Hemorphin 7), a peptide generated from hemoglobin by cathepsin D. By immunohistochemical analysis, we showed that Hemorphin 7 (H7) colocalizes with cathepsin D and cathepsin G in the luminal layer of the intraluminal thrombus. In vitro, cathepsin G was able to generate H7 peptides at pH 7.4, whereas cathepsin D was only active in acidic conditions. Finally, H7 peptides were shown to be increased 3- to 4-fold in sera of abdominal aortic aneurysm patients relative to controls, and their levels were positively correlated with the volume of the thrombus., Conclusions: Our results suggest that circulating H7 peptides may reflect proteolysis of hemoglobin in the aneurysmal intraluminal thrombus and may be used as a biological marker of pathological vascular remodeling.