From tissue iron retention to low systemic haemoglobin levels, new pathophysiological biomarkers of human abdominal aortic aneurysm.

Iron deposits are observed in tissue of abdominal aortic aneurysm (AAA) patients, although the underlying mechanisms are not completely elucidated. Therefore we explored circulating markers of iron metabolism in AAA patients, and tested if they could serve as biomarkers of AAA. Increased red blood cell (RBC)-borne iron retention and transferrin, transferrin receptor and ferritin expression was observed in AAA tissue compared to control aorta (immunohistochemistry and western blot). In contrast, decreased circulating iron, transferrin, mean corpuscular haemoglobin concentration (MCHC) and haemoglobin concentration, along with circulating RBC count, were observed in AAA patients (aortic diameter >3 cm, n=114) compared to controls (aortic diameter <3 cm, n=88) (ELISA), whereas hepcidin concentrations were increased in AAA subjects (MS/MS assay). Moreover, iron, transferrin and haemoglobin levels were negatively, and hepcidin positively, correlated with aortic diameter in AAA patients. The association of low haemoglobin with AAA presence or aortic diameter was independent of specific risk factors. Moreover, MCHC negatively correlated with thrombus area in another cohort of AAA patients (aortic diameter 3-5 cm, n=357). We found that anaemia was significantly more prevalent in AAA patients (aortic diameter >5 cm, n=8,912) compared to those in patients with atherosclerotic aorto-iliac occlusive disease (n=17,737) [adjusted odds ratio=1.77 (95% confidence interval: 1.61;1.93)]. Finally, the mortality risk among AAA patients with anaemia was increased by almost 30% [adjusted hazard ratio: 1.29 (95% confidence interval: 1.16;1.44)] as compared to AAA subjects without anaemia. In conclusion, local iron retention and altered iron recycling associated to high hepcidin and low transferrin systemic concentrations could lead to reduced circulating haemoglobin levels in AAA patients. Low haemoglobin levels are independently associated to AAA presence and clinical outcome.

Soluble TWEAK plasma levels predict expansion of human abdominal aortic aneurysms.

OBJECTIVES: Diminished soluble tumor necrosis factor-like weak inducer of apoptosis (sTWEAK) concentrations are associated with cardiovascular diseases. We have analyzed sTWEAK levels and its relation with expansion rate in subjects with abdominal aortic aneurysm (AAA). METHODS: sTWEAK levels were measured by ELISA. RESULTS: sTWEAK concentrations were diminished in small AAA (≤ 5 cm; 353 ± 12 pg/mL; n = 25, p = 0.03) and large AAA (>5 cm; 315 ± 21 pg/mL; n = 18, p = 0.004) compared with healthy subjects (411 ± 22 pg/mL; n=27). Moreover, sTWEAK concentrations were negatively associated with AAA size (r = -0.4; p = 0.008). sTWEAK was also negatively associated with AAA expansion rate with 5 years of follow-up (n = 79, r = -0.263; p = 0.031). Multivariate regression analysis revealed that sTWEAK levels were independently associated with AAA growth rate (ß = -0.208; p = 0.046). CONCLUSIONS: sTWEAK plasma levels were decreased in subjects with AAA and were independently related with AAA expansion rate indicating that this protein could be a novel diagnostic and prognostic biomarker of AAA.

Beneficial effects of prolonged systemic administration of PlGF on late outcome of post-ischaemic myocardial performance.

Recent evidence indicates that an imbalance between cardiomyocyte hypertrophy and blood vessel growth in the remote myocardium may contribute to heart failure in ischaemic heart disease. It remains, however, largely unknown which angiogenic factors are capable of stimulating vessel growth in the remote myocardium after myocardial infarction (MI) and whether systemic, rather than local, administration of such factors suffices to ameliorate post-MI cardiac recovery. We therefore analysed the effect of systemic placental growth factor (PlGF) delivery on myocardial recovery post-MI in mice. MI was induced by permanent ligation of the left anterior descending coronary (LAD) artery in C57Bl6/J mice, followed by systemic injection of a PlGF adenovirus, resulting in elevated circulating levels of PlGF for 4 weeks. Functional and morphological analysis revealed that PlGF treatment induced cardiomyocyte hypertrophy and improved cardiac recovery at day 28 post-MI. PlGF stimulated angiogenesis in the infarct border and vessel enlargement in the remote myocardium. In this mouse model, capillary-to-cardiomyocyte ratios in the remote myocardium were maintained post-MI, but PlGF increased the vascular perfusion area in balance with the cardiomyocyte hypertrophy. Overall, systemic delivery of PlGF improves cardiac performance and promotes adaptive remodelling of the post-MI heart.

Topology of the fibrinolytic system within the mural thrombus of human abdominal aortic aneurysms.

Development and progression of acquired abdominal aortic aneurysms (AAAs) involve proteolytic activity. In the present study, we investigate the distribution of fibrinolytic system components within mural thrombi of human AAAs. 20 mural thrombi and the remaining AAA walls were dissected. The luminal, intermediate and abluminal thrombus layers, and media and adventitia were separately incubated in cell culture medium. Conditioned media were then analysed for plasminogen activators (PAs), plasminogen activator inhibitor-1 (PAI-1), free-plasmin, plasmin alpha(2)-antiplasmin complexes (PAPs) and D-dimers release. In parallel, PA and PAI-1 mRNA expression analysis was performed by RT-PCR. The study was completed by immunohistochemical localization of these components in AAA, ex vivo functional imaging using (99m)Tc-aprotinin as a ligand and measurement of PAP and D-dimer plasma levels. All fibrinolytic system components were present in each aneurysmal layer. However, the mural thrombus was the main source of active serine-protease release. Interestingly, the luminal layer of the thrombus released greater amounts of PAPs and D-dimers. This paralleled the preferential immunolocalization of plasminogen and PAs, and the (99m)Tc-aprotinin scintigraphic signal observed in the luminal pole of the thrombus. In contrast, mRNA expression analysis showed an exclusive synthesis of tPA and PAI-1 within the wall, whereas uPA mRNA was also expressed within the thrombus. Taken together, these results suggest that the increased plasma concentrations of PAPs and D-dimers found in AAA patients are related to mural thrombus proteolytic activity, thus explaining their known link with AAA progression. Components of the fibrinolytic system could also represent a target for functional imaging of thrombus activities in AAA.

Potential role of oxidized lipids and lipoproteins in antioxidant defense.

The atherogenic oxidative modification of low-density lipoprotein is suggested to occur in the aortic intima. There is reasonable evidence to suggest that antioxidants might be beneficial in preventing or retarding the progression of atherosclerosis. Exercise, estrogens, and substitution of polyunsaturated fat for saturated fat are beneficial in the prevention of atherosclerosis. Yet, paradoxically, they are capable of inducing an oxidative stress. To reconcile with this paradox, we postulate that under certain conditions an oxidative stress might be beneficial by inducing antioxidant enzymes in arterial cells. However, those with genetic deficiency in antioxidant enzymes or those who poorly respond to oxidative stress or those with overwhelming plasma oxidative stress might need additional antioxidant protection.

CD40 signals apoptosis through FAN-regulated activation of the sphingomyelin-ceramide pathway.

The possibility that the sphingomyelin (SM)-ceramide pathway is activated by CD40, a transmembrane glycoprotein belonging to the tumor necrosis factor receptor superfamily and that plays a critical role in the regulation of immune responses has been investigated. We demonstrate that incubation of Epstein-Barr virus-transformed lymphoid cells with an anti-CD40 antibody acting as an agonist results in the stimulation of a neutral sphingomyelinase, hydrolysis of cellular SM, and concomitant ceramide generation. In addition, SM degradation was observed in acid sphingomyelinase-deficient cells, as well as after ligation by soluble CD40 ligand. The anti-CD40 antibody, as well as the soluble CD40 ligand induced a decrease in thymidine incorporation and morphological features of apoptosis, which were mimicked by cell-permeant or bacterial sphingomyelinase-produced ceramides. Stable expression of a dominant-negative form of the FAN protein (factor associated with neutral sphingomyelinase activation), which has been reported to mediate tumor necrosis factor-induced activation of neutral sphingomyelinase, significantly inhibited CD40 ligand-induced sphingomyelinase stimulation and apoptosis of transformed human fibroblasts. Transformed fibroblasts from FAN knockout mice were also protected from CD40-mediated cell death. Finally, anti-CD40 antibodies were able to co-immunoprecipitate FAN in control fibroblasts but not in cells expressing the dominant-negative form of FAN, indicating interaction between CD40 and FAN. Altogether, these results strongly suggest that CD40 ligation can activate via FAN a neutral sphingomyelinase-mediated ceramide pathway that is involved in the cell growth inhibitory effects of CD40.

Bcl-2 alters the balance between apoptosis and necrosis, but does not prevent cell death induced by oxidized low density lipoproteins.

Oxidized low density lipoproteins (oxLDL) participate in atherosclerosis plaque formation, rupture, and subsequent thrombosis. Because oxLDL are toxic to cultured cells and Bcl-2 protein prevents apoptosis, the present work aimed to study whether Bcl-2 may counterbalance the toxicity of oxLDL. Two experimental model systems were used in which Bcl-2 levels were modulated: 1) lymphocytes in which the (high) basal level of Bcl-2 was reduced by antisense oligonucleotides; 2) HL60 and HL60/B (transduced by Bcl-2) expressing low and high Bcl-2 levels, respectively. In cells expressing relatively high Bcl-2 levels (lymphocytes and HL60/B), oxLDL induced mainly primary necrosis. In cells expressing low Bcl-2 levels (antisense-treated lymphocytes, HL60 and ECV-304 endothelial cells), the rate of oxLDL-induced apoptosis was higher than that of primary necrosis. OxLDL evoked a sustained calcium rise, which is a common trigger to necrosis and apoptosis since both types of cell death were blocked by the calcium chelator EGTA. Conversely, a sustained calcium influx elicited by the calcium ionophore A23187 induced necrosis in cells expressing high Bcl-2 levels and apoptosis in cells expressing low Bcl-2 levels. This suggests that Bcl-2 acts downstream from the calcium peak and inhibits only the apoptotic pathway, not the necrosis pathway, thus explaining the apparent shift from oxLDL-induced apoptosis toward necrosis when Bcl-2 is overexpressed.

Activation of EGF receptor by oxidized LDL.

Oxidized low density lipoproteins (oxLDL) are thought to play a major role in atherosclerosis. OxLDL exhibit a wide variety of biological effects resulting from their ability to interfere with intracellular signaling. The cellular targets and primary signaling events of oxLDL are unknown. We report that oxLDL elicit, in intact cells, tyrosine phosphorylation of the epithelial growth factor receptor (EGFR) and activation of its signaling pathway. This activation triggered by oxLDL was associated with derivatization of reactive amino groups of EGFR and was mimicked by 4-hydroxynonenal (4-HNE, a major lipid peroxidation product of oxLDL). Immunopurified EGFR was derivatized and activated in vitro by oxLDL lipid extracts and 4-HNE, thus indicating that 1) EGFR may be a primary target of oxidized lipids and 2) EGFR derivatization may be associated with activation. The reported data suggest that EGFR acts as a sensor for oxidized lipids. We therefore propose a novel concept of the mechanism by which oxidized lipids (contained in oxLDL or more generally produced during oxidative stress) are able to activate receptor tyrosine kinase and subsequent signaling pathways, resulting finally in a gain of function.

Oxidized LDLs induce massive apoptosis of cultured human endothelial cells through a calcium-dependent pathway. Prevention by aurintricarboxylic acid.

Oxidized LDLs are thought to play a central role in atherogenesis. Among their wide variety of biological properties, oxidized LDLs exhibit a cytotoxic effect on cultured vascular cells. Toxic doses of mildly oxidized LDLs elicited massive apoptosis in both primary and immortalized cultures of endothelial cells as shown by characteristic morphological and biochemical changes. Cytoplasmic and nucleic modifications (eg, chromatin condensation and nucleus fragmentation) were visualized by using electron and fluorescence microscopy of intact cells labeled by the fluorescent DNA probe SYTO-11. DNA fragmentation was quantified by ultracentrifugation of chromatin fragments, evaluated in situ by using the TUNEL (Terminal transferase-mediated dUTP-biotin nick end labeling) procedure, and visualized by electrophoresis of radiolabeled DNA fragments showing the characteristic apoptotic ladder. Apoptotic cells became rapidly detached and underwent postapoptotic necrosis that led to cell disintegration. Apoptosis was subsequent to a sustained and delayed peak of cytosolic calcium. Both the calcium peak and apoptosis were blocked by chelating the extracellular calcium with EGTA or by inhibiting the calcium influx by the calcium-channel blockers nifedipine and nisoldipine, thus suggesting that the apoptotic process induced by oxidized LDLs is clearly calcium dependent. Aurintricarboxylic acid, an inhibitor of endonucleases, also blocked the apoptotic process without blocking the calcium peak. These results suggest that toxic doses of mildly oxidized LDLs induce massive apoptosis of endothelial cells through a calcium-dependent mechanism and that this apoptotic process can be prevented by inhibiting the rise of cytosolic calcium or by inhibiting cellular endonucleases by aurintricarboxylic acid.