Olivier Meilhac

Identification by a Differential Proteomic Approach of Heat Shock Protein 27 as a Potential Marker of Atherosclerosis

Background—We hypothesized that normal and pathological vessel walls display a differential pattern of secreted proteins. We have recently set up the conditions for comparing secretomes from carotid atherosclerotic plaques and control arteries using a proteomic approach to assess whether differentially secreted proteins could represent markers for atherosclerosis. Methods and Results—Normal endartery segments and different regions of endarterectomy pieces (noncomplicated/complicated plaques) were incubated in protein-free medium, and the released proteins were analyzed by 2D electrophoresis (2-DE). Among the differently secreted proteins, we have identified heat shock protein-27 (HSP27). Surprisingly, compared with control arteries, HSP27 release was drastically decreased in atherosclerotic plaques and barely detectable in complicated plaque supernatants. HSP27 was expressed primarily by intact vascular cells of normal arteries and carotid plaques (immunohistochemistry). Plasma detection of soluble HSP27 showed that circulating HSP27 levels are significantly decreased in the blood of patients with carotid stenosis relative to healthy subjects (0.19 [0.1 to 1.95] versus 83 [71.8 to 87.8]) ng/mL, P<0.0001). Conclusions—HSP27 secretion is decreased in complicated atherosclerotic plaques, and sHSP27 plasma levels are decreased in atherosclerotic patients compared with healthy subjects. Plasma sHSP27 levels could be a potential index of atherosclerosis, although further validation is needed in large patient cohorts.

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.

Topological Determinants and Consequences of Adventitial Responses to Arterial Wall Injury

Arteries are composed of 3 concentric tissue layers which exhibit different structures and properties. Because arterial injury is generally initiated at the interface with circulating blood, most studies performed to unravel the mechanisms involved in injury-induced arterial responses have focused on the innermost layer (intima) rather than on the outermost adventitial layer. In the present review, we focus on the involvement of the adventitia in response to various types of arterial injury leading to vascular remodeling. Physiologically, soluble vascular mediators are centrifugally conveyed by mass transport toward the adventitia. Moreover, in pathological conditions, neomediators and antigens can be generated within the arterial wall, whose outward conveyance triggers different patterns of local adventitial response. Adventitial angiogenesis, immunoinflammation, and fibrosis sequentially interact and their net balance defines the participation of the adventitial response in arterial pathology. In the present review we discuss 4 pathological entities in which the adventitial response to arterial wall injury participates in arterial wall remodeling. Hence, the adventitial adaptive immune response predominates in chronic rejection. Inflammatory phagocytic cell recruitment and initiation of a shift from innate to adaptive immunity characterize the adventitial response to products of proteolysis in abdominal aortic aneurysm. Adventitial sprouting of neovessels, leading to intraplaque hemorrhages, predominates in atherothrombosis. Adventitial fibrosis characterizes the response to mechanical stress and is responsible for the constrictive remodeling of arterial segments and initiating interstitial fibrosis in perivascular tissues. These adventitial events, therefore, have an impact not only on the vessel wall biology but also on the surrounding tissue.

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.—Suc, Isabelle, Meilhac, Olivier, Lajoie‐MAZENC, Isabelle, Vandaele, Jean, Jurgens, GüNTHER, Salvayre, Robert, NèGRE‐SALVAYRE, Anne Activation of EGF receptor by oxidized LDL. FASEB J. 12, 665–671 (1998)

Involvement of intraplaque hemorrhage in atherothrombosis evolution via neutrophil protease enrichment

The pathological remodeling of the arterial wall in atherosclerosis involves protease activities, which play a major role in complications via plaque rupture. Circulating leukocytes and particularly neutrophils have been shown to be an independent predictor of recurrent ischemic events. However, neutrophils are poorly documented within atherosclerotic plaques. We hypothesized that intraplaque hemorrhage could convey neutrophils into the lesion, spreading into the necrotic core, thus participating in its protease enrichment. One hundred human carotid endarterectomy specimens were dissected into culprit‐stenosing plaques (CPs) and adjacent noncomplicated plaques. Half of CPs exhibited hemorrhage, which was confirmed by the release of hemoglobin. Pro‐ and active forms of matrix metalloproteinase‐9 (MMP‐9) were increased in media conditioned by hemorrhagic plaques. Higher levels of lipocalin [neutrophil gelatinase‐associated lipocalin (NGAL)]/MMP‐9 complexes, specifically released by neutrophils, were also found in conditioned media from plaques with hemorrhage. Immunohistochemical analysis of the corresponding carotid samples showed that neutrophil markers such as elastase, NGAL/MMP‐9, CD66b, and proteinase 3 colocalized with blood constituents (i.e., hemoglobin, plasminogen). All markers of neutrophil degranulation were positively correlated in CP‐conditioned media (α1‐antitrypsin/elastase complexes, myeloperoxidase, and α‐defensins), and higher levels came from CPs containing intraplaque hemorrhages. Addition of an elastase inhibitor at the time of incubation led to a decrease in the proMMP‐9 activation in CPs, suggesting cross‐talk between proteases released by neutrophils. Finally, we found that neovessels observed at the interface between cap and core exhibit an activated endothelium, which may favor leukocyte diapedesis. Our study thus provides evidence for the involvement of neutrophils in plaque vulnerability.

Identification of Soluble Tumor Necrosis Factor-Like Weak Inducer of Apoptosis (sTWEAK) as a Possible Biomarker of Subclinical Atherosclerosis

Objectives—Assessment of vascular risk in asymptomatic patients and the response to medical therapy is a major challenge for prevention of cardiovascular events. Our aim was to identify proteins differentially released by healthy versus atherosclerotic arterial walls, which could be found in plasma and serve as markers of atherosclerosis. Methods and Results—We have analyzed supernatants obtained from cultured human carotid plaques and healthy arteries by surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry ProteinChip System. Surface-enhanced laser-desorption/ionization analysis unveiled an 18.4-kDa peak released in lower amount by carotid plaques than normal endarteries. This protein was identified as soluble tumor necrosis factor-like weak inducer of apoptosis (sTWEAK). To confirm that sTWEAK was the protein of interest, Western blot and enzyme-linked immunosorbent assay were performed. Both techniques confirmed that sTWEAK levels were decreased in carotid plaque supernatants. Subsequent measurement of sTWEAK in plasma showed a reduced concentration in subjects with carotid stenosis (N=30) compared with healthy subjects matched by sex and age (N=28) (P<0.001). Furthermore, in a test population of 106 asymptomatic subjects, we showed that sTWEAK concentrations negatively correlated with the carotid intima-media thickness (r=−0.4; P<0.001), an index of subclinical atherosclerosis. Conclusions—These results suggest that sTWEAK could be a potential biomarker of atherosclerosis.

Role of Leukocyte Elastase in Preventing Cellular Re-Colonization of the Mural Thrombus

To explore possible mechanisms responsible for the absence of cell re-colonization of mural thrombi in aneurysms, we analyzed the release and storage of leukocyte proteases in the most luminal layer versus intermediate and abluminal layers of 10 mural thrombi of human abdominal aortic aneurysms. The luminal layer contained many polymorphonuclear leukocytes (PMNs), which released pro-matrix metalloproteinase (MMP)-9 and MMP-8. Leukocyte elastase was also stored and released by the luminal layer (immunohistochemistry, activity on synthetic substrates, and casein zymography). Acid buffer allowed extraction of leukocyte elastase from the luminal layer, which was inhibited by elastase inhibitors. Casein zymography of luminal extracts and conditioned medium from formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMNs exhibited a similar lysis pattern, corresponding to elastase activity. Smooth muscle cell (SMC) seeding resulted in colonization of the intermediate thrombus layer ex vivo but not of the luminal layer. Extracts of the luminal layer induced loss of anchorage of both cultured human smooth muscle cells and stromal cells of bone marrow origin (anoikis). This anoikis was prevented by preincubation of the extracts with serine protease inhibitors. Moreover, adhesion of human SMCs and stromal bone marrow cells on fibrin gels was strongly inhibited when the gel was preincubated with pure elastase, medium of fMLP-stimulated PMNs, or extracts of luminal layers of mural thrombi. This loss of cell anchorage was prevented by the preincubation of the medium or extracts with alpha(1)-antitrypsin, but not when alpha(1)-antitrypsin was added after binding of elastase to the fibrin gel. In conclusion, elastase released by PMNs trapped within the mural thrombus impairs the spontaneous anchorage of mesenchymal cells to a fibrin matrix. This phenomenon could be one mechanism by which cellular healing of the mural thrombus in aneurysms is prevented.

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 α2‐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 99mTc‐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 99mTc‐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. Copyright

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 (ox‐LDL) 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.—Meilhac, O., Escargueil‐Blanc, I., Thiers, J.‐C., Salvayre, R., Nègre‐Salvayre, A. Bcl‐2 alters the balance between apoptosis and necrosis, but does not prevent cell death induced by oxidized low density lipoproteins. FASEB J. 13, 485–494 (1999)

Biological Significance of Decreased HSP27 in Human Atherosclerosis

Objective—Because culprit atherosclerotic plaques contain proteases, we hypothesized that the diminished heat shock protein 27 (HSP27) released by atherosclerotic plaques could be due to proteolysis. We assessed the role of HSP27 in human vascular smooth muscle cells (VSMCs) under proteolytic injury. Methods and Results—Active plasmin is present in culprit atherosclerotic plaques. Recombinant HSP27 was cleaved by plasmin and this effect was prevented by different inhibitors. Fragments and aggregated forms of HSP27 appeared after incubation of mammary control endarteries with plasmin. Coincubation of atherosclerotic plaques with recombinant HSP27 or mammary endarteries led to HSP27 proteolysis. After incubation of VSMCs with plasmin, HSP27 was overexpressed, phosphorylated, aggregated, and redistributed from the cytoskeleton to the cytosol, nucleus, and cell membrane. Plasmin-induced VSMC apoptosis was significantly higher in VSMCs treated by HSP27 siRNA. Immunohistochemical analysis of atherosclerotic plaques showed that plasmin(ogen) and apoptotic cells are localized in the core/shoulder whereas HSP27 and VSMCs are mainly expressed in the cap/media. Conclusions—Extracellular HSP27 can be degraded by enzymes released from atherosclerotic plaques and may reflect a proteolytic imbalance. Intracellular HSP27 downregulation decreases VSMCs resistance to proteolytically-induced apoptosis. HSP27 might play a pivotal role in the prevention of plaque instability and rupture.