Multimodality imaging assessment of the deleterious role of the intraluminal thrombus on the growth of abdominal aortic aneurysm in a rat model.

OBJECTIVES: To evaluate imaging changes occurring in a rat model of elastase-induced abdominal aortic aneurysm (AAA), with emphasis on the intraluminal thrombus (ILT) occurrence. METHODS: The post-induction growth of the AAA diameter was characterized using ultrasound in 22 rats. ILT was reported on 13 rats that underwent 14 magnetic resonance imaging (MRI) 2-18 days post-surgery, and on 10 rats that underwent 18 fluoro-deoxyglucose (FDG) positron emission tomography (PET)/microcomputed tomography examinations 2-27 days post-surgery. Logistic regressions were used to establish the evolution with time of AAA length, diameter, ILT thickness, volume, stratification, MRI and FDG PET signalling properties, and histological assessment of inflammatory infiltrates. RESULTS: All of the following significantly increased with time post-induction (p < 0.001): AAA length, AAA diameter, ILT maximal thickness, ILT volume, ILT iron content and related MRI signalling changes, quantitative uptake on FDG PET, and the magnitude of inflammatory infiltrates on histology. However, the aneurysm growth peak followed occurrence of ILT approximately 6 days after elastase infusion. CONCLUSION: Our model emphasizes that occurrence of ILT precedes AAA peak growth. Aneurysm growth is associated with increasing levels of iron, signalling properties changes in both MRI and FDG PET, relating to its biological activities. KEY POINTS: • ILT occurrence in AAA is associated with increasing FDG uptake and growth. • MRI signalling changes in ILT reflect activities such as haemorrhage and RBC trapping. • Monitoring ILT activities using MRI may require no exogenous contrast agent.

Smad2-dependent protease nexin-1 overexpression differentiates chronic aneurysms from acute dissections of human ascending aorta.

OBJECTIVE: Tissue activation of proteolysis is involved in acute intramural rupture (dissections, acute ascending aortic dissection) and in progressive dilation (aneurysms, thoracic aneurysm of the ascending aorta) of human ascending aorta. The translational aim of this study was to characterize the regulation of antiproteolytic serpin expression in normal, aneurysmal, and dissecting aorta. APPROACH AND RESULTS: We explored expression of protease nexin-1 (PN-1) and plasminogen activator inhibitor-1 and their regulation by the Smad2 signaling pathway in human tissue and cultured vascular smooth muscle cells (VSMCs) of aneurysms (thoracic aneurysm of the ascending aorta; n=46) and acute dissections (acute ascending aortic dissection; n=10) of the ascending aorta compared with healthy aortas (n=10). Both PN-1 and plasminogen activator inhibitor-1 mRNA and proteins were overexpressed in medial tissue extracts and primary VSMC cultures from thoracic aneurysm of the ascending aorta compared with acute ascending aortic dissection and controls. Transforming growth factor-ß induced increased PN-1 expression in control but not in aneurysmal VSMCs. PN-1 and plasminogen activator inhibitor-1 overexpression by aneurysmal VSMCs was associated with increased Smad2 binding on their promoters and, functionally, resulted in VSMC self-protection from plasmin-induced detachment and death. This phenomenon was restricted to aneurysms and not observed in acute dissections. CONCLUSIONS: These results demonstrate that epigenetically regulated PN-1 overexpression promotes development of an antiproteolytic VSMC phenotype and might favor progressive aneurysmal dilation, whereas absence of this counter-regulation in dissections would lead to acute wall rupture.

Thrombus versus wall biological activities in experimental aortic aneurysms.

BACKGROUND/AIM: The intraluminal thrombus (ILT) is considered to participate in abdominal aortic aneurysm (AAA) evolution. To assess whether this role proceeds via ILT influence on biological activity of the AAA wall, we studied the relationships between the levels of some relevant proteases and microparticles (MP) released by ILT versus wall in rat experimental AAAs. METHODS AND RESULTS: Two weeks after elastase perfusion, ILT and AAA wall were incubated in cell culture medium and studies were performed on conditioned media. As shown by gelatin zymography, ILT released higher amounts of MMP9 than the wall, whereas the level of MMP2 activation (active/pro) was similar. Levels of elastase and urokinase plasminogen activator, plasmin and MPs, determined, respectively, by casein zymography, substrate hydrolysis and flow cytometry, were higher in ILT than in wall. Aneurysm diameter positively correlated with wall MMP9 levels, MMP2 activation, plasmin activity and MP release. Moreover, wall and ILT levels of pro- and active forms of MMP2, elastase and plasmin were positively correlated. Wall levels of MMP2 activation and plasmin activity also correlated with ILT weight. CONCLUSION: The present data suggest that, in this experimental model, ILT may contribute to AAA evolution via its influence on the level of aneurysmal wall protease activity.

Fibrinolytic activity is associated with presence of cystic medial degeneration in aneurysms of the ascending aorta.

AIMS: Thoracic ascending aortic aneurysms (TAA) are characterized by elastic fibre breakdown and cystic medial degeneration within the aortic media, associated with progressive smooth muscle cell (SMC) rarefaction. The transforming growth factor (TGF)-ß/Smad2 signalling pathway is involved in this process. Because the pericellular fibrinolytic system activation is able to degrade adhesive proteins, activate matrix metalloproteinase (MMP), induce SMC disappearance and increase the bioavailability of TGF-ß, the aim was to investigate the plasminergic system in TAA. METHODS AND RESULTS: Ascending aortas [21 controls and 19 TAAs (of three different aetiologies)] were analysed. Immunohistochemistry showed accumulation of t-PA, u-PA and plasmin in TAAs, associated with residual SMCs. Overexpression of t-PA and u-PA was confirmed by reverse transcription-polymerase chain reaction (RT-PCR), immunoblotting and zymography on TAA extracts and culture medium conditioned by TAA. Plasminogen was present on the SMC surface and inside cytoplasmic vesicles, but plasminogen mRNA was undetectable in the TAA medial layer. Plasmin-antiplasmin complexes were detected in TAA-conditioned medium and activation of the fibrinolytic system was associated with increased fibronectin turnover. Fibronectin-related material was detected immunohistochemically in dense clumps around SMCs and colocalized with latent TGF-ß binding protein-1. CONCLUSIONS: The fibrinolytic pathway could play a critical role in TAA progression, via direct or indirect impact on ECM and consecutive modulation of TGF-ß bioavailability.

Dilation-dependent activation of platelets and prothrombin in human thoracic ascending aortic aneurysm.

OBJECTIVE: The purpose of this study was to investigate whether thoracic ascending aortic aneurysm (TAAA) induces platelet activation as mural thrombus participates in aortic dilatation in abdominal aortic aneurysm and TAAA are associated with rheological factors favoring coagulation activation. METHODS AND RESULTS: We studied the relation between coagulation activation and aortic diameter in Marfan patients (MFS) with various aortic diameters (n=52). We then studied patients presenting large aneurysms associated with bicuspid aortic valve (BAV) and degenerative form. Lastly, we used immunochemistry and biochemistry to investigate prothrombin/thrombin retention within the aortic wall. Microparticles, sGPV, tissue factor, and TAT complexes were increased in plasma from MFS with large aneurysms (>or=45 mm) compared to MFS with limited aortic dilatation (<45 mm). Similar elevations were observed in all patients with large aortic aneurysms, regardless of the etiology, the site of maximal aortic dilation, associated valvulopathy, risk factors, or treatments. P-selectin and platelet-bound fibrinogen were also increased, demonstrating platelet activation in large aneurysms. Significant increase in sCD146 plasma concentration suggested alteration of endothelium. CONCLUSIONS: Platelet activation occurs in patients with large aneurysms of the ascending aorta, is dependent on aortic dilation, and is associated with thrombin generation, part of which appears to be retained in mucoid degeneration areas.

Clearance of plasmin-PN-1 complexes by vascular smooth muscle cells in human aneurysm of the ascending aorta.

Plasminogen is a circulating zymogen which enters the arterial wall by radial, transmural hydraulic conductance, where it is converted to plasmin by tissue plasminogen activator t-PA on an activation platform involving S100A4 on the vascular smooth muscle cell (vSMC) membrane. Plasmin is involved in the progression of human thoracic aneurysm of the ascending aorta (TAA). vSMCs protect the TAA wall from plasmin-induced proteolytic injury by expressing high levels of antiproteases. Protease nexin-1 (PN-1) is a tissue antiprotease belonging to the serpin superfamily, expressed in the vascular wall, and is able to form a covalent complex with plasmin. LDL receptor-related protein-1 (LRP-1) is a scavenger receptor implicated in protease-antiprotease complex internalization. In this study, we investigated whether PN-1 and LRP-1 are involved in the inhibition and clearance of plasminogen by the SMCs of human TAA. We demonstrated an overexpression of S100A4, PN-1, and LRP-1 in the medial layer of human TAA. Plasminogen activation taking place in the media of TAA was revealed by immunohistochemical staining and plasmin activity analyses. We showed by cell biology studies that plasmin-PN-1 complexes are internalized via LRP-1 in vSMCs from healthy and TAA media. Thus, two complementary mechanisms are involved in the protective role of PN-1 in human TAA: one involving plasmin inhibition and the other involving tissue clearance of plasmin-PN1 complexes via the scavenger receptor LRP-1.

99mTc-annexin-V functional imaging of luminal thrombus activity in abdominal aortic aneurysms.

BACKGROUND: The mural thrombus of abdominal aortic aneurysms (AAA) is involved in aneurysm progression via several interdependent biological processes including platelet activation. 99mTc-annexin V (ANX) is a scintigraphic tracer that binds to phosphatidylserine exposed on activated platelets and apoptotic cells. Here, we evaluated the potential of ANX imaging to assess mural thrombus biological activity in an experimental AAA model. The clinical applicability was further tested ex vivo on human samples of excised AAA thrombi. METHODS AND RESULTS: Experimental AAA was created by infusing elastase into infrarenal abdominal aorta in 17 rats, and 6 sham-operated rats were used as controls. Abdominal ANX scintigraphy was performed 2 weeks later followed by quantitative autoradiography and histological studies. Among the 13 rats which developed AAA, 11 displayed intense ANX uptake within AAA by scintigraphy. ANX uptake in the aneurysms on planar and single-photon emission computed tomography (SPECT) imaging was higher than that observed in infrarenal aorta of sham-operated controls (target/background ratio: 5.7+/-0.9 versus 1.33+/-0.21; P<0.005 for SPECT). Aneurysm-to-background activity ratios obtained by scintigraphy correlated with ANX activity in corresponding autoradiograms (R=0.69; P<0.02). This activity was located in the thrombus area where activated platelets and polymorphonuclear leukocytes accumulated. Similar patterns were also found in all of the 7 human AAA thrombi harvested during surgery. CONCLUSIONS: ANX imaging may assess mural thrombus renewal activity linked to permanent flowing blood interface.

Erythrocyte Efferocytosis by the Arterial Wall Promotes Oxidation in Early-Stage Atheroma in Humans.

BACKGROUND: Since red blood cells (RBCs) are the predominant cellular blood component interacting with the arterial wall, we explored the role of RBCs efferocytosis by vascular smooth muscle cells (vSMCs) in the initiation of human atheroma. METHODS AND RESULTS: The comparison of human healthy aortas with aortic fatty streaks or fibroatheromas revealed that RBC angiophagy is implicated from the earliest stages of atherogenesis, as documented by the concomitant detection of redox-active iron, hemoglobin, glycophorin A, and ceroids. RBCs infiltration in the arterial wall was associated with local lipid and protein oxidation, as well as vascular response (expression of heme oxygenase-1 and of genes related to iron metabolism as well as those encoding for phagocytosis). These effects were recapitulated in vitro when vSMCs were co-cultured with phosphatidyl-exposing senescent (s) RBCs but not with fresh RBCs. VSMCs engulfing sRBC increased their intracellular iron content, accumulated hemoglobin, lipids, and activated their phagolysosomes. Strikingly, injections of sRBCs into rats promoted iron accumulation in the aortic wall. In rabbits, hypercholesterolemia increased circulating senescent RBCs and induced the subendothelial accumulation of iron-rich phagocytic foam cells. RBCs bring cholesterol and iron/heme into the vascular wall and interact with vSMCs that phagocytize them. CONCLUSION: This study presents a previously unforeseen mechanism of plaque formation that implicates intimal RBC infiltration as one of the initial triggers for foam cell formation and intimal oxidation. Pathogenic effects exerted by several metabolic and hemodynamic factors may rely on their effect on RBC biology, thereby impacting how RBCs interact with the vascular wall.

Histopathology of an iliac aneurysm in a case of Menkes disease.

In Menkes disease, arterial tortuosity is frequent, whereas true aneurysms are rare. Here, we report aneurysmal pathology occurring in an infant with Menkes disease. An iliac aneurysm was diagnosed in a 2-month-old boy and attributed to Menkes syndrome on the basis of plasma copper deficiency. Samples of the aneurysmal wall were taken during surgery for histopathological analysis. As in other forms of aneurysm, the arterial wall was characterized by smooth muscle cell (SMC) disappearance, linked to SMC apoptosis and oxidative stress, areas of mucoid degeneration, and extracellular matrix breakdown, including disappearance of elastic fibers and presence of abnormal collagen.

Mediators of neutrophil recruitment in human abdominal aortic aneurysms.

AIMS: Neutrophils/platelet interactions are involved in abdominal aortic aneurysm (AAA). The intraluminal thrombus (ILT) is a human model of platelet/neutrophil interactions. The present study focused on mediators involved in neutrophil recruitment in AAA. METHODS AND RESULTS: Conditioned media from luminal, intermediate, and abluminal layers of 29 human ILTs were analysed for neutrophil markers [elastase/alpha1-antitrypsin and MMP9/NGAL complexes, myeloperoxidase (MPO), and alpha-defensin peptides], RANTES, platelet factor 4 (PF4), and interleukin-8 (IL-8). Their time-dependent release into serum from clots generated in vitro and their plasma concentrations in AAA patients and controls were determined. Immunohistochemistry for neutrophils, platelets, IL-8, PF4, and RANTES on AAA sections was performed; and molecules involved in ILT neutrophil chemotactic function were analysed in vitro. Neutrophils and platelets colocalized in the luminal layer of the thrombus. Consistently, neutrophil markers and platelet-derived RANTES and PF4 were released predominantly by the luminal thrombus pole, where their concentrations were significantly correlated. The luminal ILT layer was also the main source of IL-8, whose immunostaining colocalized with neutrophils. All were also released time dependently from clots and were increased in plasma of AAA patients. Luminal ILT layers displayed potent neutrophil chemotactic activity in vitro, which was inhibited by RANTES- and IL-8-blocking antibodies as well as by reparixin, an antagonist of the IL-8 receptors CXCR1 and CXCR2. CONCLUSION: Taken together, these results suggest that platelet-derived RANTES and neutrophil-derived IL-8 are involved in attracting neutrophils to the luminal layer of AAA ILT.

Tissue diffusion and retention of metalloproteinases in ascending aortic aneurysms and dissections.

Histopathological alterations in human aneurysms and dissections of the thoracic ascending aorta include areas of mucoid degeneration within the medial layer, colocalized with areas of cell disappearance and disruption of extracellular matrix elastic and collagen fibers. We studied the presence of matrix metalloproteinases in relation to their capacity to diffuse through the tissue or to be retained in areas of mucoid degeneration in aneurysms and dissections of the ascending aorta. Ascending aortas from 9 controls, 33 patients with aneurysms, and 14 with acute dissections, all collected at surgery, were analyzed. The morphological aspect was similar whatever the etiology or phenotypic expression of the pathological aortas, involving areas of extracellular matrix breakdown and cell rarefaction associated with mucoid degeneration. Release of proMMP-2, constitutively expressed by smooth muscle cells, was not different between controls and aneurysmal aortas, whereas the aneurysmal aortas released more of the active form. Release of pro and active MMP-9 was also similar between controls and aneurysmal aortas. Immunohistochemical staining of MMP-2 and MMP-9 was weak in both control and pathological aortas. In contrast, released MMP-7 (matrilysin) and MMP-3 (stromelysin-1) could not be detected in conditioned media but were present in tissue extracts with no detectable quantitative difference between controls and pathological aortas. Immunohistochemical staining of MMP-7 and MMP-3 revealed their retention in areas of mucoid degeneration, and semiquantitative evaluation of immunostaining showed more MMP-7 in pathological aortas than in controls. In conclusion, areas of mucoid degeneration, the hallmark of aneurysms, and dissections of thoracic ascending aortas, whatever their etiology, are not inert and can retain specific proteases.

A new macromolecular paramagnetic MR contrast agent binds to activated human platelets.

A new functionalized macromolecular magnetic resonance (MR) contrast agent has been developed from a carboxymethyldextran-Gd(DOTA) devoid of biospecificity. The functionalized contrast agent was synthesized in order to mimic PSGL-1, the main ligand of P-selectin, a glycoprotein mainly expressed on the surface of activated platelets. The starting compound, CM1, was first carboxymethylated by monochloroacetic acid leading to a series of 10 derivatives varying in their carboxymethyl content. CM8 derivative, with a degree of substitution in carboxymethyl of 0.84, was chosen for subsequent fluorolabeling and sulfation to give CM8FS. CM8FS has an average number molecular weight of 27 000 +/- 500 g/mol, a hydrodynamic radius of 5.7 +/- 0.2 nm and a high relaxivity (r(1) = 11.2/mM (Gd)/s at 60 MHz). Flow cytometry experiments on whole human blood or on isolated platelets evidenced in vitro a preferential binding of CM8FS on TRAP-activated human platelets. Interestingly, CM8FS did not bind to other blood cells or to resting platelets. Pellets of TRAP-activated human platelets have also been imaged in tubes with a 1.5 T MR imager. A MR signal was observed for activated platelets incubated with CM8FS. Altogether, these in vitro results evidenced the recognition of activated human platelets by a fluorescent paramagnetic contrast agent grafted with carboxyl and sulfate groups. This biomimetic approach associated with the versatile macromolecular platform appears promising for the development of new contrast agents for molecular imaging of activated platelets in cardiovascular diseases such as atherosclerosis and aneurysms.

Renewal of mural thrombus releases plasma markers and is involved in aortic abdominal aneurysm evolution.

Human abdominal aortic aneurysm (AAA) expansion has been linked to the presence of a mural thrombus. Here we explored the mechanism of the continual luminal renewal of this thrombus and its ability to release biological markers potentially detectable in plasma. We also explored the ability of platelet inhibition to pacify the thrombus and to limit aneurysm progression in an experimental model. Blood samples and mural thrombi were collected in 20 AAA patients. In parallel, segments of sodium dodecyl sulfate-decellularized guinea pig aorta were xenografted onto the abdominal aorta of 30 rats to induce aneurysms. Fifteen rats received abciximab treatment and fifteen received irrelevant immunoglobulins. Procoagulant activity and platelet activation markers (microparticles, sP-selectin, sGPV, sCD40L) were increased threefold to fivefold in eluates from the luminal thrombus layer compared to other layers. All these markers were increased twofold to fivefold in patients' plasma compared to matched controls (P < 0.005). In the rat model, abciximab reduced both thrombus area and aneurysmal enlargement (P < 0.05). Platelet aggregation is probably responsible for the renewal of the thrombus in AAA. The luminal thrombus released markers of platelet activation that could easily be detected in plasma. Platelet inhibition limited aortic aneurysm expansion in a rat model, providing new therapeutic perspectives in the prevention of AAA enlargement.

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.