In vivo targeted molecular imaging for activated platelets by mri using USPIO-fucoidan in rat abdominal aortic aneuryms model

POSTER PRESENTATIONInternational audienc

Fibrosis of the left atria during progression of heart failure is associated with increased matrix metalloproteinases in the rat

AbstractObjectivesThe purpose of this study was to determine the pathogenic factors and molecular mechanisms involved in fibrosis of the atria.BackgroundFibrosis is an important component of the pathophysiology of atrial fibrillation, especially when the arrhythmia is associated with heart failure (HF) or atrial dilation.MethodsWe used a rat model of myocardial infarction (MI) complicated by various degrees of left ventricular dysfunction and atrial dilation to study fibrosis and matrix metalloproteinase (MMP) activity in the left atrial (LA) myocardium by means of histologic, Western blot, zymographic, and immunohistologic techniques.ResultsThree months after surgical ligature of the left coronary artery, 27 rats had a large MI, 12 were in mild HF, and 15 in severe HF. Both groups had LA enlargement at the echocardiography. Masson’s trichrome and picrosirius staining of tissue sections revealed marked fibrosis at the periphery of trabeculae and also surrounding myolytic myocytes, in both mild and severe HF. In mild HF, the activity and expression of the matrilysin MMP-7 were increased (122%), whereas in severe HF, both MMP-7 (211%) and the gelatinase MMP-2 (187%) were up-regulated. There were no changes in the expression or activity of MMP inhibitors, TIMP-1, -2, and -4. Immunostaining of cryosections showed that MMP-2 was present in the interstitial spaces, whereas MMP-7 accumulated in myolytic myocytes.ConclusionsHemodynamic overload of the atria is an important pathogenic factor of fibrosis; MMP-7 appears to be involved in the early stage of this tissue remodeling process

High Density Lipoproteinspotentiate α-1 antitrypsin therapy in elastase-induced pulmonary emphysema

Several studies report that high-density lipoproteins (HDLs) can carry α1-antitrypsin (AAT; an elastase inhibitor). We aimed to determine whether injection of exogenous HDL, enriched or not in AAT, may have protective effects against pulmonary emphysema. After tracheal instillation of saline or elastase, mice were randomly treated intravenously with saline, human plasma HDL (75 mg apolipoprotein A1/kg), HDL-AAT (75 mg apolipoprotein A1–3.75 mg AAT/kg), or AAT alone (3.75 mg/kg) at 2, 24, 48, and 72 hours. We have shown that HDL-AAT reached the lung and prevented the development of pulmonary emphysema by 59.3% at 3 weeks (alveoli mean chord length, 22.9 ± 2.8 μm versus 30.7 ± 4.5 μm; P < 0.001), whereas injection of HDL or AAT alone only showed a moderate, nonsignificant protective effect (28.2 ± 4.2 μm versus 30.7 ± 5 μm [P = 0.23] and 27.3 ± 5.66 μm versus 30.71 ± 4.96 μm [P = 0.18], respectively). Indeed, protection by HDL-AAT was significantly higher than that observed with HDL or AAT (P = 0.006 and P = 0.048, respectively). This protective effect was associated (at 6, 24, and 72 h) with: (1) a reduction in neutrophil and macrophage number in the bronchoalveolar lavage fluid; (2) decreased concentrations of IL-6, monocyte chemoattractant protein-1, and TNF-α in both bronchoalveolar lavage fluid and plasma; (3) a reduction in matrix metalloproteinase-2 and matrix metalloproteinase-9 activities; and (4) a reduction in the degradation of fibronectin, a marker of tissue damage. In addition, HDL-AAT reduced acute cigarette smoke–induced inflammatory response. Intravenous HDL-AAT treatment afforded a better protection against elastase-induced pulmonary emphysema than AAT alone, and may represent a significant development for the management of emphysema associated with AAT deficiency

In vivo and in vitro evidence for the role of elastase shedding of CD163 in human atherothrombosis

Aims: CD163 is a macrophage receptor for haptoglogin/hemoglobin (Hp-Hb) complexes, responsible for the clearance of hemoglobin. We hypothesized that production of soluble CD163 may be due to proleolytic shedding of membrane CD163 by neutrophil elastase, reported to be increased in culprit atherosclerotic plaques. We analyzed the relationship between CD163 solubilization and elastase in vitro, in macrophage culture, ex vivo in human atherosclerotic plaque samples, and in vivo plasma of patients with coronary artery disease. Methods and Results: Neutrophil elastase was shown to enhance CD163 shedding and decreased the uptake of hemoglobin-haptoblobin (Hb-Hp) complexes by cultured macrophages. In addition, cultured carotid endarterectomy samples showing features of intra-plaque hemorrhage released more sCD163 and elastase/α1-antitrypsin (α1-AT) complexes than non-hemorrhagic plaques (n=44). Plasma levels of sCD163 and neutrophil elastase (complexed with α1-AT) were measured in patients with an acute coronary syndrome (ACS, n=42), stable angina pectoris (SAP, n=28), or normal coronary angiograms without subclinical atherosclerosis (n=21). ACS patients had higher sCD163 and elastase/α1-AT complexes plasma concentrations than subjects without coronary atherosclerosis. Circulating sCD163 and elastase/α1-AT complexes were positively correlated in patients with ACS (r=0.51, p<0.001) and SAP (r=0.62, p<0.001). Conclusion: Our results suggest that neutrophil elastase promotes CD163 shedding, resulting in a decreased clearance of Hb by macrophages, which may favor plaque destabilization. This may be reflected by increased plasma levels of sCD163 and elastase/α1-AT complexes which are positively correlated in patients with coronary artery disease

255 In vivo detection of non-occlusive thrombi in drug-eluting stents by scintigraphy and radio-labelled annexin V in a rabbit model

IntroductionThrombi in contact with non re-endothelialized stent struts associated with drug-eluting stent (DES) thrombosis. Hence, detection of thrombi in DES could help to evaluate the risk of DES thrombosis. Annexin V radio-labelled with 99mTechnetium (99mTc) is a radio-tracer with a high affinity for activated platelets.ObjectivesOur objectives were: 1) to develop an animal model of non-occlusive thrombosis of stents, 2) to evaluate the ability of annexin V 99mTc for the detection of in-stent thrombi using scintigraphy.MethodsRight carotid arteries of NZW rabbits (n=14) fed a high cholesterol diet were implanted with overlapping DES (n=7) or bare-metal stents (BMS; n=7). Four weeks after stent implantation, rabbits underwent a first scintigraphy 3 hours after injection of 200 MBq of radio-labelled annexin V 99mTc. At the end of the first scintigraphy, a suture was placed surgically proximal to the stented carotid arteries in order to induce a thrombus-prone flow limiting stenosis. Four days later, a second scintigraphy was performed. After the second scintigraphy, stents were excised, imaged ex vivo and then fixed for histological examination and scanning electron microscopy (SEM).ResultsActivities measured in vivo in the stented carotid arteries after injection of annexin V 99mTc were higher on the second scintigraphy after creation of a surgical stenosis as compared to the first scintigraphy (0.24 vs. 0.15 counts/pixel/MBq, respectively; p<0.05). On the second scintigraphy, activities were higher in DES vs. BMS (0.26 vs. 0.19 counts/pixel/MBq, respectively; p < 0.005). High activities measured in stents in vivo were associated with the detection of thrombi on corresponding histological sections and SEM.ConclusionsIn this work, we developed a rabbit model of non-occlusive thrombosis of stents in carotid arteries. In this model, in-stent thrombi could be detected using annexin V 99mTc scintigraphy

In vitro and in vivo evaluation of a dextran-graft-polybutylmethacrylate copolymer coated on CoCr metallic stent

International audienceIntroduction: The major complications of stent implantation are restenosis and late stent thrombosis. PBMA polymers are used for stent coating because of their mechanical properties. We previously synthesized and characterized Dextrangraft-polybutylmethacrylate copolymer (Dex-PBMA) as a potential stent coating. In this study, we evaluated the haemocompatibility and biocompatibility properties of Dex-PBMA in vitro and in vivo.Methods: Here, we investigated: (1) the effectiveness of polymer coating under physiological conditions and its ability to release Tacrolimus®, (2) the capacity of Dex-PBMA to inhibit Staphylococcus aureus adhesion, (3) the thrombin generation and the human platelet adhesion in static and dynamic conditions, (4) thebiocompatibility properties in vitro on human endothelial colony forming cells (ECFC) and on mesenchymal stem cells (MSC) and in vivo in rat models, and (5) we implanted Dex-PBMA and Dex-PBMA TAC coated stents in neointimal hyperplasia restenosis rabbit model. Results: Dex-PBMA coating efficiently prevented bacterial adhesion and release Tacrolimus®. Dex-PBMA exhibit haemocompatibility properties under flow and ECFC and MSC compatibility. In vivo, no pathological foreign body reaction was observed neither after intramuscular nor intravascular aortic implantation. After Dex-PBMA and Dex-PBMATAC coated stents 30 days implantation in a restenosis rabbit model, an endothelial cell coverage was observed and the lumenpatency was preserved.Conclusion: Based on our findings, Dex-PBMA exhibited vascular compatibility and can potentially be used as a coating for metallic coronary stents

Peristrut microhemorrhages: a possible cause of in-stent neoatherosclerosis?

AbstractBackgroundIn-stent neoatherosclerosis is characterized by the delayed appearance of markers of atheroma in the subintima, but the pathophysiology underlying this new disease entity remains unclear.Methods and resultsWe collected 20 human coronary artery stents by removal from explanted hearts. The mean duration of stent implantation was 34 months. In all samples, neoatherosclerosis was detected, particularly in peristrut areas. It consisted of foam cells and cholesterol clefts, with or without calcification, associated with neovascularization. Iron and glycophorin-A were present in peristrut areas, as well as autofluorescent ceroids. Moreover, in response to neoatherosclerosis, tertiary lymphoid organs (tissue lymphoid clusters) often developed in the adventitia. Some of these features could be reproduced in an experimental carotid stenting model in rabbits fed a high-cholesterol diet. Foam cells were present in all samples, and peristrut red blood cells (RBCs) were also detected, as shown by iron deposits and Bandeiraea simplicifiola isolectin-B4 staining of RBC membranes. Finally, in silico models were used to evaluate the compliance mismatch between the rigid struts and the distensible arterial wall using finite element analysis. They show that stenting approximately doubles the local von Mises stress in the intimal layer.ConclusionsWe show here that stent implantation both in human and in rabbit arteries is characterized by local peristrut microhemorrhages and finally by both cholesterol accumulation and oxidation, triggering together in-stent neoatherosclerosis. Our data indicate that these processes are likely initiated by an increased mechanical stress due to the compliance mismatch between the rigid stent and the soft wall

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

BackgroundSince 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 resultsThe 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.ConclusionThis 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