Thrombi and neutrophils
Michel JB, Ho-Tin-Noé B

2015 • Circ Res • [pdf] (Editorial to this paper)


Bizzozero’s drawings showing developmental stages of red blood cells.
Series a shows red blood cells in the bone marrow of the frog, series b shows red blood cells in the bone marrow of humans, and d and e show macrophages that have ingested erythrocytes.

More than a century ago, in 1881, Bizzozero discovered platelets and their role in primary hemostasis. Since this founding study, it has been shown that, in addition to the scaffold of platelets and fibrin, other proteins and cells play an important role in thrombus formation and stabilization.

[…]

Intravascular thrombi, whatever their initial cause and localization, are highly pathogenic because they not only induce downstream tissue ischemia by occluding arteries but also participate in injury of the surrounding tissue by releasing proteolytic and oxidative enzymes. Because of the various mechanisms of resistance to thrombolysis by r-tPA, new therapeutic approaches for the treatment of occlusive thrombosis are being actively investigated. In this context, the study of Mangold et al is of critical interest as it designates NETs as a source of new biomarkers to define the biological process of acute coronary syndrome and DNA as a therapeutic target for increasing the efficacy of r-tPA–induced thrombus lysis. Furthermore, this study strengthens the idea that periodontal diseases can affect the development and evolution of atherothrombotic diseases.

Clinical evidence for oral antiplatelet therapy in acute coronary syndromes
Wiviott SD, Steg PG

2015 • Lancet • [pdf]

Platelet-mediated thrombosis is a major pathophysiological mechanism that underlies acute coronary syndromes, and therefore, antiplatelet therapy is an important foundation in the treatment and prevention of recurrence of these syndromes. Nearly 30 years ago, aspirin was the first agent to show a benefit for acute coronary syndromes and is still a key therapeutic agent. The landmark CURE trial showed that the addition of a P2Y12 antagonist, clopidogrel, to aspirin was beneficial in the treatment of acute coronary syndromes. Despite substantial benefits with clopidogrel, limitations include the slow speed of onset, variable response, and a modest antiplatelet effect. Next-generation P2Y12 antagonists, prasugrel and ticagrelor, overcome these limitations and have been shown, in large-scale clinical trials for acute coronary syndromes, to reduce ischaemic events more than clopidogrel, at the expense of an increase in bleeding. Additional agents that target platelets by alternate mechanisms, including the protease-activated receptor-1 antagonist vorapaxar, have shown ischaemic benefit. These large-scale trials inform treatment decisions that need to balance ischaemic benefit and bleeding risk in patients with acute coronary syndromes. This Series paper describes major trial results, implications for clinical practice, and summarises continuing controversy.

Control of the Tfh-GC B Cell Axis by CD8+ Tregs Limits Atherosclerosis and Tertiary Lymphoid Organ Development
Clement M, Guedj K, Andreata F, Morvan M, Bey L, Khallou-Laschet J, Gaston AT, Delbosc S, Alsac JM, Bruneval P, Deschildre C, Le Borgne M, Castier Y, Kim HJ, Cantor H, Michel JB, Caligiuri G, Nicoletti A

2015 • Circulation • [pdf] • Editorial

BACKGROUND: -The atheromodulating activity of B cells during the development of atherosclerosis is well documented, but the mechanisms by which these cells are regulated have not been investigated.
METHODS AND RESULTS: -Here, we analyzed the contribution of Qa-1-restricted CD8+ regulatory T cells (CD8+ Tregs) to the control of the T follicular helper (Tfh)-germinal center (GC) B cell axis during atherogenesis. Genetic disruption of CD8+ Treg function in atherosclerosis-prone apolipoprotein E knockout (ApoE degrees ) mice resulted in overactivation of this axis in secondary lymphoid organs (SLOs), led to the increased development of tertiary lymphoid organs (TLOs) in the aorta, and enhanced disease development. In contrast, restoring control of the Tfh-GC B cell axis by blocking the ICOS-ICOSL pathway reduced the development of atherosclerosis and the formation of TLOs. Moreover, an analysis of human atherosclerotic aneurysmal arteries by flow cytometry, gene expression analysis, and immunofluorescence confirmed the presence of Tfh cells within TLOs.
CONCLUSIONS: -This study is the first to demonstrate that the Tfh-GC B cell axis is pro-atherogenic and that CD8+ Tregs control the GC reaction in both SLOs and TLOs. Therefore, disrupting this axis represents an innovative therapeutic approach.

MFAP5 Loss-of-Function Mutations Underscore the Involvement of Matrix Alteration in the Pathogenesis of Familial Thoracic Aortic Aneurysms and Dissections
Barbier M, Gross MS, Aubart M, Hanna N, Kessler K, Guo DC, Tosolini L, Ho-Tin-Noe B, Regalado E, Varret M, Abifadel M, Milleron O, Odent S, Dupuis-Girod S, Faivre L, Edouard T, Dulac Y, Busa T, Gouya L, Milewicz DM, Jondeau G, Boileau C

2014 • Am J Hum Genet • [pdf]

Thoracic aortic aneurysm and dissection (TAAD) is an autosomal-dominant disorder with major life-threatening complications. The disease displays great genetic heterogeneity with some forms allelic to Marfan and Loeys-Dietz syndrome, and an important number of cases still remain unexplained at the molecular level. Through whole-exome sequencing of affected members in a large TAAD-affected family, we identified the c.472C>T (p.Arg158( *)) nonsense mutation in MFAP5 encoding the extracellular matrix component MAGP-2. This protein interacts with elastin fibers and the microfibrillar network. Mutation screening of 403 additional probands identified an additional missense mutation of MFAP5 (c.62G>T [p.Trp21Leu]) segregating with the disease in a second family. Functional analyses performed on both affected individual's cells and in vitro models showed that these two mutations caused pure or partial haploinsufficiency. Thus, alteration of MAGP-2, a component of microfibrils and elastic fibers, appears as an initiating mechanism of inherited TAAD.

Platelets in Inflammation: Regulation of Leukocyte Activities and Vascular Repair
Gros A, Ollivier V, Ho-Tin-Noé B

2015 • Frontiers in immunology • [pdf]

There is now a large body of evidence that platelets are central actors of inflammatory reactions. Indeed, platelets play a significant role in a variety of inflammatory diseases. These diseases include conditions as varied as atherosclerosis, arthritis, dermatitis, glomerulonephritis, or acute lung injury. In this context, one can note that inflammation is a convenient but imprecise catch-all term that is used to cover a wide range of situations. Therefore, when discussing the role of platelets in inflammation, it is important to clearly define the pathophysiological context and the exact stage of the reaction. Inflammatory reactions are indeed multistep processes that can be either acute or chronic, and their sequence can vary greatly depending on the situation and organ concerned. Here, we focus on how platelets contribute to inflammatory reactions involving recruitment of neutrophils and/or macrophages. Specifically, we review past and recent data showing that platelets intervene at various stages of these reactions to regulate parameters such as endothelial permeability, the recruitment of neutrophils and macrophages and their effector functions, as well as inflammatory bleeding.The mechanisms underlying these various modulating effect of platelets are also discussed.

Upholding the T cell immune-regulatory function of CD31 inhibits the formation of T/B immunological synapses in vitro and attenuates the development of experimental autoimmune arthritis in vivo
Clement M, Fornasa G, Loyau S, Morvan M, Andreata F, Guedj K, Khallou-Laschet J, Larghi P, Le Roux D, Bismuth G, Chiocchia G, Hivroz C, Newman DK, Nicoletti A, Caligiuri G

2014 • J Autoimmun • [pdf]

CD31, a trans-homophilic inhibitory receptor expressed on both T- and B-lymphocytes, drives the mutual detachment of interacting leukocytes. Intriguingly, T cell CD31 molecules relocate to the immunological synapse (IS), where the T and B cells establish a stable interaction. Here, we show that intact CD31 molecules, which are able to drive an inhibitory signal, are concentrated at the periphery of the IS but are excluded from the center of the IS. At this site, were the cells establish the closest contact, the CD31 molecules are cleaved, and most of the extracellular portion of the protein, including the trans-homophilic binding sites, is shed from the cell surface. T cells lacking CD31 trans-homophilic binding sites easily establish stable interactions with B cells; at the opposite, CD31 signaling agonists inhibit T/B IS formation as well as the ensuing helper T cell activation and function. Confocal microscopy and flow cytometry analysis of experimental T/B IS shows that the T cell inhibitory effects of CD31 agonists depend on SHP-2 signaling, which reduces the phosphorylation of ZAP70. The analysis of synovial tissue biopsies from patients affected by rheumatoid arthritis showed that T cell CD31 molecules are excluded from the center of the T/B cell synapses in vivo. Interestingly, the administration of CD31 agonists in vivo significantly attenuated the development of the clinical signs of collagen-induced arthritis in DBA1/J mice. Altogether, our data indicate that the T cell co-inhibitory receptor CD31 prevents the formation of functional T/B immunological synapses and that therapeutic strategies aimed at sustaining CD31 signaling will attenuate the development of autoimmune responses in vivo.

Increased expression of protease nexin-1 in fibroblasts during idiopathic pulmonary fibrosis regulates thrombin activity and fibronectin expression
Francois D, Venisse L, Marchal-Somme J, Jandrot-Perrus M, Crestani B, Arocas V, Bouton MC

2014 • Lab Invest • [pdf]

Idiopathic pulmonary fibrosis (IPF) is a chronic diffuse lung disease characterized by an accumulation of excess fibrous material in the lung. Protease nexin-1 (PN-1) is a tissue serpin produced by many cell types, including lung fibroblasts. PN-1 is capable of regulating proteases of both coagulation and fibrinolysis systems, by inhibiting, respectively, thrombin and plasminergic enzymes. PN-1 is thus a good candidate for regulating tissue remodeling occurring during IPF. We demonstrated a significant increase of PN-1 expression in lung tissue extracts, lung fibroblasts and bronchoalveolar lavage fluids of patients with IPF. The increase of PN-1 expression was reproduced after stimulation of control lung fibroblasts by transforming growth factor-beta, a major pro-fibrotic cytokine involved in IPF. Another serpin, plasminogen activator inhibitor-1 (PAI-1) is also overexpressed in fibrotic fibroblasts. Unlike PAI-1, cell-bound PN-1 as well as secreted PN-1 from IPF and stimulated fibroblasts were shown to inhibit efficiently thrombin activity, indicating that both serpins should exhibit complementary roles in IPF pathogenesis, via their different preferential antiprotease activities. Moreover, we observed that overexpression of PN-1 induced by transfection of control fibroblasts led to increased fibronectin expression, whereas PN-1 silencing induced in fibrotic fibroblasts led to decreased fibronectin expression. Overexpression of PN-1 lacking either its antiprotease activity or its binding capacity to glycosaminoglycans had no effect on fibronectin expression. These novel findings suggest that modulation of PN-1 expression in lung fibroblasts may also have a role in the development of IPF by directly influencing the expression of extracellular matrix proteins. Our data provide new insights into the role of PN-1 in the poorly understood pathological processes involved in IPF and could therefore give rise to new therapeutic approaches.

Hemostatic disorders in a JAK2V617F-driven mouse model of myeloproliferative neoplasm
Lamrani L, Lacout C, Ollivier V, Denis CV, Gardiner E, Ho Tin Noe B, Vainchenker W, Villeval JL, Jandrot-Perrus M

2014 • Blood • [pdf]

Thrombosis is common in patients suffering from myeloproliferative neoplasm (MPN), whereas bleeding is less frequent. JAK2(V617F), the main mutation involved in MPN, is considered as a risk factor for thrombosis, although the direct link between the mutation and hemostatic disorders is not strictly established. We investigated this question using conditional JAK2(V617F) knock-in mice with constitutive and inducible expression of JAK2(V617F) in hematopoietic cells, which develop a polycythemia vera (PV)-like disorder evolving into myelofibrosis. In vitro, thrombosis was markedly impaired with an 80% decrease in platelet-covered surface, when JAK2(V617F) blood was perfused at arterial shear over collagen. JAK2(V617F) platelets presented only a moderate glycoprotein (GP) VI deficiency not responsible for the defective platelet accumulation. In contrast, a decreased proportion of high-molecular-weight von Willebrand factor multimers could reduce platelet adhesion. Accordingly, the tail bleeding time was prolonged. In the FeCl3-induced thrombosis model, platelet aggregates formed rapidly but were highly unstable. Interestingly, vessels were considerably dilated. Thus, mice developing PV secondary to constitutive JAK2(V617F) expression exhibit a bleeding tendency combined with the accelerated formation of unstable clots, reminiscent of observations made in patients. Hemostatic defects were not concomitant with the induction of JAK2(V617F) expression, suggesting they were not directly caused by the mutation but were rather the consequence of perturbations in blood and vessel homeostasis.

Collagen Can Selectively Trigger a Platelet Secretory Phenotype via Glycoprotein VI
Ollivier V, Syvannarath V, Gros A, Butt A, Loyau S, Jandrot-Perrus M, Ho-Tin-Noe B

2014 • PLoS One • [pdf]

Platelets are not only central actors of hemostasis and thrombosis but also of other processes including inflammation, angiogenesis, and tissue regeneration. Accumulating evidence indicates that these "non classical" functions of platelets do not necessarily rely on their well-known ability to form thrombi upon activation. This suggests the existence of non-thrombotic alternative states of platelets activation. We investigated this possibility through dose-response analysis of thrombin- and collagen-induced changes in platelet phenotype, with regards to morphological and functional markers of platelet activation including shape change, aggregation, P-selectin and phosphatidylserine surface expression, integrin activation, and release of soluble factors. We show that collagen at low dose (0.25 microg/mL) selectively triggers a platelet secretory phenotype characterized by the release of dense- and alpha granule-derived soluble factors without causing any of the other major platelet changes that usually accompany thrombus formation. Using a blocking antibody to glycoprotein VI (GPVI), we further show that this response is mediated by GPVI. Taken together, our results show that platelet activation goes beyond the mechanisms leading to platelet aggregation and also includes alternative platelet phenotypes that might contribute to their thrombus-independent functions.

CD31 is a key coinhibitory receptor in the development of immunogenic dendritic cells
Clement M, Fornasa G, Guedj K, Ben Mkaddem S, Gaston AT, Khallou-Laschet J, Morvan M, Nicoletti A, Caligiuri G

2014 • Proc Natl Acad Sci USA • [pdf]

CD31 is a transhomophilic tyrosine-based inhibitory motif receptor and is expressed by both dendritic cells (DCs) and T lymphocytes. Previous studies have established that the engagement of CD31 drives immune-inhibitory signaling in T lymphocytes, but the effect exerted by CD31 signaling in DCs remains elusive. Here, we show that CD31 is a key coinhibitory receptor on stimulated DCs, favoring the development of tolerogenic functions and finally resulting in T-cell tolerance. The disruption of CD31 signaling favored the immunogenic maturation and migration of resident DCs to the draining lymph nodes. In contrast, sustaining the CD31/SHP-1 signaling during DC maturation resulted in reduced NF-kappaB nuclear translocation, expression of costimulatory molecules, and production of immunogenic cytokines (e.g., IL-12, IL-6), whereas the expression of TGF-beta and IL-10 were increased. More importantly, CD31-conditioned DCs purified from the draining lymph nodes of ovalbumin-immunized mice favored the generation of antigen-specific regulatory T cells (CD25(+) forkhead box P3(+)) at the expense of effector (IFN-gamma(+)) cells upon coculture with naive ovalbumin-specific CD4(+) T lymphocytes ex vivo. Finally, the adoptive transfer of CD31-conditioned myelin oligodendrocyte glycoprotein-loaded DCs carried immune tolerance against the subsequent development of MOG-induced experimental autoimmune encephalomyelitis in vivo. The key coinhibitory role exerted by CD31 on DCs highlighted by the present study may have important implications both in settings where the immunogenic function of DCs is desirable, such as infection and cancer, and in settings where tolerance-driving DCs are preferred, such as autoimmune diseases and transplantation.

Organ Repair, Hemostasis, and In Vivo Bonding of Medical Devices by Aqueous Solutions of Nanoparticles
Meddahi-Pelle A, Legrand A, Marcellan A, Louedec L, Letourneur D, Leibler L

2014 • Angew Chem Int Ed Engl • [pdf] • Video

Sutures are traumatic to soft connective tissues, such as liver or lungs. Polymer tissue adhesives require complex in vivo control of polymerization or cross-linking reactions and currently suffer from being toxic, weak, or inefficient within the wet conditions of the body. Herein, we demonstrate using Stober silica or iron oxide nanoparticles that nanobridging, that is, adhesion by aqueous nanoparticle solutions, can be used in vivo in rats to achieve rapid and strong closure and healing of deep wounds in skin and liver. Nanoparticles were also used to fix polymer membranes to tissues even in the presence of blood flow, such as occurring after liver resection, yielding permanent hemostasis within a minute. Furthermore, medical devices and tissue engineering constructs were fixed to organs such as a beating heart. The simplicity, rapidity, and robustness of nanobridging bode well for clinical applications, surgery, and regenerative medicine.

Pathology of human plaque vulnerability: Mechanisms and consequences of intraplaque haemorrhages
Michel JB, Martin-Ventura JL, Nicoletti A, Ho-Tin-Noe B

2014 • Atherosclerosis • [pdf]

Atherothrombotic diseases are still major causes of inability and mortality and fighting atherothrombosis remains a public health priority. The involvement of repeated intraplaque haemorrhages (IPH) in the evolution of atherothrombotic lesions towards complications was proposed as early as 1936. This important topic has been recently revisited and reviewed. Histological observations have been corroborated by magnetic resonance imaging (MRI) of human carotid atheroma, identifying IPH as the main determinant of plaque evolution towards rupture. Beside the intimal integration of asymptomatic luminal coagulum, inward sprouting of neovessels from the adventitia towards the plaque, is one source of IPH in human atheroma. We recently described that directed neo-angiogenesis from the adventitia towards the plaque, across the media, is initiated by lipid mediators generated by the plaque on the luminal side, outwardly convected to the medial VSMCs. Subsequent stimulation of VSMC PPAR-gamma receptors induces VEGF expression which causes centripetal sprouting of adventitial vessels. However, this neovascularization is considered to be immature and highly susceptible to leakage. The main cellular components of IPH are Red Blood Cells (RBCs), which with their haemoglobin content and their cell membrane components, particularly enriched in unesterified cholesterol, participate in both the oxidative process and cholesterol accumulation. The presence of iron, glycophorin A and ceroids provides evidence of RBCs. IPH also convey blood leukocytes and platelets and are sites prone to weak pathogen contamination. Therefore prevention and treatment of the biological consequences of IPH pave the way to innovative preventive strategies and improved therapeutic options in human atherothrombotic diseases.

Polysaccharide Nanosystems for Future Progress in Cardiovascular Pathologies
Karine Andriola Silva A, Letourneur D, Chauvierre C

2014 • Theranostics • [pdf] • Cover of the Journal

Natural polysaccharides have received a lot of attention in the biomedical field. Indeed, sources of polysaccharides, extracted or produced from plants, bacteria, fungi or algae, are diverse and renewable. Moreover, recent progresses in polysaccharide chemistry and nanotechnologies allow elaborating new dedicated nanosystems. Polysaccharide-based nanosystems may be designed for interacting in several biological processes. In particular, the atherothrombotic pathology is highly concerned by polysaccharide-mediated recognition. Atherothrombotic diseases, regardless of the anatomical localization, remain the main causes of morbidity and mortality in the industrialized world. This review intends to provide an overview on polysaccharide-based nanosystems as drug delivery systems and targeted contrast agents for molecular imaging with an emphasis on the treatment and imaging of cardiovascular pathologies.

Deciphering the stromal and hematopoietic cell network of the adventitia from non-aneurysmal and aneurysmal human aorta
Dutertre CA, Clement M, Morvan M, Schakel K, Castier Y, Alsac JM, Michel JB, Nicoletti A

2014 • PLoS One • [pdf]

Aneurysm is associated to a complex remodeling of arteries that affects all their layers. Although events taking place in the intima and the media have received a particular attention, molecular and cellular events taking place in the adventitia have started to be deciphered only recently. In this study, we have precisely described the composition and distribution of stromal and hematopoietic cells in human arterial adventitia, both at steady state and in the setting of aortic aneurysm. Using polychromatic immunofluorescent and flow cytometry analyses, we observed that unlike the medial layer (which comprises mostly macrophages and T cells among leukocytes), the adventitia comprises a much greater variety of leukocytes. We observed an altered balance in macrophages subsets in favor of M2-like macrophages, an increased proliferation of macrophages, a greater number of all stromal cells in aneurysmal aortas. We also confirmed that in this pathological setting, adventitia comprised blood vessels and arterial tertiary lymphoid organs (ATLOs), which contained also M-DC8(+) dendritic cells (slanDCs) that could participate in the induction of T-cell responses. Finally, we showed that lymphatic vessels can be detected in aneurysmal adventitia, the functionality of which will have to be evaluated in future studies. All together, these observations provide an integrative outlook of the stromal and hematopoietic cell network of the human adventitia both at steady state and in the context of aneurysm.

Decreased PGE2 Content Reduces MMP-1 Activity and Consequently Increases Collagen Density in Human Varicose Vein
Gomez I, Benyahia C, Louedec L, Leseche G, Jacob MP, Longrois D, Norel X

2014 • PLoS One • [pdf]

Varicose veins are elongated and dilated saphenous veins. Despite the high prevalence of this disease, its pathogenesis remains unclear. AIMS: In this study, we investigated the control of matrix metalloproteinases (MMPs) expression by prostaglandin (PG)E2 during the vascular wall remodeling of human varicose veins. METHODS AND RESULTS: Varicose (small (SDv) and large diameter (LDv)) and healthy saphenous veins (SV) were obtained after surgery. Microsomal and cytosolic PGE-synthases (mPGES and cPGES) protein and mRNA responsible for PGE2 metabolism were analyzed in all veins. cPGES protein was absent while its mRNA was weakly expressed. mPGES-2 expression was similar in the different saphenous veins. mPGES-1 mRNA and protein were detected in healthy veins and a significant decrease was found in LDv. Additionally, 15-hydroxyprostaglandin dehydrogenase (15-PGDH), responsible for PGE2 degradation, was over-expressed in varicose veins. These variations in mPGES-1 and 15-PGDH density account for the decreased PGE2 level observed in varicose veins. Furthermore, a significant decrease in PGE2 receptor (EP4) levels was also found in SDv and LDv. Active MMP-1 and total MMP-2 concentrations were significantly decreased in varicose veins while the tissue inhibitors of metalloproteinases (TIMP -1 and -2), were significantly increased, probably explaining the increased collagen content found in LDv. Finally, the MMP/TIMP ratio is restored by exogenous PGE2 in varicose veins and reduced in presence of an EP4 receptor antagonist in healthy veins. CONCLUSIONS: In conclusion, PGE2 could be responsible for the vascular wall thickening in human varicose veins. This mechanism could be protective, strengthening the vascular wall in order to counteract venous stasis.

An immunologist's guide to CD31 function in T-cells
Marelli-Berg FM, Clement M, Mauro C, Caligiuri G

2013 • J Cell Sci • [pdf]

Although it is expressed by all leukocytes, including T-, B-lymphocytes and dendritic cells, the immunoglobulin-like receptor CD31 is generally regarded by immunologists as a marker of endothelial cell lineage that lacks an established functional role in adaptive immunity. This perception has recently been challenged by studies that reveal a key role for this molecule in the regulation of T-cell homeostasis, effector function and trafficking. The complexity of the biological functions of CD31 results from the integration of its adhesive and signaling functions in both the immune and vascular systems. Signaling by means of CD31 is induced by homophilic engagement during the interactions of immune cells and is mediated by phosphatase recruitment or activation through immunoreceptor tyrosine inhibitory motifs (ITIMs) that are located in its cytoplasmic tail. Loss of CD31 function is associated with excessive immunoreactivity and susceptibility to cytotoxic killing. Here, we discuss recent findings that have brought to light a non-redundant, complex role for this molecule in the regulation of T-cell-mediated immune responses, with large impact on our understanding of immunity in health and disease.

M1 macrophages act as LTßR-independent lymphoid tissue inducer cells during atherosclerosis-related lymphoid neogenesis
Guedj K, Khallou-Laschet J, Clement M, Morvan M, Gaston AT, Fornasa G, Dai J, Gervais-Taurel M, Eberl G, Michel JB, Caligiuri G, Nicoletti A

2013 • Cardiovasc Res • [pdf]

AIMS: The goal of this study was to characterize the role of inflammatory macrophages in the induction of the vascular smooth muscle cell (VSMC)-mediated formation of aortic tertiary lymphoid organs (TLO).
Methods and Results: Mouse bone marrow-derived M1 macrophages acted as lymphoid tissue inducer cells. Indeed, they expressed high levels of TNF-alpha and LT-alpha, two inducing cytokines that triggered expression of the chemokines CCL19, CCL20, and CXCL16, as did M1 supernatant. The blockade of LTbetaR signaling with LTbetaR-Ig had no effect while the blockade of TNFR1/2 signaling reduced chemokine expression by VSMCs in both WT and LTbetaR KO mice, demonstrating that LTbetaR signaling is dispensable for the M1-inducing effect. This effect was corroborated by the development of TLOs observed in LTbetaR KO->ApoE KO aortic segments after orthotopic transplantation. Furthermore, treatment of ApoE KO mice with anti TNF-alpha antibody decreased the number and incidence of aortic TLOs. Finally, lymphoid nodules composed of T and B cells formed in in vivo-implanted scaffolds seeded with VSMCs previously stimulated ex vivo by M1 conditioned medium.
Conclusions: These results are the first to identify M1 macrophages as inducer cells that trigger the expression of chemokines by VSMCs independently of LTbetaR signaling. We propose that the dialogue between macrophages and VSMCs - established across the vascular wall - contributes to the formation of aortic TLOs.

Bivalirudin started during emergency transport for primary PCI
Steg PG, van 't Hof A, Hamm CW, Clemmensen P, Lapostolle F, Coste P, Ten Berg J, Van Grunsven P, Eggink GJ, Nibbe L, Zeymer U, Campo dell' Orto M, Nef H, Steinmetz J, Soulat L, Huber K, Deliargyris EN, Bernstein D, Schuette D, Prats J, Clayton T, Pocock S, Hamon M, Goldstein P, Investigators E

2013 • N Engl J Med • [pdf]

BACKGROUND: Bivalirudin, as compared with heparin and glycoprotein IIb/IIIa inhibitors, has been shown to reduce rates of bleeding and death in patients undergoing primary percutaneous coronary intervention (PCI). Whether these benefits persist in contemporary practice characterized by prehospital initiation of treatment, optional use of glycoprotein IIb/IIIa inhibitors and novel P2Y12 inhibitors, and radial-artery PCI access use is unknown.
METHODS: We randomly assigned 2218 patients with ST-segment elevation myocardial infarction (STEMI) who were being transported for primary PCI to receive either bivalirudin or unfractionated or low-molecular-weight heparin with optional glycoprotein IIb/IIIa inhibitors (control group). The primary outcome at 30 days was a composite of death or major bleeding not associated with coronary-artery bypass grafting (CABG), and the principal secondary outcome was a composite of death, reinfarction, or non-CABG major bleeding.
RESULTS: Bivalirudin, as compared with the control intervention, reduced the risk of the primary outcome (5.1% vs. 8.5%; relative risk, 0.60; 95% confidence interval [CI], 0.43 to 0.82; P=0.001) and the principal secondary outcome (6.6% vs. 9.2%; relative risk, 0.72; 95% CI, 0.54 to 0.96; P=0.02). Bivalirudin also reduced the risk of major bleeding (2.6% vs. 6.0%; relative risk, 0.43; 95% CI, 0.28 to 0.66; P<0.001). The risk of acute stent thrombosis was higher with bivalirudin (1.1% vs. 0.2%; relative risk, 6.11; 95% CI, 1.37 to 27.24; P=0.007). There was no significant difference in rates of death (2.9% vs. 3.1%) or reinfarction (1.7% vs. 0.9%). Results were consistent across subgroups of patients.
CONCLUSIONS: Bivalirudin, started during transport for primary PCI, improved 30-day clinical outcomes with a reduction in major bleeding but with an increase in acute stent thrombosis. (Funded by the Medicines Company; EUROMAX ClinicalTrials.gov number, NCT01087723.).

The role of prostaglandin E2 in human vascular inflammation
Gomez I, Foudi N, Longrois D, Norel X

2013 • Prostaglandins Leukot Essent Fatty Acids • [pdf]

Prostaglandins (PG) are the product of a cascade of enzymes such as cyclooxygenases and PG synthases. Among PG, PGE2 is produced by 3 isoforms of PGE synthase (PGES) and through activation of its cognate receptors (EP1-4), this PG is involved in the pathophysiology of vascular diseases. Some anti-inflammatory drugs (e.g. glucocorticoids, nonsteroidal anti-inflammatory drugs) interfere with its metabolism or effects. Vascular cells can initiate many of the responses associated with inflammation. In human vascular tissue, PGE2 is involved in many physiological processes, such as increasing vascular permeability, cell proliferation, cell migration and control of vascular smooth muscle tone. PGE2 has been shown to contribute to the pathogenesis of atherosclerosis, abdominal aortic aneurysm but also in physiologic/adaptive processes such as angiogenesis. Understanding the roles of PGE2 and its cognate receptors in vascular diseases could help to identify diagnostic and prognostic biomarkers. In addition, from these recent studies new promising therapeutic approaches like mPGES-1 inhibition and/or EP4-antagonism should be investigated.

Design of biomimetic vascular grafts with magnetic endothelial patterning
Fayol D, Le Visage C, Ino J, Gazeau F, Letourneur D, Wilhelm C

2013 • Cell Transplant • [pdf] • Cover of the Journal

The development of small diameter vascular grafts with a controlled pluricellular organization is still needed for effective vascular tissue engineering. Here, we describe a technological approach combining a tubular scaffold and magnetically labeled cells to create a pluricellular and organized vascular graft, the endothelialization of which could be monitored by MRI prior to transplantation. A novel type of scaffold was developed with a tubular geometry and a porous bulk structure enabling the seeding of cells in the scaffold pores. A homogeneous distribution of human mesenchymal stem cells in the macroporous structure was obtained by seeding the freeze-dried scaffold with the cell suspension. The efficient covering of the luminal surface of the tube was then made possible thanks to the implementation of a magnetic-based patterning technique. Human endothelial cells or endothelial progenitors were magnetically labeled with iron oxide nanoparticles and successfully attracted to the 2-mm lumen where they attached and formed a continuous endothelium. The combination of imaging modalities [fluorescence imaging, histology, and 3D magnetic resonance imaging (MRI)] evidenced the integrity of the vascular construct. In particular, the observation of different cell organizations in a vascular scaffold within the range of resolution of single cells by 4.7 T MRI is reported.

Modifications of chromatin dynamics control smad2 pathway activation in aneurysmal smooth muscle cells
Gomez D, Kessler K, Michel JB, Vranckx R

2013 • Circ Res • [pdf] • Editorial

Rationale: The activation of the Smad2 signaling pathway is thought to play an important role in human aneurysmal diseases as described by an important body of research. We previously showed that constitutive Smad2 activation is associated with Smad2 mRNA overexpression in aneurysmal vascular smooth muscle cells (VSMCs), which is dependent on epigenetic regulation of the SMAD2 promoter involving histone modifications. However, the underlying molecular mechanisms controlling Smad2 overexpression are currently unknown.
Objective: The aim of the present study is to understand the mechanisms regulating the constitutive Smad2 overexpression in VSMCs by identification of the histone-modifying enzymes, transcription factors, and cofactors responsible for Smad2 promoter activation in aneurysmal disease.
Methods and Results: This study was performed on medial tissue extracts and primary cultures of VSMCs of human thoracic aneurysms (n=17) and normal thoracic aortas (n=10). Here, we demonstrate that the activation of SMAD2 promoter is driven by the recruitment of a multipartner complex, including the transcription factor p53 and histone acetyltransferases. Remarkably, the transcriptional regulatory network of the SMAD2 promoter is dramatically altered in human aneurysmal VSMCs in vitro and in situ with a switch from Myc-dependent repression of SMAD2 in normal vessel to a p53-dependent activation of SMAD2 in aneurysms. Furthermore, histone acetyltransferases p300 and P300/CBP-associated protein play a major role in SMAD2 promoter activation by acting on histone acetylation, p53 recruitment, and acetylation.
Conclusions: These results provide evidence for a major role of p53 and the complex composed of p300 and p300/CBP-associated protein in Smad2 activation in human aneurysmal VSMCs.

Impaired high-density lipoprotein anti-oxidant capacity in human abdominal aortic aneurysm
Delbosc S, Diallo D, Dejouvencel T, Lamiral Z, Louedec L, Martin-Ventura JL, Rossignol P, Leseche G, Michel JB, Meilhac O

2013 • Cardiovasc Res • [pdf]

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.

Biomechanical factors in the biology of aortic wall and aortic valve diseases
Back M, Gasser TC, Michel JB, Caligiuri G

2013 • Cardiovasc Res • [pdf] • Cover of the Journal

The biomechanical factors that result from the haemodynamic load on the cardiovascular system are a common denominator of several vascular pathologies. Thickening and calcification of the aortic valve will lead to reduced opening and the development of left ventricular outflow obstruction, referred to as aortic valve stenosis. The most common pathology of the aorta is the formation of an aneurysm, morphologically defined as a progressive dilatation of a vessel segment by more than 50% of its normal diameter. The aortic valve is exposed to both haemodynamic forces and structural leaflet deformation as it opens and closes with each heartbeat to assure unidirectional flow from the left ventricle to the aorta. The arterial pressure is translated into tension-dominated mechanical wall stress in the aorta. In addition, stress and strain are related through the aortic stiffness. Furthermore, blood flow over the valvular and vascular endothelial layer induces wall shear stress. Several pathophysiological processes of aortic valve stenosis and aortic aneurysms, such as macromolecule transport, gene expression alterations, cell death pathways, calcification, inflammation, and neoangiogenesis directly depend on biomechanical factors.

Smad2-Dependent Protease Nexin-1 Overexpression Differentiates Chronic Aneurysms From Acute Dissections of Human Ascending Aorta
Gomez D, Kessler K, Borges LF, Richard B, Touat Z, Ollivier V, Mansilla S, Bouton MC, Alkoder S, Nataf P, Jandrot-Perrus M, Jondeau G, Vranckx R, Michel JB

2013 • Arterioscler Thromb Vasc Biol • [pdf]

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-beta 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.

Endothelial Protease Nexin-1 Is a Novel Regulator of A Disintegrin and Metalloproteinase 17 Maturation and Endothelial Protein C Receptor Shedding via Furin Inhibition
Boulaftali Y, Francois D, Venisse L, Jandrot-Perrus M, Arocas V, Bouton MC

2013 • Arterioscler Thromb Vasc Biol • [pdf]

OBJECTIVE: Human protein C is a plasma serine protease that plays a key role in hemostasis, and activated protein C (aPC) is known to elicit protective responses in vascular endothelial cells. This cytoprotective activity requires the interaction of the protease with its cell membrane receptor, endothelial protein C receptor. However, the mechanisms regulating the beneficial cellular effects of aPC are not well known. We aimed to determine whether a serine protease inhibitor called protease nexin-1 (PN-1) or serpinE2, expressed by vascular cells, can modulate the effect of aPC on endothelial cells.
APPROACH AND RESULTS: We found that vascular barrier protective and antiapoptotic activities of aPC were reduced both in endothelial cells underexpressing PN-1 and in endothelial cells whose PN-1 function was blocked by a neutralizing antibody. Our in vitro data were further confirmed in vivo. Indeed, we found that vascular endothelial growth factor-mediated hyperpermeability in the skin of mice was markedly reduced by local intradermal injection of aPC in wild-type mice but not in PN-1-deficient mice. Furthermore, we demonstrated a previously unknown protective role of endothelial PN-1 on endothelial protein C receptor shedding. We provided evidence that PN-1 inhibits furin, a serine protease that activates a disintegrin and metalloproteinase 17 involved in the shedding of endothelial protein C receptor. We indeed evidenced a direct interaction between PN-1 and furin in endothelial cells.
CONCLUSIONS: Our results thus demonstrate an original role of PN-1 as a furin convertase inhibitor, providing new insights for understanding the regulation of endothelial protein C receptor-dependent aPC endothelial protective effects.

Supported protein G on gold electrode: Characterization and immunosensor application
Hafaiedh I, Chammem H, Abdelghani A, Ait E, Feldman L, Meilhac O, Mora L

2013 • Talanta • [pdf]

In this work, we study the electrochemical properties of protein layer grafted on gold electrode for C-reactive protein detection. Two CRP-antibody immobilization methods were used: the first method is based on direct physisorption of CRP-antibody onto the gold surface and the second method is based on oriented CRP-antibody with protein G intermediate layer. The two developed immunosensors were tested against CRP antigen in phosphate buffer saline solution and in human plasma. The electrochemical characterization of each immobilized layers was achieved by cyclic voltammetry and impedance spectroscopy. The morphology of the deposited biomolecules was observed by Atomic Force Microscopy and the roughness was measured. Moreover, contact angle measurement was used for wettability studies. The response of the developed immunosensors was reproducible, rapid, and highly stable and a detection limit of 100 fg/mL and 10 pg/mL antigen was observed with and without protein G respectively. The developed immunosensors was used for CRP detection in human plasma.

HDL and endothelial protection
Tran-Dinh A, Diallo D, Delbosc S, Varela-Perez LM, Dang Q, Lapergue B, Burillo E, Michel J, Levoye A, Martin-Ventura J, Meilhac O

2013 • Br J Pharmacol • [pdf]

High-density lipoproteins (HDLs) represent a family of particles characterized by the presence of apolipoprotein A-I (apoA-I) and by their ability to transport cholesterol from peripheral tissues back to the liver. In addition to this function, HDLs display pleiotropic effects including antioxidant, anti-apoptotic, anti-inflammatory, anti-thrombotic or anti-proteolytic properties that account for their protective action on endothelial cells. Vasodilatation via production of nitric oxide is also a hallmark of HDL action on endothelial cells. Endothelial cells express receptors for apoA-I and HDLs that mediate intracellular signalling and potentially participate in the internalization of these particles. In this review, we will detail the different effects of HDLs on the endothelium in normal and pathological conditions with a particular focus on the potential use of HDL therapy to restore endothelial function and integrity.

A nano-hydroxyapatite--pullulan/dextran polysaccharide composite macroporous material for bone tissue engineering
Fricain JC, Schlaubitz S, Le Visage C, Arnault I, Derkaoui SM, Siadous R, Catros S, Lalande C, Bareille R, Renard M, Fabre T, Cornet S, Durand M, Leonard A, Sahraoui N, Letourneur D, Amedee J

2013 • Biomaterials • [pdf]

Research in bone tissue engineering is focused on the development of alternatives to allogenic and autologous bone grafts that can stimulate bone healing. Here, we present scaffolds composed of the natural hydrophilic polysaccharides pullulan and dextran, supplemented or not with nanocrystalline hydroxyapatite particles (nHA). In vitro studies revealed that these matrices induced the formation of multicellular aggregates and expression of early and late bone specific markers with human bone marrow stromal cells in medium deprived of osteoinductive factors. In absence of any seeded cells, heterotopic implantation in mice and goat, revealed that only the composite macroporous scaffold (Matrix + nHA) (i) retained subcutaneously local growth factors, including Bone Morphogenetic Protein 2 (BMP2) and VEGF165, (ii) induced the deposition of a biological apatite layer, (iii) favored the formation of a dense mineralized tissue subcutaneously in mice, as well osteoid tissue after intramuscular implantation in goat. The composite scaffold was thereafter implanted in orthotopic preclinical models of critical size defects, in small and large animals, in three different bony sites, i.e. the femoral condyle of rat, a transversal mandibular defect and a tibial osteotomy in goat. The Matrix + nHA induced a highly mineralized tissue in the three models whatever the site of implantation, as well as osteoid tissue and bone tissue regeneration in direct contact to the matrix. We therefore propose this composite matrix as a material for stimulating bone cell differentiation of host mesenchymal stem cells and bone formation for orthopedic and maxillofacial surgical applications.

Predominant Role of Host Proteases in Myocardial Damage Associated with Infectious Endocarditis Induced by Enterococcus faecalis in a Rat Model
Augustin P, Alsalih G, Launey Y, Delbosc S, Louedec L, Ollivier V, Chau F, Montravers P, Duval X, Michel JB, Meilhac O

2013 • Infect Immun • [pdf]

Infective endocarditis (IE) remains a life-threatening infectious disease with high morbidity and mortality. The objectives of the present study are to assess the host proteolytic activities of the vegetations and their cytotoxic potential in a rat model of experimental IE. Rats were infected with a strain of Enterococcus faecalis of particularly low virulence and weak protease expression. We tested the presence of proteases released by infiltrated leukocytes (matrix metalloproteinases and elastase) or produced in situ within the septic vegetation, such as those linked to the fibrinolytic system (plasmin and plasminogen activators). We also assessed the tissue damage induced by the infective thrombus in vitro and ex vivo. The model of IE was characterized by larger and more extensive vegetations in infected than in nonseptic rats and by an intense neutrophil infiltrate interfacing with the injured underlying tissue. Neutrophil extracellular DNA was shown to trap bacteria and to produce increased levels of cell-free DNA in plasma. Matrix metalloproteinase-9, elastase, and plasminogen activators were increased in septic versus nonseptic vegetations (as shown by zymography and immunohistology). Finally, proteolysis of the extracellular matrix and apoptosis were shown to be associated with host proteases. Bacteria exhibited no detectable proteolytic activity or direct cytotoxic effects. Bacterial membranes/dead bacteria were sufficient to induce leukocyte recruitment and activation that could promote vegetation formation and growth. Our results suggest that, despite the lack of bacterial proteases, the continuous attractant signals coming from bacterial colonies may lead to a chronic and deleterious aggression toward myocardial/valvular tissues by host proteases.

RANTES/CCL5-induced pro-angiogenic effects depend on CCR1, CCR5 and glycosaminoglycans
Suffee N, Hlawaty H, Meddahi-Pelle A, Maillard L, Louedec L, Haddad O, Martin L, Laguillier C, Richard B, Oudar O, Letourneur D, Charnaux N, Sutton A

2012 • Angiogenesis • [pdf]

Atherosclerosis involves angiogenesis and inflammation with the ability of endothelial cells and monocytes to respond to chemokines. We addressed here by in vitro and in vivo approaches, the role of the chemokine Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES)/CCL5 on angiogenesis through its receptors CCR1, CCR5, syndecan-1 (SDC-1), syndecan-4 (SDC-4) and CD-44. Our data demonstrate that RANTES/CCL5 is pro-angiogenic in a rat subcutaneous model. This RANTES/CCL5-activity may be related to the in vitro promotion of endothelial cell migration, spreading and neo-vessel formation. RANTES/CCL5-mediated angiogenesis depends at least partly on Vascular Endothelial Growth Factor (VEGF) secretion by endothelial cells, since this effect is decreased when endothelial cells are incubated with anti-VEGF receptor antibodies. RANTES/CCL5-induced chemotaxis is mediated by matrix metalloproteinase-9. We demonstrate that specific receptors of RANTES/CCL5 such as G protein-coupled receptors CCR1 and CCR5, and heparan sulfate proteoglycans, SDC-1, SDC-4 or CD-44, play a major role in RANTES/CCL5-induced angiogenic effects. By the use of two RANTES/CCL5 mutants, [E66A]-RANTES/CCL5 with impaired ability to oligomerize, and [44AANA47]-RANTES/CCL5 mutated in the main RANTES/CCL5-glycosaminoglycan (GAG) binding site, we demonstrate that chemokine oligomerization and binding to GAGs are essential in RANTES/CCL5-induced angiogenic effects. According to these results, new therapeutic strategies based on RANTES/CCL5 can be proposed for neo-angiogenesis after vascular injury. Mutants of RANTES/CCL5 may also represent an innovative approach to prevent the angiogenesis associated with the formation of atherosclerotic plaque.

Discs large 1 (Dlg1) scaffolding protein participates with clathrin and adaptator protein complex 1 (AP-1) in forming Weibel-Palade bodies of endothelial cells
Philippe M, Leger T, Desvaux R, Walch L

2013 • J Biol Chem • [pdf]

Weibel-Palade bodies (WPBs) are specific cigar-shaped granules that store von Willebrand factor (VWF) for its regulated secretion by endothelial cells. The first steps of the formation of these granules at the trans-Golgi network (TGN) specifically require VWF aggregation and an external scaffolding complex that contains the adaptator protein complex 1 (AP-1) and clathrin. Discs large 1 (Dlg1) is generally considered to be a modular scaffolding protein implicated in the control of cell polarity in a large variety of cells by specific recruiting of receptors, channels or signaling proteins to specialized zones of the plasma membrane. We propose here that in endothelial cells, Dlg1 in a complex with AP-1 and clathrin participates in the biogenesis of WPBs. Supporting data show that Dlg1 colocalizes with microtubules, intermediate filaments and Golgi markers. Tandem mass spectrometry experiments led to the identification of clathrin as a Dlg1-interacting partner, interaction was confirmed by in situ proximity ligation assays. Furthermore, AP-1 and VWF immunoprecipitate and colocalize with Dlg1 in the juxta-nuclear zone. Finally, Dlg1 depletion by siRNA duplexes disrupts TGN morphology and WPB formation. Our results provide the first evidence for an unexpected role of Dlg1 in controlling the formation of specific secretory granules involved in VWF exocytosis in endothelial cells.

High-density Lipoprotein-based Therapy Reduces the Hemorrhagic Complications Associated With Tissue Plasminogen Activator Treatment in Experimental Stroke
Lapergue B, Dang BQ, Desilles JP, Ortiz-Munoz G, Delbosc S, Loyau S, Louedec L, Couraud PO, Mazighi M, Michel JB, Meilhac O, Amarenco P

2013 • Stroke • [pdf] • Cover of the Journal

BACKGROUND AND PURPOSE: We have previously reported that intravenous injection of high-density lipoproteins (HDLs) was neuroprotective in an embolic stroke model. We hypothesized that HDL vasculoprotective actions on the blood-brain barrier (BBB) may decrease hemorrhagic transformation-associated with tissue plasminogen activator (tPA) administration in acute stroke.
METHODS: We used tPA alone or in combination with HDLs in vivo in 2 models of focal middle cerebral artery occlusion (MCAO) (embolic and 4-hour monofilament MCAO) and in vitro in a model of BBB. Sprague-Dawley rats were submitted to MCAO, n=12 per group. The rats were then randomly injected with tPA (10 mg/kg) or saline with or without human plasma purified-HDL (10 mg/kg). The therapeutic effects of HDL and BBB integrity were assessed blindly 24 hours later. The integrity of the BBB was also tested using an in vitro model of human cerebral endothelial cells under oxygen-glucose deprivation.
RESULTS: tPA-treated groups had significantly higher mortality and rate of hemorrhagic transformation at 24 hours in both MCAO models. Cotreatment with HDL significantly reduced stroke-induced mortality versus tPA alone (by 42% in filament MCAO, P=0.009; by 73% in embolic MCAO, P=0.05) and tPA-induced intracerebral parenchymal hematoma (by 92% in filament MCAO, by 100% in embolic MCAO; P<0.0001). This was consistent with an improved BBB integrity. In vitro, HDLs decreased oxygen-glucose deprivation-induced BBB permeability (P<0.05) and vascular endothelial cadherin disorganization.
CONCLUSIONS: HDL injection decreased tPA-induced hemorrhagic transformation in rat models of MCAO. Both in vivo and in vitro results support the vasculoprotective action of HDLs on BBB under ischemic conditions.

La protéase nexine-1 : une serpine clé dans l’hémostase et la biologie vasculaire
Boulaftali Y, Richard B, Arocas V, Bouton MC

2013 • Hématologie • [pdf]

The balance between proteases and anti-proteases plays an important role in many biological processes. Serine proteases are mainly regulated by inhibitors belonging to the serpin superfamily. Protease nexin-1 (PN-1) is a tissue serpin, whose role in hemostasis and thrombosis has emerged during the last decade. PN-1 is present in the vascular wall and is expressed by blood cells (platelets, leukocytes). It is the most effective tissue inhibitor of thrombin and also inhibits enzymes of the plasminergic system (plasminogen activators and plasmin). Its expression is increased in pathological conditions in the arterial wall, especially during hypertension, the development of atherosclerotic plaque or thoracic aortic aneurysm. Modulation of PN-1 expression has an impact on the general characteristics of vascular cell motility and on cell responses to thrombin or plasminergic enzymes. The presence of a membrane and intracellular pool of PN-1 has been demonstrated in platelets. This platelet PN-1 secreted during their activation has an ambivalent characteristic since it has both anticoagulant properties through its ability to inhibit thrombin, and antifibrinolytic properties through its ability to inhibit plasminogen activators and plasmin. The role of PN-1 in vascular pathophysiology has been up to now largely underestimated. This article reviews recent data on PN-1 which open new perspectives regarding its involvement in the development of drugs and treatment strategies to prevent or limit thrombotic disorders.

High-density lipoproteins limit neutrophil-induced damage to the blood-brain barrier in vitro
Bao Dang Q, Lapergue B, Tran-Dinh A, Diallo D, Moreno JA, Mazighi M, Romero IA, Weksler B, Michel JB, Amarenco P, Meilhac O

2013 • J Cerebral Blood Flow Metab • [pdf]

Breakdown of the blood-brain barrier (BBB) is a key step associated with ischemic stroke and its increased permeability causes extravasation of plasma proteins and circulating leukocytes. Polymorphonuclear neutrophil (PMN) proteases may participate in BBB breakdown. We investigated the role of PMNs in ischemic conditions by testing their effects on a model of BBB in vitro, under oxygen-glucose deprivation (OGD) to mimic ischemia, supplemented or not with high-density lipoproteins (HDLs) to assess their potential protective effects. Human cerebral endothelial cells cultured on transwells were incubated for 4 hours under OGD conditions with or without PMNs and supplemented or not with HDLs or alpha-1 antitrypsin (AAT, an elastase inhibitor). The integrity of the BBB was then assessed and the effect of HDLs on PMN-induced proteolysis of extracellular matrix proteins was evaluated. The release of myeloperoxidase and matrix metalloproteinase 9 (MMP-9) by PMNs was quantified. Polymorphonuclear neutrophils significantly increased BBB permeability under OGD conditions via proteolysis of extracellular matrix proteins. This was associated with PMN degranulation. Addition of HDLs or AAT limited the proteolysis and associated increased permeability by inhibiting PMN activation. Our results suggest a deleterious, elastase-mediated role of activated PMNs under OGD conditions leading to BBB disruption that could be inhibited by HDLs.

The future of glycoprotein VI as an antithrombotic target
Zahid M, Mangin P, Loyau S, Hechler B, Billiald P, Gachet C, Jandrot-Perrus M

2012 • J Thromb Haemost • [pdf]

The treatment of acute coronary syndromes has been considerably improved in recent years with the introduction of highly efficient antiplatelet drugs. However, there are still significant limitations: the recurrence of adverse vascular events remains a problem, and the improvement in efficacy is counterbalanced by an increased risk of bleeding, which is of particular importance in patients at risk of stroke. One of the most attractive targets for the development of new molecules with potential antithrombotic activity is platelet glycoprotein (GP)VI, because its blockade appears to ideally combine efficacy and safety. This review summarizes current knowledge on GPVI regarding its structure, its function, and its role in physiologic hemostasis and thrombosis. Strategies for inhibiting GPVI are presented, and evidence of the antithrombotic efficacy and safety of GPVI antagonists is provided.

Role of vegetation-associated protease activity in valve destruction in human infective endocarditis
Al-Salih G, Al-Attar N, Delbosc S, Louedec L, Corvazier E, Loyau S, Michel JB, Pidard D, Duval X, Meilhac O

2012 • PLoS ONE • [pdf]

AIMS: Infective endocarditis (IE) is characterized by septic thrombi (vegetations) attached on heart valves, consisting of microbial colonization of the valvular endocardium, that may eventually lead to congestive heart failure or stroke subsequent to systemic embolism. We hypothesized that host defense activation may be directly involved in tissue proteolytic aggression, in addition to pathogenic effects of bacterial colonization.
METHODS AND RESULTS: IE valve samples collected during surgery (n = 39) were dissected macroscopically by separating vegetations (VG) and the surrounding damaged part of the valve from the adjacent, apparently normal (N) valvular tissue. Corresponding conditioned media were prepared separately by incubation in culture medium. Histological analysis showed an accumulation of platelets and polymorphonuclear neutrophils (PMNs) at the interface between the VG and the underlying tissue. Apoptotic cells (PMNs and valvular cells) were abundantly detected in this area. Plasminogen activators (PA), including urokinase (uPA) and tissue (tPA) types were also associated with the VG. Secreted matrix metalloproteinase (MMP) 9 was also increased in VG, as was leukocyte elastase and myeloperoxidase (MPO). The presence of neutrophil extracellular traps (NETs) associating MPO and externalized nucleosomes, was shown by immunostaining in the VG. Both MPO and cell-free DNA were released in larger amounts by VG than N samples, suggesting bacterial activation of PMNs within the vegetation. Finally, evidence of proteolytic tissue damage was obtained by the release of fragments of extracellular matrix components such as fibrinogen and fibronectin, as well as protease-sensitive receptors such as the uPA receptor.
CONCLUSION: Our data obtained using human IE valves suggest that septic vegetations represent an important source of proteases originating from massive leukocyte recruitment and activation of the host plasminergic system. The latter forms a potential therapeutic target to minimize valvular tissue degradation independently from that induced by bacterial proteases.

Prostaglandin E(2) induced contraction of human intercostal arteries is mediated by the EP(3) receptor
Longrois D, Gomez I, Foudi N, Topal G, Dhaouadi M, Kotelevets L, Chastre E, Norel X

2012 • Eur J Pharmacol • [pdf]

Arterial vascularization of the spinal cord may be mechanically or functionally altered during thoraco-abdominal surgery/intravascular procedures. Increased arterial pressure has been shown to restore spinal perfusion and function probably by increasing the blood flow through the intercostal arteries. The regulation of human intercostal artery (HICA) vascular tone is not well documented. Prostaglandin (PG)E(2) concentration is increased during inflammatory conditions and has been shown to regulate vascular tone in many preparations. In this context, the pharmacological response of HICA to PGE(2) and the characterization of the PGE(2) receptor subtypes (EP(1), EP(2), EP(3) or EP(4)) involved are of importance and that is the aim of this study. Rings of HICA were prepared from 29 patients and suspended in organ baths for isometric recording of tension. Cumulative concentration-response curves were performed in these preparations with various EP receptor agonists in the absence or presence of different receptor antagonists or inhibitors. PGE(2) induced the contraction of HICA (E(max)=7.28 +/- 0.16 g; pEC(50) value=0.79 +/- 0.18; n=17); contractions were also observed with the EP(3) receptor agonists, sulprostone, 17-phenyl-PGE(2), misoprostol or ONO-AE-248. In conclusion, PGE(2) induced vasoconstriction of HICA via EP(3) receptor subtypes and this result was confirmed by the use of selective EP receptor antagonists (L-826266, ONO-8713, SC-51322) and by a strong detection of EP(3) mRNA. These observations suggest that in the context of perioperative inflammation, increased PGE(2) concentrations could trigger vasoconstriction of HICA and possibly alter spinal vascularization.

TGFB2 mutations cause familial thoracic aortic aneurysms and dissections associated with mild systemic features of Marfan syndrome
Boileau C, Guo DC, Hanna N, Regalado ES, Detaint D, Gong L, Varret M, Prakash SK, Li AH, d'Indy H, Braverman AC, Grandchamp B, Kwartler CS, Gouya L, Santos-Cortez RL, Abifadel M, Leal SM, Muti C, Shendure J, Gross MS, Rieder MJ, Vahanian A, Nickerson DA, Michel JB, Jondeau G, Milewicz DM

2012 • Nat Genet • [pdf]

A predisposition for thoracic aortic aneurysms leading to acute aortic dissections can be inherited in families in an autosomal dominant manner. Genome-wide linkage analysis of two large unrelated families with thoracic aortic disease followed by whole-exome sequencing of affected relatives identified causative mutations in TGFB2. These mutations-a frameshift mutation in exon 6 and a nonsense mutation in exon 4-segregated with disease with a combined logarithm of odds (LOD) score of 7.7. Sanger sequencing of 276 probands from families with inherited thoracic aortic disease identified 2 additional TGFB2 mutations. TGFB2 encodes transforming growth factor (TGF)-beta2, and the mutations are predicted to cause haploinsufficiency for TGFB2; however, aortic tissue from cases paradoxically shows increased TGF-beta2 expression and immunostaining. Thus, haploinsufficiency for TGFB2 predisposes to thoracic aortic disease, suggesting that the initial pathway driving disease is decreased cellular TGF-beta2 levels leading to a secondary increase in TGF-beta2 production in the diseased aorta.

In vitro and in vivo Anti-Angiogenic Properties of the Serpin Protease Nexin-1
Selbonne S, Azibani F, Iatmanen S, Boulaftali Y, Richard B, Jandrot-Perrus M, Bouton MC, Arocas V

2012 • Mol Cell Biol • [pdf]

The serpin protease nexin-1 (PN-1) is expressed by vascular cells, and secreted by platelets upon activation, and is known to interact with several modulators of angiogenesis such as proteases, matrix proteins or glycosaminoglycans. We therefore investigated the impact of PN-1 on endothelial cell angiogenic responses in vitro and ex vivo and in vivo in PN-1 deficient mice. We found that PN-1 is anti-angiogenic in vitro: it inhibited VEGF-induced endothelial cell responses including proliferation, migration and capillary tube formation, and decreased cell spreading on vitronectin. These effects do not require the anti-protease activity of PN-1 but involve PN-1 binding to glycosaminoglycans. In addition, our results indicate that PN-1 does not act by blocking VEGF binding to its heparan sulfate proteoglycans coreceptors. The results obtained in vitro were supported ex vivo in PN-1-deficient mice, where the microvascular network sprouting from aortic rings was significantly enhanced. Moreover, in vivo, neovessel formation was promoted in the Matrigel plug assay, in PN-1 deficient mice compared to WT mice, and these effects were reversed by the addition of recombinant PN-1. Taken together, our results demonstrate that PN-1 has direct anti-angiogenic properties and is a yet unrecognized player in the angiogenic balance.

The mouse dorsal skinfold chamber as a model for the study of thrombolysis by intravital microscopy
Boulaftali Y, Lamrani L, Rouzaud MC, Loyau S, Jandrot-Perrus M, Bouton MC, Ho-Tin-Noe B

2012 • Thromb Haemost • [pdf]

Although intravital microscopy models of thrombosis in mice have contributed to dissect the mechanisms of thrombus formation and stability, they have not been well adapted to study long-term evolution of occlusive thrombi. Here, we assessed the suitability of the dorsal skinfold chamber (DSC) for the study of thrombolysis and testing of thrombolytic agents by intravital microscopy. We show that induction of FeCl3-induced occlusive thrombosis is achievable in microvessels of DSCs, and that thrombi formed in DSCs can be visualised by intravital microscopy using brightfield transmitted light, or fluorescent staining of thrombus components such as fibrinogen, platelets, leukocytes, and von Willebrand factor. Direct application of control saline or recombinant tissue-plasminogen activator (rtPA) to FeCl3-produced thrombi in DSCs did not affect thrombus size or induce recanalisation. However, in the presence of hirudin, rtPA treatment caused a rapid dose-dependent lysis of occlusive thrombi, resulting in recanalisation within 1 hour after treatment. Skin haemorrhage originating from vessels located inside and outside the FeCl3-injured area was also observed in DSCs of rtPA-treated mice. We further show that rtPA-induced thrombolysis was enhanced in plasminogen activator inhibitor-1-deficient (PAI-1-/-) mice, and dropped considerably as the time between occlusion and treatment application increased. Together, our results show that by allowing visualization and measurement of thrombus lysis and potential bleeding complications of thrombolytic treatments, the DSC provides a model for studying endogenous fibrinolysis and for first-line screening of thrombolytic agents. Furthermore, using this system, we found that thrombin and clot aging impair the thrombolytic action of rtPA towards FeCl3-produced thrombi.

A CD31-derived peptide prevents angiotensin II-induced atherosclerosis progression and aneurysm formation
Fornasa G, Clement M, Groyer E, Gaston AT, Khallou-Laschet J, Morvan M, Guedj K, Kaveri SV, Tedgui A, Michel JB, Nicoletti A, Caligiuri G

2012 • Cardiovasc Res • [pdf]

AIMS: The loss of the inhibitory receptor CD31 on peripheral T lymphocytes is associated with the incidence of atherosclerotic complications such as abdominal aortic aneurysms (AAA) in patients and plaque thrombosis in mice. However, we have recently discovered that a small fragment of extracellular CD31 remains expressed on the surface of the apparently "CD31-negative" T-cells and that it is possible to restore the CD31-mediated T-cell inhibition in vivo, with using a synthetic CD31-derived peptide. Here we wanted to evaluate the therapeutic potential of the peptide in an experimental model of accelerated atherosclerosis and AAA formation.
METHODS AND RESULTS: The effect of the murine CD31-derived peptide (aa 551-574, 1.5 mg/Kg/day, s.c.) was evaluated on the extent of atherosclerotic plaques and the incidence of AAA in 28 week-old apolipoprotein E knockout mice (male, n>/=8/group) submitted to chronic angiotensin II infusion. The therapeutic mechanisms of the peptide were assessed by evaluating its effect on immune cell functions in vivo and in vitro. The prevalence of angiotensin II-induced AAA correlated with the loss of extracellular CD31 on T-cells. CD31-peptide treatment reduced both aneurysm formation and plaque size (p<0.05 vs control). Protection was associated with reduced perivascular leukocyte infiltration and T-cell activation in vivo. Functional in vitro studies showed that the peptide is able to suppress both T-cell and macrophage activation.
CONCLUSION: CD31 peptides could represent a new class of drugs envisaged to prevent the inflammatory cell processes, such as those underlying progression of atherosclerosis and development of AAA.

Cardiac FKBP12.6 overexpression protects against triggered ventricular tachycardia in pressure overloaded mouse hearts
Vinet L, Pezet M, Bito V, Briec F, Biesmans L, Rouet-Benzineb P, Gellen B, Previlon M, Chimenti S, Vilaine JP, Charpentier F, Sipido KR, Mercadier JJ

2011 • Basic Res Cardiol • [pdf]

Alterations in RyR2 function have been proposed as a major pathophysiological mechanism of arrhythmias and heart failure (HF). Cardiac FKBP12.6 overexpression protects against myocardial infarction-induced HF and catecholamine-promoted ventricular arrhythmias. We tested the hypothesis that FKBP12.6 overexpression protects against maladaptive LVH and triggered ventricular arrhythmias following transverse aorta constriction (TAC) in the mouse. The TAC-associated mortality rate was significantly lower in male transgenic (DT) than in Ctr mice (p < 0.05). TAC-associated maladaptive hypertrophy was blunted in DT mice especially 1 month post-TAC and their SERCA2a/PLB ratio remained unchanged 1 and 2 months post-TAC. Two months after TAC, trains of 30 stimuli (burst pacing) performed following isoproterenol injection (0.2 mg/kg, ip), induced VT in 50% of the TAC-Ctr and in none of the TAC-DT mice (p = 0.022). The increase in myocyte shortening and Ca(2+) spark frequency observed in sham-operated Ctr mice in response to 50 nM isoproterenol was reduced in DT mice, and abolished in TAC-DT mice. NCX1 function was reduced in Sham-DT and TAC-DT compared with Sham-Ctr and TAC-Ctr mice, respectively (p < 0.05 for the 2 comparisons). In mice killed after isoproterenol injection and burst pacing, RyR2 S2814 phosphorylation was decreased by 50% in TAC-DT versus TAC-Ctr mice (p < 0.05), with no change in RyR2 S2808 and PLB S16 and T17 phosphorylation. Cardiac FKBP12.6 overexpression in the mouse blunts pressure overload-induced maladaptive LV remodelling and protects against catecholamine-promoted burst pacing-induced ventricular tachycardia by decreasing cardiac sensitivity to adrenergic stress and RyR2 S2814 phosphorylation, and decreasing NCX1 activity.

Platelet Glycoprotein VI Dimerization, an Active Process Inducing Receptor Competence, Is an Indicator of Platelet Reactivity
Loyau S, Dumont B, Ollivier V, Boulaftali Y, Feldman L, Ajzenberg N, Jandrot-Perrus M

2011 • Arterioscler Thromb Vasc Biol • [pdf]

OBJECTIVE: The immune receptor homologue glycoprotein VI (GPVI)/FcRgamma complex is primarily responsible for platelet activation by collagen. There is growing evidence that optimal binding of GPVI to collagen depends on the assembly of GPVI dimers. The valence of GPVI on resting platelets needs to be clearly established because platelet avidity for collagen would be greater if GPVI is constitutively expressed as a dimer than as a monomer.
METHODS AND RESULTS: Using a monoclonal antibody (9E18) that preferentially binds to GPVI dimers, we found that GPVI was maintained in a monomeric form on human resting platelets under the control of intraplatelet cAMP concentration. Activation by soluble agonists or von Willebrand factor induced a shift toward GPVI dimerization related to increased platelet adhesion to collagen. A correlation between platelet binding of 9E18 and P-selectin exposure was observed in patients experiencing coronary artery disease, and antagonists of the ADP receptor P2Y(12) limited ADP-induced GPVI dimerization.
CONCLUSIONS: The rapid assembly of highly competent dimers of GPVI at sites of vascular lesion represents an important step in the sequence of events leading to platelet activation by collagen. GPVI dimers could represent a new marker to analyze platelet reactivity.

Aortic Event Rate in the Marfan Population: A Cohort Study
Jondeau G, Detaint D, Tubach F, Arnoult F, Milleron O, Raoux F, Delorme G, Mimoun L, Krapf L, Hamroun D, Beroud C, Roy C, Vahanian A, Boileau C

2011 • Circulation • [pdf]

BACKGROUND: -Optimal management, including timing of surgery, remains debated in Marfan syndrome (MFS), due to lack of data on aortic risk associated with this disease.
METHODS AND RESULTS: -We used our database to evaluate aortic risk associated with standardised care. Patients who fulfilled the international criteria, had not had previous aortic surgery or dissection and who came to our centre at least twice were included. Aortic measurements were made using echocardiography (every 2 years); patients were given systematic beta-blockade and advice about sports activities. Prophylactic aortic surgery was proposed when the maximal aortic diameter reached 50 mm. 732 patients with MFS were followed up for a mean of 6.6 years. Five deaths and two dissections of the ascending aorta occurred during follow-up. Event rate (death/aortic dissection) was 0.17% per year. Risk rose with increasing aortic diameter measured within 2 years of the event: from 0.09% per year (95% confidence interval 0.00-0.20) when the aortic diameter was <40 mm, to 0.3% (0.00-0.71) with diameters of 45-49 mm, and 1.33% (0.00-3.93) with diameters of 50-54 mm: the risk increased 4-times above 50 mm. The annual risk dropped below 0.05% when the aortic diameter was <50 mm, after exclusion of a neonatal patient, a woman who became pregnant against our recommendation, and a 72-year-old woman with previous myocardial infarction.
CONCLUSIONS: -Risk of sudden death or aortic dissection remains low in patients with MFS and aortic diameter between 45 and 49 mm. 50 mm appears to be a reasonable threshold for prophylactic surgery.

In vitro Studies and Preliminary In vivo Evaluation of Silicified Concentrated Collagen Hydrogels
Desimone MF, Helary C, Quignard S, Rietveld IB, Bataille I, Copello GJ, Mosser G, Giraud-Guille MM, Livage J, Meddahi-Pelle A, Coradin T

2011 • ACS Appl Mater Interfaces • [pdf]

Hybrid and nanocomposite silica-collagen materials derived from concentrated collagen hydrogels were evaluated in vitro and in vivo to establish their potentialities for biological dressings. Silicification significantly improved the mechanical and thermal stability of the collagen network within the hybrid systems. Nanocomposites were found to favor the metabolic activity of immobilized human dermal fibroblasts while decreasing the hydrogel contraction. Cell adhesion experiments suggested that in vitro cell behavior was dictated by mechanical properties and surface structure of the scaffold. First-to-date in vivo implantation of bulk hydrogels in subcutaneous sites of rats was performed over the vascular inflammatory period. These materials were colonized and vascularized without inducing strong inflammatory response. These data raise reasonable hope for the future application of silica-collagen biomaterials as biological dressings.

How platelets safeguard vascular integrity
Ho-Tin-Noe B, Demers M, Wagner DD

2011 • J Thromb Haemost • [pdf]

The haemostatic role of platelets was established in the 1880s by Bizzozero who observed their ability to adhere and aggregate at sites of vascular injury. It was only some 80 years later that the function of platelets in maintaining the structural integrity of intact blood vessels was reported by Danielli. Danielli noted that platelets help preserve the barrier function of endothelium during organ perfusion. Subsequent studies have demonstrated further that platelets are continuously needed to support intact mature blood vessels. More recently, platelets were shown to safeguard developing vessels, lymphatics, as well as the microvasculature at sites of leukocyte infiltration, including inflamed organs and tumours. Interestingly, from a mechanistic point of view, the supporting role of platelets in these various vessels does not necessarily involve the well-understood process of platelet plug formation but, rather, may rely on secretion of the various platelet granules and their many active components. The present review focuses on these nonconventional aspects of platelet biology and function by presenting situations in which platelets intervene to maintain vascular integrity and discusses possible mechanisms of their actions. We propose that modulating these newly described platelet functions may help treat haemorrhage as well as treat cancer by increasing the efficacy of drug delivery to tumours.

Early Atheroma-Derived Agonists of Peroxisome Proliferator-Activated Receptor-{gamma} Trigger Intramedial Angiogenesis in a Smooth Muscle Cell-Dependent Manner
Ho-Tin-Noe B, Le Dall J, Gomez D, Louedec L, Roger V, El-Bouchtaoui M, Legres L, Meilhac O, Michel JB

2011 • Circ Res • [pdf]

Rationale: Neovascularization favors intraplaque hemorrhage and plaque rupture. Development of therapeutic strategies against atheromatous angiogenesis requires elucidation of its initiating factors.
Objective: We investigated the contribution of smooth muscle cells (SMCs) and atheroma-derived lipids to the initiation of atheroma-associated neoangiogenesis.
Methods and Results: Forty human aortic segments, each harvested from a different donor, were classified as healthy or as bearing early atheromatous lesions, including fatty streaks and fibrolipidic atheroma, according to their histological features. Immunostaining for blood vessels and vascular endothelial growth factor-A (VEGF-A), as well as measurement of VEGF-A protein and mRNA levels by ELISA and real-time PCR, revealed that angiogenesis and VEGF-A production were enhanced in the medial layer of atheromatous aortas. The intramedial vessel density and invasiveness and the production of VEGF-A by medial SMCs were indeed increased in atheromatous aortas compared with healthy aortas. Furthermore, intimal layers of atheromatous aortas were enriched in soluble lipid mediators capable of inducing a sustained increase in VEGF-A production by medial SMCs, turning these cells into potent inducers of angiogenesis when incorporated into mouse Matrigel implants. Both effects were inhibited by the peroxisome proliferator-activated receptor-gamma inhibitor GW9662 and mimicked by its agonist, rosiglitazone.
Conclusions: We show that VEGF-A production is upregulated in medial SMCs of human atheromatous aortas and that peroxisome proliferator-activated receptor-gamma agonists derived from early intimal lesions are likely to contribute to this phenotypic change. Our findings suggest that medial SMCs are central organizers of an angiogenic response initiated by the subendothelial accumulation of atherogenic lipids.

Heat-shock proteins in cardiovascular disease
Madrigal-Matute J, Martin-Ventura JL, Blanco-Colio LM, Egido J, Michel JB, Meilhac O

2011 • Adv Clin Chem • [pdf]

Heat-shock proteins (HSPs) belong to a group of highly conserved families of proteins expressed by all cells and organisms and their expression may be constitutive or inducible. They are generally considered as protective molecules against different types of stress and have numerous intracellular functions. Secretion or release of HSPs has also been described, and potential roles for extracellular HSPs reported. HSP expression is modulated by different stimuli involved in all steps of atherogenesis including oxidative stress, proteolytic aggression, or inflammation. Also, antibodies to HSPs may be used to monitor the response to different types of stress able to induce changes in HSP levels. In the present review, we will focus on the potential implication of HSPs in atherogenesis and discuss the limitations to the use of HSPs and anti-HSPs as biomarkers of atherothrombosis. HSPs could also be considered as potential therapeutic targets to reinforce vascular defenses and delay or avoid clinical complications associated with atherothrombosis.

Radiolabeled Fucoidan as a P-Selectin Targeting Agent for In Vivo Imaging of Platelet-Rich Thrombus and Endothelial Activation
Rouzet F, Bachelet-Violette L, Alsac JM, Suzuki M, Meulemans A, Louedec L, Petiet A,
Jandrot-Perrus M, Chaubet F, Michel JB, Le Guludec D, Letourneur D

2011 • J Nucl Med • [pdf]

P-selectin expression is involved in the pathophysiology of biologically active arterial thrombus and endothelial activation after a transient ischemic event. Fucoidan is a polysaccharidic ligand of P-selectin, with a nanomolar affinity. In the present study, we propose a new approach of P-selectin molecular imaging based on radiolabeled fucoidan.
METHODS: Two kinds of experimental models were selected to evaluate the ability of radiolabeled fucoidan to detect P-selectin expression: platelet-rich arterial thrombi (vegetations of infective endocarditis and arterial mural thrombus) and myocardial ischemia-reperfusion. These 2 settings were chosen because they were clinically relevant, and both were associated with an important overexpression of platelet and endothelial P-selectin, respectively.
RESULTS: (99m)Tc-fucoidan SPECT was able to detect the presence of platelet-rich arterial thrombi in all animals, with a median target-to-background ratio of 5.2 in vegetations of endocarditis and 3.6 in mural aneurysmal thrombus, and to detect a persistent endothelial activation at 2 h after reperfusion. In this latter model, the magnitude of the signal was correlated with the extent of myocardium that underwent transient ischemia. The sensitivity of selectivity of the uptake and retention of (99m)Tc-fucoidan in both settings was excellent.
CONCLUSION: This study supports (99m)Tc-fucoidan as a relevant imaging agent for in vivo detection of biologic activities associated with P-selectin overexpression, such as arterial thrombus and ischemic memory. Given the reported wide availability at a low cost, and its low toxicity, fucoidan seems to overcome some of the limitations of previous P-selectin-targeted imaging agents.

Doxorubicin release triggered by alginate embedded magnetic nanoheaters: a combined therapy
Brule S, Levy M, Wilhelm C, Letourneur D, Gazeau F, Menager C, Le Visage C

2011 • Adv Mater • [pdf]

In recent years, local heat generation (hyperthermia) has been tested as a means of destroying malignant tumors. Hyperthermia is a very promising treatment for cancer based on the hypothesis that cancerous cells are more sensitive to an increase of temperature than normal cells. When exposed to a high-frequency magnetic field, magnetic nanoparticles generate heat through oscillation of their magnetic moment due to Neel and Brownian relaxations. Direct injection of magnetic nanoparticles into solid tumors, followed by exposure to an alternating magnetic field, has been shown to be capable of inducing tumor regression. Ideally, the particle-induced magnetic heating should be precisely controlled, highly localized, and should result in no systemic effects and significantly reduced side effects in patients. On the other side, nanotechnology applications for anticancer drug delivery have been extensively explored, hoping to improve the efficacy and to reduce side effects of chemotherapy. Once at the target site, the drug is released from the carrier creating a high local concentration in the tumor tissue. Nanoparticles can stimulate drug uptake by cancer cells by locally providing high extracellular concentrations of the drug and/or by direct action on the permeability of cell membranes. Recently, Ciofani and coauthors hypothesized that polymeric nanoparticles with a core of magnetite and carrying specific drugs could be investigated for simultaneous drug targeting and hyperthermia application but they did not investigate the potential application of this system. Remote-controlled degradation of degradable nanocomposite hydrogels was also obtained by application of an alternating magnetic field. Here, we designed microbeads loaded with both magnetic nanoparticles (or nanoheaters) and doxorubicin to combine magnetic control of heating and drug delivery. We demonstrate that this new nano-in-micro platform greatly enhances the performance of encapsulated doxorubicin and yields substantial promise for the development of highly efficient anticancer treatment.

Magnetic resonance imaging tracking of human adipose derived stromal cells within three-dimensional scaffolds for bone tissue engineering
Lalande C, Miraux S, Derkaoui SM, Mornet S, Bareille R, Fricain JC, Franconi JM, Le Visage C, Letourneur D, Amedee J, Bouzier-Sore AK

2011 • Eur Cell Mater • [pdf]

For bone tissue engineering, human Adipose Derived Stem Cells (hADSCs) are proposed to be associated with a scaffold for promoting bone regeneration. After implantation, cellularised scaffolds require a non-invasive method for monitoring their fate in vivo. The purpose of this study was to use Magnetic Resonance Imaging (MRI)-based tracking of these cells, labelled with magnetic agents for in vivo longitudinal assessment. hADSCs were isolated from adipose tissue and labelled with USPIO-rhodamine (Ultrasmall SuperParamagnetic Iron Oxide). USPIO internalisation, absence of toxicity towards hADSCs, and osteogenic differentiation of the labelled cells were evaluated in standard culture conditions. Labelled cells were then seeded within a 3D porous polysaccharide-based scaffold and imaged in vitro using fluorescence microscopy and MRI. Cellularised scaffolds were implanted subcutaneously in nude mice and MRI analyses were performed from 1 to 28 d after implantation. In vitro, no effect of USPIO labelling on cell viability and osteogenic differentiation was found. USPIO were efficiently internalised by hADSCs and generated a high T2* contrast. In vivo MRI revealed that hADSCs remain detectable until 28 d after implantation and could migrate from the scaffold and colonise the area around it. These data suggested that this scaffold might behave as a cell carrier capable of both holding a cell fraction and delivering cells to the site of implantation. In addition, the present findings evidenced that MRI is a reliable technique to validate cell-seeding procedures in 3D porous scaffolds, and to assess the fate of hADSCs transplanted in vivo.

Physiological Induction of Regulatory Qa-1-Restricted CD8+ T Cells Triggered by Endogenous CD4+ T Cell Responses
Varthaman A, Clement M, Khallou-Laschet J, Fornasa G, Gaston AT, Dussiot M, Caligiuri G, Cantor H, Kaveri S, Nicoletti A

2011 • PLoSONE • [pdf]

T-cell-dependent autoimmune diseases are characterized by the expansion of T cell clones that recognize immunodominant epitopes on the target antigen. As a consequence, for a given autoimmune disorder, pathogenic T cell clones express T cell receptors with a limited number of variable regions that define antigenic specificity. Qa-1, a MHC class I-like molecule, presents peptides from the variable region of TCRs to Qa-1-restricted CD8+ T cells. The induction of Vß-specific CD8+ T cells has been harnessed in an immunotherapeutic strategy known as the “T cell vaccination” (TCV) that comprises the injection of activated and attenuated CD4+ T cell clones so as to induce protective CD8+ T cells. We hypothesized that Qa-1-restricted CD8+ regulatory T cells could also constitute a physiologic regulatory arm of lymphocyte responses upon expansion of endogenous CD4+ T cells, in the absence of deliberate exogenous T cell vaccination. We immunized mice with two types of antigenic challenges in order to sequentially expand antigen-specific endogenous CD4+ T cells with distinct antigenic specificities but characterized by a common Vß chain in their TCR. The first immunization was performed with a non-self antigen while the second challenge was performed with a myelin-derived peptide known to drive experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. We show that regulatory Vß-specific Qa-1-restricted CD8+ T cells induced during the first endogenous CD4+ T cell responses are able to control the expansion of subsequently mobilized pathogenic autoreactive CD4+ T cells. In conclusion, apart from the immunotherapeutic TCV, Qa-1-restricted specialized CD8+ regulatory T cells can also be induced during endogenous CD4+ T cell responses. At variance with other regulatory T cell subsets, the action of these Qa-1-restricted T cells seems to be restricted to the immediate re-activation of CD4+ T cells.

In vitro and in vivo evidence for the role of elastase shedding of CD163 in human atherothrombosis
Moreno JA, Ortega-Gomez A, Delbosc S, Beaufort N, Sorbets E, Louedec L, Esposito-Farese M, Tubach F, Nicoletti A, Steg PG, Michel JB, Feldman L, Meilhac O

2011 • Eur Heart J • [pdf]

Aims CD163 is a macrophage receptor for haemoglobin-haptoglobin (Hb-Hp) complexes, responsible for the clearance of haemoglobin. We hypothesized that production of soluble CD163 (sCD163) may be due to proleolytic shedding of membrane CD163 by neutrophil elastase, reported to be increased in culprit atherosclerotic plaques. We analysed the relationship between CD163 solubilization and elastase in vitro, in macrophage culture, ex vivo in human atherosclerotic plaque samples, and in vivo, in plasma of patients with coronary artery disease.
Methods and results Neutrophil elastase was shown to enhance CD163 shedding and to decrease the uptake of Hb-Hp complexes by cultured macrophages. In addition, cultured carotid endarterectomy samples showing features of intraplaque haemorrhage released more sCD163 and elastase/alpha1-antitrypsin (alpha1-AT) complexes than non-haemorrhagic plaques (n= 44). Plasma levels of sCD163 and neutrophil elastase (complexed with alpha1-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). Acute coronary syndrome patients had higher sCD163 and elastase/alpha1-AT complexes plasma concentrations than subjects without coronary atherosclerosis. Circulating sCD163 and elastase/alpha1-AT complexes were positively correlated in patients with ACS (r = 0.56, P< 0.0002) and SAP (r = 0.62, P< 0.0005).
Conclusion Our results suggest that neutrophil elastase promotes CD163 shedding, resulting in a decreased clearance of Hb by macrophages, which may favour plaque destabilization. This may be reflected by increased plasma levels of sCD163 and elastase/alpha1-AT complexes which are positively correlated in patients with coronary artery disease.

Porphyromonas gingivalis Participates in Pathogenesis of Human Abdominal Aortic Aneurysm by Neutrophil Activation. Proof of Concept in Rats
Delbosc S, Alsac JM, Journe C, Louedec L, Castier Y, Bonnaure-Mallet M, Ruimy R, Rossignol P, Bouchard P, Michel JB, Meilhac O

2011 • PLoS ONE • [pdf]

BACKGROUND: Abdominal Aortic Aneurysms (AAAs) represent a particular form of atherothrombosis where neutrophil proteolytic activity plays a major role. We postulated that neutrophil recruitment and activation participating in AAA growth may originate in part from repeated episodes of periodontal bacteremia.
METHODS AND FINDINGS: Our results show that neutrophil activation in human AAA was associated with Neutrophil Extracellular Trap (NET) formation in the IntraLuminal Thrombus, leading to the release of cell-free DNA. Human AAA samples were shown to contain bacterial DNA with high frequency (11/16), and in particular that of Porphyromonas gingivalis (Pg), the most prevalent pathogen involved in chronic periodontitis, a common form of periodontal disease. Both DNA reflecting the presence of NETs and antibodies to Pg were found to be increased in plasma of patients with AAA. Using a rat model of AAA, we demonstrated that repeated injection of Pg fostered aneurysm development, associated with pathological characteristics similar to those observed in humans, such as the persistence of a neutrophil-rich luminal thrombus, not observed in saline-injected rats in which a healing process was observed.
CONCLUSIONS: Thus, the control of periodontal disease may represent a therapeutic target to limit human AAA progression.

The Thermolysin-Like Metalloproteinase and Virulence Factor LasB from Pathogenic Pseudomonas aeruginosa Induces Anoikis of Human Vascular Cells
Beaufort N, Corvazier E, Hervieu A, Choqueux C, Dussiot M, Louedec L, Cady A, de Bentzmann S, Michel JB, Pidard D

2011 • Cell Microbiol • [pdf]

Disruption of cell/ECM interactions resulting from uncontrolled pericellular proteolysis leads to detachment-induced cell apoptosis (anoikis), contributing to the morbid evolution of inflammatory vascular diseases. During cardiovascular infections, bacterial proteinases might induce vascular cells to enter a similar pathway. We focused on LasB, the predominant metalloproteinase secreted by the hematotropic pathogen Pseudomonas aeruginosa. While the exosecretome of the LasB-deficient pseudomonal strain PAO1lasBDelta had limited impact on human vascular cell adherence and viability, secretomes from the LasB-expressing reference strain, PAO1, or clinical isolates from patients with cardiac infection all induced anoikis, as did purified LasB. Immunofluorescence and/or immunoblotting analysis of heart valve myofibroblast cultures or whole tissue revealed an extensive, LasB-dependent degradation of ECM-associated fibronectin and vitronectin, that preceded cell de-adherence, whereas type I collagen showed limited degradation. Moreover, LasB produced a rapid endoproteolysis of the cell-associated urokinase receptor/uPAR, leaving a truncated receptor that is unable to support cell adherence and survival via interactions with vitronectin and integrins. Conversely, major myofibroblast integrins showed no or only minor alterations. Thus, among P. aeruginosa-secreted metalloproteinases, LasB can induce vascular cell anoikis through simultaneous proteolysis of ECM components and cell receptors, suggesting the uPAR-vitronectin axis as a major target in this process.

Platelet Protease Nexin-1, a Serpin That Strongly Influences Fibrinolysis and Thrombolysis
Boulaftali Y, Ho-Tin-Noe B, Pena A, Loyau S, Venisse L, Francois D, Richard B, Arocas V, Collet JP, Jandrot-Perrus M, Bouton MC

2011 • Circulation • [pdf]

Background- Protease nexin-1 (PN-1) is a serpin that inhibits plasminogen activators, plasmin, and thrombin. PN-1 is barely detectable in plasma, but we have shown recently that PN-1 is present within the alpha-granules of platelets.
Methods and Results- In this study, the role of platelet PN-1 in fibrinolysis was investigated with the use of human platelets incubated with a blocking antibody and platelets from PN-1-deficient mice. We showed by using fibrin-agar zymography and fibrin matrix that platelet PN-1 inhibited both the generation of plasmin by fibrin-bound tissue plasminogen activator and the activity of fibrin-bound plasmin itself. Rotational thromboelastometry and laser scanning confocal microscopy were used to demonstrate that PN-1 blockade or deficiency resulted in increased clot lysis and in an acceleration of the lysis front. Protease nexin-1 is thus a major determinant of the lysis resistance of platelet-rich clots. Moreover, in an original murine model in which thrombolysis induced by tissue plasminogen activator can be measured directly in situ, we observed that vascular recanalization was significantly increased in PN-1-deficient mice. Surprisingly, general physical health, after tissue plasminogen activator-induced thrombolysis, was much better in PN-1-deficient than in wild-type mice.
Conclusions- Our results reveal that platelet PN-1 can be considered as a new important regulator of thrombolysis in vivo. Inhibition of PN-1 is thus predicted to promote endogenous and exogenous tissue plasminogen activator-mediated fibrinolysis and may enhance the therapeutic efficacy of thrombolytic agents.

Intraplaque haemorrhages as the trigger of plaque vulnerability
Michel JB, Virmani R, Arbustini E, Pasterkamp G

2011 • Eur Heart J • [pdf]

Atherothrombosis remains one of the main causes of morbidity and mortality in the western countries. Human atherothrombotic disease begins early in life in relation to circulating lipid retention in the inner vascular wall. Risk factors enhance the progression towards clinical expression: dyslipidaemia, diabetes, smoking, hypertension, ageing, etc. The evolution from the initial lipid retention in the arterial wall to clinical events is a continuum of increasingly complex biological processes. Current strategies to fight the consequences of atherothrombosis are orientated either towards the promotion of a healthy life style(1) and preventive treatment of risk factors, or towards late interventional strategies.(2) Despite this therapeutic arsenal, the incidence of clinical events remains dramatically high,(3) dependent, at least in part, on the increasing frequency of type 2 diabetes and ageing. But some medical treatments, focusing only on prevention of the metabolic risk, have failed to reduce cardiovascular mortality, thus illustrating that our understanding of the pathophysiology of human atherothrombosis leading to clinical events remain incomplete. New paradigms are now emerging which may give rise to novel experimental strategies to improve therapeutic efficacy and prediction of disease progression. Recent studies strengthen the concept that the intraplaque neovascularization and bleeding (Figure 1, upper panel) are events that could play a major role in plaque progression and leucocyte infiltration, and may also serve as a measure of risk for the development of future events. The recent advances in our understanding of IntraPlaque Hemorrhage as a critical event in triggering acute clinical events have important implications for clinical research and possibly future clinical practice.

Diagnostic and therapeutic strategies for small abdominal aortic aneurysms
Klink A, Hyafil F, Rudd J, Faries P, Fuster V, Mallat Z, Meilhac O, Mulder WJ, Michel JB, Ramirez F, Storm G, Thompson R, Turnbull IC, Egido J, Martin-Ventura JL, Zaragoza C, Letourneur D, Fayad ZA

2011 • Nat Rev Cardiol • [pdf]

Abdominal aortic aneurysms (AAA) affect 5% of the population in developed countries and are characterized by progressive aortic dilatation with an unpredictable time course. This condition is more common in men than in women, and in smokers than in nonsmokers. If left untreated, AAA can result in aortic rupture and death. Pathologically, aortic extracellular matrix degradation, inflammation, and neovascularization are hallmarks of AAA. Diagnosis of AAA and subsequent surveillance utilize established aortic imaging methods, such as ultrasound, CT, and MRI. More-speculative diagnostic approaches include molecular and cellular imaging methods that interrogate the underlying pathological processes at work within the aneurysm. In this Review, we explore the current diagnostic and therapeutic strategies for the management of AAA. We also describe the diagnostic potential of new imaging techniques and therapeutic potential of new treatments for the management of small AAA.

Solid-phase hexapeptide ligand libraries open up new perspectives in the discovery of biomarkers in human plasma
Leger T, Lavigne D, Le Caer JP, Guerrier L, Boschetti E, Fareh J, Feldman L, Laprevote O, Meilhac O

2011 • Clin Chim Acta • [pdf]

BACKGROUND: Pre-treatment of plasma with hexapeptide ligand libraries prior to proteomic analysis is well documented. However, the maintenance of biomarker abundance throughout the different pre-analytical steps is required for a potential application of differential proteomics in clinical studies.
METHODS: We combined the use of an amino-terminal hexapeptide ligand library and its carboxyl-terminal version with a sequential elution strategy of the proteins/peptides bound to the beads, followed by either mass spectrometry or 2D electrophoresis analyses.
RESULTS: We show the maintenance of C-reactive protein abundance (a marker of inflammation) throughout the process (including hexapeptide bead treatment and proteomic analysis) in patients presenting high and low levels of this protein. In parallel, we assessed the contribution of this workflow to increasing the number of potential biomarkers detected and its suitability for a clinical study on approximately a hundred samples, as well as the reproducibility of the process.
CONCLUSIONS: Pre-treatment with hexapeptide ligand librairies opens up new perspectives in the discovery of biomarkers in human plasma by improving the detection of new species while maintaining their original differential abundance. This approach is also suitable for an application in a clinical proteomic study of at least 100 samples.

Development of a new polypropylene-based suture: Plasma grafting, surface treatment, characterization, and biocompatibility studies
Saxena S, Ray AR, Kapil A, Pavon-Djavid G, Letourneur D, Gupta B, Meddahi-Pelle A

2010 • Macromol Biosci • [pdf]

Polypropylene sutures (PP) are already used in surgery. Because microbial infection leads to complications, we developed antimicrobial PP suture by plasma-induced graft polymerization of acrylic acid followed by chitosan binding on the remaining carboxyl groups. Mechanical properties and surface morphologies were analyzed on these sutures. Tetracycline hydrochloride (TC) or nanosilver (NS) was then immobilized to PP. The resulting PP sutures evidenced drug release properties and antimicrobial activity in vitro. PP implanted in vivo for 30 days in the muscle of rats showed the absence of adverse effects and a tissue organization. This new polypropylene suture with suitable antimicrobial features appears to be a promising macromolecular material for clinical and cosmetic applications.

Fibrinolytic activity is associated with presence of cystic medial degeneration in aneurysms of the ascending aorta
Borges LF, Gomez D, Quintana M, Touat Z, Jondeau G, Leclercq A, Meilhac O, Jandrot-Perrus M, Gutierrez PS, Freymuller E, Vranckx R, Michel JB

2010 • Histopathology • [pdf]

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)-beta/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-beta, 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 immunohistochamically in dense clumps around SMCs and colocalized with latent TGF-beta 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-beta bioavailability.

Des fonctions plaquettaires aux implications thérapeutiques
Jandrot-Perrus M, Nurden P

2010 • La Revue de Médecine Interne • [pdf]



Nouvelles molécules cibles et modulation des fonctions plaquettaires: anticiper, démontrer, gérer, utiliser
Jandrot-Perrus M, Gratacap MP, Lhermusier T, Sié P, Payrastre B
2010 • Hématologie • [pdf]

Epigenetic control of vascular smooth muscle cells in Marfan and non-Marfan thoracic aortic
Gomez D, Coyet A, Ollivier V, Jeunemaitre X, Jondeau G, Michel JB, Vranckx R

2010 • Cardiovasc Res • [pdf]

AIMS: Human thoracic aortic aneurysms (TAAs) are characterized by extracellular matrix breakdown associated with progressive smooth muscle cell (SMC) rarefaction. These features are present in all types of TAA: monogenic forms [mainly Marfan syndrome (MFS)], forms associated with bicuspid aortic valve (BAV), and degenerative forms. Initially described in a mouse model of MFS, the transforming growth factor-β1 (TGF-β1)/Smad2 signalling pathway is now assumed to play a role in TAA of various aetiologies. However, the relation between the aetiological diversity and the common cell phenotype with respect to TGF-β signalling remains unexplained.
METHODS AND RESULTS: This study was performed on human aortic samples, including TAA [MFS, n = 14; BAV, n = 15; and degenerative, n = 19] and normal aortas (n = 10) from which tissue extracts and human SMCs and fibroblasts were obtained. We show that all types of TAA share a complex dysregulation of Smad2 signalling, independent of TGF-β1 in TAA-derived SMCs (pharmacological study, qPCR). The Smad2 dysregulation is characterized by an SMC-specific, heritable activation and overexpression of Smad2, compared with normal aortas. The cell specificity and heritability of this overexpression strongly suggest the implication of epigenetic control of Smad2 expression. By chromatin immunoprecipitation, we demonstrate that the increases in H3K9/14 acetylation and H3K4 methylation are involved in Smad2 overexpression in TAA, in a cell-specific and transcription start site-specific manner.
CONCLUSION: Our results demonstrate the heritability, the cell specificity, and the independence with regard to TGF-β1 and genetic backgrounds of the Smad2 dysregulation in human thoracic aneurysms and the involvement of epigenetic mechanisms regulating histone marks in this process.

Novel aspects of the pathogenesis of Aneurysms of the Abdominal Aorta in humans
Michel JB, Martin Ventura JL, Egido J , Sakalihasan N, Treska V, Lindholt J, Allaire E, Thorsteinsdottir U, Cocker G, Swedenborg J
For the FAD EU consortium

2010 • Cardiovasc Res • [pdf]

AAA is a particular, specifically localized form of atherothrombosis, providing a unique human model of this disease. AAA pathogenesis is characterized by a breakdown of the extracellular matrix due to an excessive proteolytic activity, leading to potential arterial wall rupture. The roles of matrix metalloproteinases and plasmin generation in AAA progression have been demonstrated both in animal models and clinical studies. In the present review, we highlight recent studies addressing the role of the hemoglobin-rich, intraluminal thrombus (ILT) and the adventitial response in the development of human AAA. The ILT exerts its pathogenic effect through platelet activation, fibrin formation, binding of plasminogen and its activators and trapping of erythrocytes and neutrophils, leading to oxidative and proteolytic injury of the arterial wall. These events occur mainly at the ILT/circulating blood interface and pathologic mediators are conveyed outwards where they promote matrix degradation of the arterial wall. In response, neo-angiogenesis, phagocytosis by mononuclear cells, and a shift from innate to adaptive immunity in the adventitia are observed. AAA thus represents an accessible spatiotemporal model of human atherothrombotic progression towards clinical events, the study of which should allow further understanding of its pathogenesis and the translation of pathogenic biological activities into diagnostic and therapeutic applications.

European Perspectives in Cardiology
Steg G

2010 • Circulation • [pdf]



“Jean-Baptiste Michel Has Been an Incredible Leader in the Growth of Cardiovascular Research at Our Site”



“We’re Continuing to Witness Reductions in Mortality and Improvement in Clinical Outcomes. I Think It’s an Exciting Time”

Immune Responses Elicited in Tertiary Lymphoid Tissues Display Distinctive Features
Thaunat O, Graff-Dubois S, Brouard S, Gautreau C, Varthaman A, Fabien N, Field AC, Louedec L, Dai J, Joly E, Morelon E, Soulillou JP, Michel JB, Nicoletti A

2010 • PLoS ONE • [pdf]

During chronic inflammation, immune effectors progressively organize themselves into a functional tertiary lymphoid tissue (TLT) within the targeted organ. TLT has been observed in a wide range of chronic inflammatory conditions but its pathophysiological significance remains unknown. We used the rat aortic interposition model in which a TLT has been evidenced in the adventitia of chronically rejected allografts one month after transplantation. The immune responses elicited in adventitial TLT and those taking place in spleen and draining lymph nodes (LN) were compared in terms of antibody production, T cell activation and repertoire perturbations. The anti-MHC humoral response was more intense and more diverse in TLT. This difference was associated with an increased percentage of activated CD4+ T cells and a symmetric reduction of regulatory T cell subsets. Moreover, TCR repertoire perturbations in TLT were not only increased and different from the common pattern observed in spleen and LN but also "stochastic"€ since each recipient displayed a specific pattern. We propose that the abnormal activation of CD4+ T cells promotes the development of an exaggerated pathogenic immune humoral response in TLT. Preliminary findings suggest that this phenomenon i) is due to a defective immune regulation in this non-professional inflammatory-induced lymphoid tissue, and ii) also occurs in human chronically rejected grafts.

Chronic rejection triggers the development of an aggressive intragraft immune response through recapitulation of lymphoid organogenesis
Thaunat O, Patey N, Caligiuri G, Gautreau C, Mamani-Matsuda M, Mekki Y, Dieu-Nosjean MC, Eberl G, Ecochard R, Michel JB, Graff-Dubois S, Nicoletti A

2010 • J Immunol • [pdf]

The unwarranted persistence of the immunoinflammatory process turns this critical component of the body's natural defenses into a destructive mechanism, which is involved in a wide range of diseases, including chronic rejection. Performing a comprehensive analysis of human kidney grafts explanted because of terminal chronic rejection, we observed that the inflammatory infiltrate becomes organized into an ectopic lymphoid tissue, which harbors the maturation of a local humoral immune response. Interestingly, intragraft humoral immune response appeared uncoupled from the systemic response because the repertoires of locally produced and circulating alloantibodies only minimally overlapped. The organization of the immune effectors within adult human inflamed tissues recapitulates the biological program recently identified in murine embryos during the ontogeny of secondary lymphoid organs. When this recapitulation was incomplete, intragraft B cell maturation was impeded, limiting the aggressiveness of the local humoral response. Identification of the molecular checkpoints critical for completion of the lymphoid neogenesis program should help develop innovative therapeutic strategies to fight chronic inflammation.

Control of T cell reactivation by regulatory Qa-1-restricted CD8+ T cells
Varthaman A, Khallou-Laschet J, Clement M, Fornasa G, Kim HJ, Gaston AT, Dussiot M, Caligiuri G, Herbelin A, Kaveri S, Cantor H, Nicoletti A

2010 • J Immunol • [pdf]

Administration of attenuated pathogenic T cell clones, a procedure known as T cell vaccination, induces CD8+ T cells specific for peptides derived from the Vbeta-chain of the TCR presented by the MHC class Ib molecule, Qa-1 expressed on the vaccine cells. These regulatory CD8+ T cells have the capacity to control the activation of endogenous T cells expressing the same TCR Vbeta-chain as the vaccinating cells. We hypothesized that vaccination with NKT cells could also induce Qa-1-restricted CD8+ T cells that would control NKT cell activation. We tested this hypothesis in a murine model of Con A-induced hepatitis that is induced by NKT cells. Vaccination with NKT cells effectively induced protective Qa-1-restricted CD8+ T cells that prevented hepatitis. Surprisingly, upon vaccination with T cells expressing Vbeta-chains irrelevant to NKT cells, we discovered that the specificity of vaccine-induced Qa-1-restricted CD8+ T cells was not limited to the Vbeta-chain of the vaccinating cells. We further show that these regulatory Qa-1-restricted CD8+ T cells arise spontaneously upon polyclonal activation of T cells in the absence of deliberate T cell vaccination. These experiments provide new insight into a CD8+ T cell compartment that regulates the immediate reactivation of conventional T cells and NKT cells.

Protective Effect of High-Density Lipoprotein-Based Therapy in a Model of Embolic Stroke
Lapergue B, Moreno JA, Dang BQ, Coutard M, Delbosc S, Raphaeli G, Auge N, Klein I, Mazighi M, Michel JB, Amarenco P, Meilhac O

2010 • Stroke • [pdf]

BACKGROUND AND PURPOSE: High-density lipoprotein (HDL) levels are inversely associated with stroke incidence, suggesting a protective effect. Using a rat model, we tested the hypothesis that HDL exerts direct vasculo-/neuroprotective effects when administered during the acute phase of embolic stroke.
METHODS: After embolic occlusion, Sprague-Dawley rats were randomly treated intravenously with purified HDL versus saline immediately (2, 10 mg/kg) or 3 or 5 hours (10 mg/kg) after stroke. The effects of HDL were assessed blindly 24 hours later by evaluating neurological deficit score and measuring the infarct volume and blood-brain barrier breakdown. Protease activities and neutrophil infiltration were also evaluated.
RESULTS: HDL injection immediately after stroke (10 mg/kg) reduced by 68% the mortality at 24 hours (P=0.015). HDL administration immediately or at 3 or 5 hours after stroke also reduced cerebral infarct volume by 74%, 68%, and 70.7%, respectively (P=0.0003, P=0.011, and P=0.019; n=17 per group). The neurological deficit at 24 hours in the HDL-treated group was decreased versus the saline-treated group (P=0.015). Ischemia-induced blood-brain barrier breakdown was significantly reduced in HDL-treated rats versus controls (P=0.0045). Neuroprotective effects of HDL were associated with decreased neutrophil recruitment in the infarct area (P=0.0027) accompanied by reduced matrix metalloproteinase gelatinase activity. Immunostaining showed that HDL was associated with endothelial and glial cells, and also that intercellular adhesion molecule-1 expression was decreased in vessels within the infarct area.
CONCLUSIONS: Administration of HDL is neuroprotective when performed up to 5 hours after experimental stroke. This effect may be attributed to the ability of HDL to protect the blood-brain barrier and limit neutrophil recruitment.

TCR Stimulation Drives Cleavage and Shedding of the ITIM Receptor CD31
Fornasa G, Groyer E, Clement M, Dimitrov J, Compain C, Gaston AT, Varthaman A, Khallou-Laschet J, Newman DK, Graff-Dubois S, Nicoletti A, Caligiuri G

2010 • J Immunol • [pdf]

CD31 is a transmembrane molecule endowed with T cell regulatory functions owing to the presence of 2 immunotyrosine-based inhibitory motifs. For reasons not understood, CD31 is lost by a portion of circulating T lymphocytes, which appear prone to uncontrolled activation. In this study, we show that extracellular T cell CD31 comprising Ig-like domains 1 to 5 is cleaved and shed from the surface of human T cells upon activation via their TCR. The shed CD31 can be specifically detected as a soluble, truncated protein in human plasma. CD31 shedding results in the loss of its inhibitory function because the necessary cis-homo-oligomerization of the molecule, triggered by the trans-homophilic engagement of the distal Ig-like domain 1, cannot be established by CD31(shed) cells. However, we show that a juxta-membrane extracellular sequence, comprising part of the domain 6, remains expressed at the surface of CD31(shed) T cells. We also show that the immunosuppressive CD31 peptide aa 551-574 is highly homophilic and possibly acts by homo-oligomerizing with the truncated CD31 remaining after its cleavage and shedding. This peptide is able to sustain phosphorylation of the CD31 ITIM(686) and of SHP2 and to inhibit TCR-induced T cell activation. Finally, systemic administration of the peptide in BALB/c mice efficiently suppresses Ag-induced T cell-mediated immune responses in vivo. We conclude that the loss of T cell regulation caused by CD31 shedding driven by TCR stimulation can be rescued by molecular tools able to engage the truncated juxta-membrane extracellular molecule that remains exposed at the surface of CD31(shed) cells.

Macrophage plasticity in experimental atherosclerosis
Khallou-Laschet J, Varthaman A, Fornasa G, Compain C, Gaston A-T, Clement M, Dussiot M, Levillain O, Graff-Dubois S, Nicoletti A, Caligiuri G

2010 • PloS ONE • [pdf]

As in human disease, macrophages (MØ) are central players in the development and progression of experimental atherosclerosis. In this study we have evaluated the phenotype of MØ associated with progression of atherosclerosis in the apolipoprotein E (ApoE) knockout (KO) mouse model.

We found that bone marrow-derived MØ submitted to M1 and M2 polarization specifically expressed arginase (Arg) II and Arg I, respectively. This distinct arginase expression was used to evaluate the frequency and distribution of M1 and M2 MØ in cross-sections of atherosclerotic plaques of ApoE KO mice. Early lesions were infiltrated by Arg I+ (M2) MØ. This type of MØ favored the proliferation of smooth muscle cells, in vitro. Arg II+ (M1) MØ appeared and prevailed in lesions of aged ApoE KO mice and lesion progression was correlated with the dominance of M1 over the M2 MØ phenotype. In order to address whether the M2->M1 switch could be due to a phenotypic switch of the infiltrated cells, we performed in vitro repolarization experiments. We found that fully polarized MØ retained their plasticity since they could revert their phenotype. The analysis of the distribution of Arg I- and Arg II-expressing MØ also argued against a recent recruitment of M1 MØ in the lesion. The combined data therefore suggest that the M2->M1 switch observed in vivo is due to a conversion of cells already present in the lesion.

Our study suggests that interventional tools able to revert the MØ infiltrate towards the M2 phenotype may exert an atheroprotective action.

Anticoagulant and antithrombotic properties of platelet protease nexin-1
Boulaftali Y, Adam F, Venisse L, Ollivier V, Richard B, Taieb S, Monard D, Favier R, Alessi MC, Bryckaert M, Arocas V, Jandrot-Perrus M, Bouton MC

2010 • Blood • [pdf]

Protease nexin-1 (PN-1) is a serpin that inhibits plasminogen activators, plasmin, and thrombin. PN-1 is barely detectable in plasma but is expressed by platelets. Here, we studied platelet PN-1 in resting and activated conditions and its function in thrombosis. Studies on human platelets from healthy donors and from patients with a Gray platelet syndrome demonstrate that PN-1 is present both at the platelet surface and in alpha-granules. The role of PN-1 was investigated in vitro using human platelets incubated with a blocking antibody and using platelets from PN-1-deficient mice. Both approaches indicate that platelet PN-1 is active on thrombin and urokinase-type plasminogen activator. Blockade and deficiency of platelet PN-1 result in accelerated and increased tissue factor-induced thrombin generation as indicated by calibrated automated thrombography. Moreover, platelets from PN-1-deficient mice respond to subthreshold doses of thrombin, as assessed by P-selectin expression and platelet aggregation. Thrombus formation, induced ex vivo by collagen in blood flow conditions and in vivo by FeCl(3)-induced injury, is significantly increased in PN-1-deficient mice, demonstrating the antithrombotic properties of platelet PN-1. Platelet PN-1 is thus a key player in the thrombotic process, whose negative regulatory role has been, up to now, markedly underestimated.

Magnetic micro-manipulations to probe the local physical properties of porous scaffolds and to confine stem cells
Robert D, Fayol D, Le Visage C, Frasca G, Brule S, Menager C, Gazeau F, Letourneur D, Wilhelm C

2010 • Biomaterials • [pdf]



The in vitro generation of engineered tissue constructs involves the seeding of cells into porous scaffolds. Ongoing challenges are to design scaffolds to meet biochemical and mechanical requirements and to optimize cell seeding in the constructs. In this context, we have developed a simple method based on a magnetic tweezer set-up to manipulate, probe, and position magnetic objects inside a porous scaffold. The magnetic force acting on magnetic objects of various sizes serves as a control parameter to retrieve the local viscosity of the scaffolds internal channels as well as the stiffness of the scaffolds pores. Labeling of human stem cells with iron oxide magnetic nanoparticles makes it possible to perform the same type of measurement with cells as probes and evaluate their own microenvironment. For 18 mum diameter magnetic beads or magnetically labeled stem cells of similar diameter, the viscosity was equivalently equal to 20 mPa s in average. This apparent viscosity was then found to increase with the magnetic probes sizes. The stiffness probed with 100 mum magnetic beads was found in the 50 Pa range, and was lowered by a factor 5 when probed with cells aggregates. The magnetic forces were also successfully applied to the stem cells to enhance the cell seeding process and impose a well defined spatial organization into the scaffold.

Hemorphin 7 reflects hemoglobin proteolysis in abdominal aortic aneurysm
Dejouvencel T, Feron D, Rossignol P, Sapoval M, Kauffmann C, Piot JM, Michel JB, Fruitier-Arnaudin I, Meilhac O

2009 • ATVB • [pdf]

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 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.

Development of a functionalized polymer for stent coating in the arterial delivery of small interfering RNA
San Juan A, Bala M, Hlawaty H, Portes P, Vranckx R, Feldman LJ, Letourneur D

2009 • Biomacromolecules • [pdf]

In patients receiving drug eluting stents, there is a growing concern about both the long-term toxicity/degradability of the polymers used for the coating, and the nature of the therapeutic agents. We hypothesized that the use of a functionalized biocompatible polymer for a stent coating could be appropriate for local arterial therapy. A cationized pullulan hydrogel was thus prepared to cover bare metal stents that could be further loaded with small interfering RNA (siRNA) targeted at MMP2 for gene silencing in vascular cells. The efficient coverage of the stent struts by a smooth polymeric layer, which can withstand the crimping of the stent on a balloon-catheter and its deployment, was demonstrated by fluorescence microscopy, scanning electron microscopy, and atomic force microscopy. The release of siRNA from the stents was modulated by the presence of the cationic groups, as compared to noncationized pullulan hydrogel. In vivo implantation of coated stents was successful and cationized pullulan-based hydrogels loaded with siRNA in rabbit balloon-injured carotid arteries induced an uptake of siRNA into the arterial wall and a decrease of pro-MMP2 activity. These results suggest that cationized pullulan-based hydrogel could be used as a new biocompatible and biodegradable stent coating for local gene therapy in the arterial wall.

Concentrated collagen hydrogels as dermal substitutes
Helary C, Bataille I, Abed A, Illoul C, Anglo A, Louedec L, Letourneur D, Meddahi-Pelle A, Giraud-Guille MM

2009 • Biomaterials • [pdf]

Collagen hydrogels first appeared promising for skin repair. Unfortunately, their extensive contraction and their poor mechanical properties constituted major disadvantages toward their utilization as permanent graft. The present study has investigated a way to correct these drawbacks by increasing the collagen concentration in controlled conditions. Concentrated collagen hydrogels (CCH) at 1.5, 3 and 5mg/ml were obtained. The effect of raised collagen concentration on contraction, cell growth and remodeling activities was evaluated for 21 days in culture. Subsequently, in vivo integration of CCH and normal collagen hydrogels (NCH) was assessed. Compared to NCH, CCH contraction was delayed and smaller. At day 21, surface area of CCH at 3mg/ml was 18 times more important than that of NCH. Whatever the initial fibroblast density, CCH favored cell growth that reached about 10 times the initial cell number at day 21; cell proliferation was inhibited in NCH. Gelatinase A activities appeared lower in CCH than within NCH. In vivo studies in rats revealed a complete hydrolysis of NCH 15 days after implantation. In contrast, CCH at 3mg/ml was still present after 30 days. Moreover, CCH showed cell colonization, neovascularization and no severe inflammatory response. Our results demonstrate that concentrated collagen hydrogels can be considered as new candidates for dermal substitution because they are easy to handle, do not contract drastically, favor cell growth, and can be quickly integrated in vivo.

An expansible aortic ring for a physiological approach to conservative aortic valve surgery
Lansac E, Di Centa I, Raoux F, Bulman-Fleming N, Ranga A, Abed A, Ba M, Paolitto A, Letourneur D, Meddahi-Pellé A

2009 • J Thorac Cardiovasc Surg • [pdf]

OBJECTIVE: Dystrophic aortic insufficiency is characterized by dilation of the aortic annular base and sinotubular junction diameters preventing coaptation of thin and pliable cusps amenable to valve repair. An expansible aortic ring was designed to reduce dilated aortic root diameters to increase valvular coaptation height while maintaining root dynamics. The properties of the device were tested in vitro and in vivo in an ovine model.
METHODS: Expansible rings were composed of an elastomer core covered by polyester fabric. After in vitro analysis of their mechanical properties, the rings were implanted in 6 sheep at both the level of the annular base and sinotubular junction (double subvalvular and supravalvular external aortic annuloplasty). Root dynamics were assessed by using intracardiac ultrasonography before surgical intervention and at 6 months. Histologic, scanning electron microscopic, and mechanical studies were then performed on explanted samples.
RESULTS: The expansible ring produced a significant reduction of the aortic annular base and sinotubular junction diameters. Coaptation height was increased from 2.5 +/- 0.7 mm to 6.2 +/- 1.1 mm (P < .001). Mechanical testing on 6-month explanted samples revealed no significant differences in elastic modulus. Dynamics of the root were well preserved. Histomorphologic studies showed incorporation of the material without degradation.
CONCLUSIONS: Expansible aortic ring implantation produces a significant annuloplasty that increases coaptation height while preserving the dynamics of the aortic root. The effectiveness of the device in treating aortic insufficiency is currently being evaluated in the prospective Conservative Aortic Valve surgery for aortic Insufficiency and Aneurysm of the Aortic Root trial comparing conservative aortic valve surgery versus mechanical valve replacement.

Immaturity of microvessels in hemorrhagic plaques is associated with proteolytic degradation of angiogenic factors
Le Dall J, Ho-Tin-Noe B, Louedec L, Meilhac O, Roncal C, Carmeliet P, Germain S, Michel JB, Houard X

2009 • Cardiovasc Res • [pdf]

AIMS: We investigated the causes of microvessel immaturity and destabilization in human atherosclerotic lesions.
METHODS: Human atherosclerotic carotid plaques (n=24) were classified as non-hemorrhagic (NH) or hemorrhagic (HEM), according to their macroscopic aspect and hemoglobin content. Plaque microvessel density and maturity were quantified by immunohistochemistry. Expression of angiogenic factors was studied by immunohistochemistry, in situ hybridization and ELISA. Plaque-conditioned media were tested for plasmin and elastase activities, and for their ability to degrade angiogenic factors and to induce smooth muscle cell migration.
RESULTS: Microvessel density and leukocyte infiltration were increased in HEM compared to NH plaques. Plaque vasculature appeared vulnerable as indicated by the absence of alpha-actin-positive mural cells in most plaque vessels. Despite increased numbers of angiogenic factor-expressing microvessels and leukocytes in HEM plaques, lower levels of vascular endothelial growth factor, placental growth factor and angiopoietin-1 were found in conditioned media from HEM plaques. However, NH and HEM plaques released similar levels of the vascular destabilizing factor, angiopoietin-2. Addition of recombinant angiogenic factors to plaque extracts showed that all factors but angiopoietin-2 were selectively degraded by plasmin and/or elastase released from HEM plaques. Furthermore, conditioned media from HEM plaques showed a reduced ability to induce smooth muscle cell migration.
CONCLUSIONS: Our results provide evidence that immaturity of plaque vessels is associated with degradation of angiogenic factors by hemorrhage-conveyed leukocytes and proteases.

Design and humanization of a murine scFv that blocks human platelet glycoprotein VI in vitro
Muzard J, Bouabdelli M, Zahid M, Ollivier V, Lacapere JJ, Jandrot-Perrus M, Billiald P

2009 • FEBS J • [pdf]

Platelet adhesion and aggregation at the site of vascular injury is essential for hemostasis, but can also lead to arterial occlusion in thrombotic disorders. Glycoprotein (GP) VI is the major platelet membrane receptor that interacts directly with collagen, the most thrombogenic compound in the blood vessels. GPVI could therefore be a major therapeutic target. Fab fragments of the anti-GPVI murine monoclonal IgG 9O12 have previously been shown to completely block collagen-induced platelet aggregation, to inhibit the procoagulant activity of collagen-stimulated platelets, and to prevent thrombus formation under arterial flow conditions without significantly prolonging the bleeding time. Here, we engineered recombinant scFvs that preserve the functional properties of 9O12, and could constitute building blocks for designing new compounds with potentially therapeutic antithrombotic properties. First, the 9O12 variable domains were cloned, sequenced, and expressed as a recombinant murine scFv, which was fully characterized. This scFv preserved all the characteristics that make 9O12 Fab potentially useful for therapeutic applications, including its high affinity for GPVI, ability to inhibit platelet adhesion, and aggregation with collagen under arterial flow conditions. A humanized version of this scFv was also designed after complementarity-determining region grafting and structural refinements using homology-based modeling. The final product was produced in recombinant bacteria. It retained GPVI-binding specificity and high affinity, which are the main parameters usually impaired by humanization procedures. This is a simple, efficient and straightforward method that could also be used for humanizing other antibodies.

Absence of collagen-induced platelet activation caused by compound heterozygous GPVI mutations
Dumont B, Lasne D, Rothschild C, Bouabdelli M, Ollivier V, Oudin C, Ajzenberg N, Grandchamp B, Jandrot-Perrus M

2009 • Blood • [pdf]

The GPVI/FcRgamma complex is a key receptor for platelet activation by collagen. We describe for the first time two genetic abnormalities in one patient. This 10 year-old girl presented ecchymoses since infancy, a prolonged bleeding time despite a normal platelet count and no antiplatelet antibodies. Collagen-induced platelet activation was null whereas GPVI quantification by flow cytometry evidenced an incomplete deficiency. Immunoblotting showed an abnormal migration of residual GPVI, and no FcRgamma defect. GPVI DNA sequencing revealed (i) a R38C mutation in exon 3 of one allele (ii) an insertion of 5 nucleotides in exon 4 of the other allele, leading to a premature nonsense codon and absence of the corresponding mRNA. Introduction of the R38C mutation into recombinant GPVI-Fc resulted in abnormal protein migration and a loss of collagen binding. Thus, this composite genetic GPVI deficiency and dysfunction causes absence of platelet responses to collagen and a mild bleeding phenotype.

Mediators of neutrophil recruitment in human abdominal aortic aneurysms
Houard X, Touat Z, Ollivier V, Louedec L, Philippe M, Sebbag U, Meilhac O, Rossignol P, Michel JB

2009 • Cardiovasc Res • [pdf]

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.

Non-Invasive Molecular Imaging of Fibrosis Using a Collagen-Targeted Peptidomimetic of the Platelet Collagen Receptor Glycoprotein VI
Muzard J, Sarda-Mantel L, Loyau S, Meulemans A, Louedec L, Bantsimba-Malanda C, Hervatin F, Marchal-Somme J, Michel JB, Le Guludec D, Billiald P, Jandrot-Perrus M

2009 • PLoS One • [pdf]

BACKGROUND: Fibrosis, which is characterized by the pathological accumulation of collagen, is recognized as an important feature of many chronic diseases, and as such, constitutes an enormous health burden. We need non-invasive specific methods for the early diagnosis and follow-up of fibrosis in various disorders. Collagen targeting molecules are therefore of interest for potential in vivo imaging of fibrosis. In this study, we developed a collagen-specific probe using a new approach that takes advantage of the inherent specificity of Glycoprotein VI (GPVI), the main platelet receptor for collagens I and III.
METHODOLOGY/PRINCIPAL FINDINGS: An anti-GPVI antibody that neutralizes collagen-binding was used to screen a bacterial random peptide library. A cyclic motif was identified, and the corresponding peptide (designated collagelin) was synthesized. Solid-phase binding assays and histochemical analysis showed that collagelin specifically bound to collagen (Kd 10(-7) M) in vitro, and labelled collagen fibers ex vivo on sections of rat aorta and rat tail. Collagelin is therefore a new specific probe for collagen. The suitability of collagelin as an in vivo probe was tested in a rat model of healed myocardial infarctions (MI). Injecting Tc-99m-labelled collagelin and scintigraphic imaging showed that uptake of the probe occurred in the cardiac area of rats with MI, but not in controls. Post mortem autoradiography and histological analysis of heart sections showed that the labeled areas coincided with fibrosis. Scintigraphic molecular imaging with collagelin provides high resolution, and good contrast between the fibrotic scars and healthy tissues. The capacity of collagelin to image fibrosis in vivo was confirmed in a mouse model of lung fibrosis.
CONCLUSION/SIGNIFICANCE: Collagelin is a new collagen-targeting agent which may be useful for non-invasive detection of fibrosis in a broad spectrum of diseases.

HDL antielastase activity prevents smooth muscle cell anoikis, a potential new antiatherogenic property
Ortiz-Munoz G, Houard X, Martín-Ventura JL, Ishida BY, Loyau S, Rossignol P, Moreno JA, Kane JP, Chalkley RJ, Burlingame AL, Michel JB, Meilhac O

2009 • FASEB J • [pdf]

Various studies using proteomic approaches have shown that HDL can carry many proteins other than its constitutive apolipoprotein A-I (apoA-I). Using mass spectrometry and Western blotting, we showed the presence of alpha1-antitrypsin (AAT) (SERPINA1, serpin peptidase inhibitor, clade A, an elastase inhibitor) in HDL, isolated either by ultracentrifugation or by selected-affinity immunosorption using an anti-apoA-I column. Furthermore, we report that HDL possesses potent antielastase activity. We further showed that only HDL but not LDL is able to bind AAT. HDL-associated AAT was able to inhibit extracellular matrix degradation, cell detachment, and apoptosis induced by elastase in human vascular smooth muscle cells (VSMCs) and in mammary artery cultured ex vivo. Degradation of fibronectin by elastase used as a marker of pericellular proteolysis was prevented by addition of HDL. Elastase present in aortic abdominal aneurysm (AAA) thrombus samples was also able to induce apoptosis of VSMCs in culture. This phenomenon was prevented by addition of HDL but not of LDL. Finally, we report that the proportion of AAT in HDL isolated from patients with an AAA is decreased relative to that from matched control subjects, suggesting a reduced capacity of HDL to inhibit elastase in these patients. In conclusion, our data provide evidence of a new potential antiatherogenic property of HDL attributable to AAT and its antielastase activity.

Comparative studies on the mechanisms of action of four polysaccharides on arterial restenosis
Deux JF, Meddahi-Pelle A, Bree F, Bataille I, Michel JB, Letourneur D

2009 • J Biomater Sci Polym Ed • [pdf]

Percutaneous coronary interventions play a major role in the management of patients affected by coronary artery diseases. However, their efficiency is impaired by restenosis, defined as a reduction of the vessel lumen, occurring a few months after the procedure. A low-molecular-weight fraction of fucoidan, a vegetal heparin-like sulphated polysaccharide, was recently shown to greatly reduce in-stent restenosis after angioplasty in rabbits. To better understand the in vivo anti-restenotic effects of this polymer, we used fractions of fucoidan and compared to heparin and dextran of different sizes. We carried out in vitro growth inhibition experiments on vascular smooth muscle cells, performed an in vivo pharmacokinetic study, and locally delivered fluorescently-labeled polysaccharides in rabbit iliac arteries after angioplasty with a non-occlusive catheter. The results indicated that (i) preparation of well-characterized fractions from natural fucoidan is compulsory for in vitro and in vivo studies, (ii) antiproliferative activity of sulphated polysaccharides on cultured smooth muscle cells is not a major predictive factor for the reduction of restenosis in vivo and (iii) pharmacokinetic parameters and binding of low-molecular-weight fucoidan on angioplasty-induced injured vascular walls are important local and general factors controlling its mechanisms of action.

Syndromic and non-syndromic aneurysms of the human ascending aorta share activation of the Smad2 pathway
Gomez D, Al Haj Zen A, Borges LF, Philippe M, Gutierrez PS, Jondeau G, Michel JB, Vranckx R

2009 • J Pathol • [pdf]

Common features such as elastic fibre destruction, mucoid accumulation, and smooth muscle cell apoptosis are co-localized in aneurysms of the ascending aorta of various aetiologies. Recent experimental studies reported an activation of TGF-beta in aneurysms related to Marfan (and Loeys-Dietz) syndrome. Here we investigate TGF-beta signalling in normal and pathological human ascending aortic wall in syndromic and non-syndromic aneurysmal disease. Aneurysmal ascending aortic specimens, classified according to aetiology: syndromic MFS (n = 15, including two mutations in TGFBR2), associated with BAV (n = 15) or degenerative forms (n = 19), were examined. We show that the amounts of TGF-beta1 protein retained within and released by aneurysmal tissue were greater than for control aortic tissue, whatever the aetiology, contrasting with an unchanged TGF-beta1 mRNA level. The increase in stored TGF-beta1 was associated with enhanced LTBP-1 protein and mRNA levels. These dysregulations of the extracellular ligand are associated with higher phosphorylated Smad2 and Smad2 mRNA levels in the ascending aortic wall from all types of aneurysm. This activation correlated with the degree of elastic fibre fragmentation. Surprisingly, there was no consistent association between the nuclear location of pSmad2 and extracellular TGF-beta1 and LTBP-1 staining and between their respective mRNA expressions. In parallel, decorin was focally increased in aneurysmal media, whereas biglycan was globally decreased in aneurysmal aortas. In conclusion, this study highlights independent dysregulations of TGF-beta retention and Smad2 signalling in syndromic and non-syndromic aneurysms of the ascending aorta.

Biomimetic MRI Contrast Agent for Imaging of Inflammation in Atherosclerotic Plaque of ApoE-/- Mice: A Pilot Study
Alsaid H, De Souza G, Bourdillon MC, Chaubet F, Sulaiman A, Desbleds-Mansard C, Chaabane L, Zahir C, Lancelot E, Rousseaux O, Corot C, Douek P, Briguet A, Letourneur D, Canet-Soulas E

2009 • Invest Radiol • [pdf]

OBJECTIVE: Atherosclerosis involves an inflammatory process characterized by cellular and molecular responses. A slow-clearance blood-pool paramagnetic agent (CMD-A2-Gd-DOTA: P717) chemically modified to create a functionalized product (F-P717) for targeting inflammation in vessel walls was evaluated in vivo in mice.
METHODS AND RESULTS: Carboxylate and sulfate groups were grafted onto the macromolecular paramagnetic Gd-DOTA-dextran backbone. Products were also fluorescently labeled with rhodamine isothiocyanate. Pre- and postcontrast MRI was performed on a 2-Tesla magnet in ApoE and control C57BL/6 mice after P717 or F-P717 injection at a dose of 60 mumol Gd/kg. Axial T1-weighted images of the abdominal aorta were obtained using a 2D multislice spin-echo sequence. F-P717 significantly enhanced the magnetic resonance imaging (MRI) signal in the abdominal aortic wall of ApoE mice (>50% signal-to-noise ratio increase between 10 and 30 minutes), but not of control mice. P717 produced only moderate (<20%) MRI signal enhancement within the same time frame. The MRI data were correlated to histopathology. Immunofluorescence in ApoE mice colocalized F-P717 but not P717 with the inflammatory area revealed by P-selectin labeling.
CONCLUSION: This study demonstrates the efficacy of F-P717 as a new molecular imaging agent for noninvasive in vivo MRI location of inflammatory vascular tree lesions in ApoE mice.

Differential inflammatory activity across human abdominal aortic aneurysms reveals neutrophil-derived leukotriene B4 as a major chemotactic factor released from the intraluminal thrombus
Houard X, Ollivier V, Louedec L, Michel JB, Back M

2009 • FASEB J • [pdf]

Development and progression of acquired abdominal aortic aneurysms (AAAs) have been associated with different inflammatory mediators. The aim of the present study was to elucidate the topology and the potential mechanisms linking the leukotriene pathway to human AAAs.Human aneurysmal lesions were obtained from 24 patients undergoing surgery, and the intraluminal thrombus was separated from the vascular wall. Histological examination revealed major expression of the leukotriene-producing enzymes 5-lipoxygenase and LTA4 hydrolase, as well as the two receptors for leukotriene B4 (BLT1R and BLT2R), corresponding to neutrophils in the luminal part of the thrombus. In contrast, in the vascular wall, the leukotriene pathway mainly localized in macrophage-rich adventitial areas. Furthermore, conditioned media of the intraluminal thrombus contained significantly higher concentrations of leukotriene B4 than that derived from the vascular wall, which were significantly correlated to other neutrophil-derived mediators, such as elastase/alpha1-antitrypsin complexes, myeloperoxidase, and MMP9/NGAL complexes. Finally, the neutrophil-chemotactic activity of the conditioned media from the intraluminal thrombus exhibited major inhibition by antagonists of the leukotriene B4 receptors.Taken together, these results indicate neutrophil-derived leukotriene B4 as a major neutrophil chemotactic factor released from the intraluminal thrombus of human AAAs and suggest that targeting BLT receptors may represent a potential medical therapeutic strategy in the prevention of AAA progression in humans.

Antiangiogenic treatment prevents adventitial constrictive remodeling in graft arteriosclerosis
Thaunat O, Louedec L, Graff-Dubois S, Dai J, Groyer E, Yacoub-Youssef H, Mandet C, Bruneval P, Kaveri S, Caligiuri G, Germain S, Michel JB, Nicoletti A

2008 • Tranplantation • [pdf]

Background. Lumen loss in graft arteriosclerosis is the consequence of the development of a thick neointima and constrictive arterial remodeling. The latter is due to adventitial chronic inflammation and excessive perivascular collagen deposition. We reasoned that blockade of the portal of entry of inflammatory effectors may constitute a strategy to prevent constrictive arterial remodeling.
Methods and Results. We found that an anti-angiogenic therapy (ABT-510 nonapeptide), devoid of direct immunomodulatory properties, dramatically reduced adventitial angiogenesis by 66% (P<0.0001) in the rat aortic interposition model of graft arteriosclerosis. The associated decreased entry of inflammatory cells (44%; P
<0.0001) resulted in drastic reduction of collagen deposition (57%; P<0.0001) thereby preventing subsequent adventitial constrictive remodeling and reduction of lumen surface area (5260.74 vs. 8582.48 µ
m2; Control vs. ABT-510-treated rats; P<0.0001). ABT-510 had no effect on the development of the neointima.
Conclusion. This work supports the idea that targeting angiogenesis may act synergistically with conventional immunosuppressive therapy in preventing graft arteriosclerosis, a crucial feature of chronic graft rejection.

Technetium 99m-labeled annexin V scintigraphy of platelet activation in vegetations of experimental endocarditis
Rouzet F, Dominguez Hernandez M, Hervatin F, Sarda-Mantel L, Lefort A, Duval X, Louedec L, Fantin B, Le Guludec D, Michel JB

2007 • Circ • [pdf]

BACKGROUND: The pathophysiology of infective endocarditis involves a pathogen/host tissue interaction, leading to formation of infected thrombotic vegetations. Annexin V is a ligand of phosphatidylserines exposed by activated platelets and apoptotic cells. Because vegetations are platelet-fibrin clots in which platelet proaggregant activity is enhanced by bacterial colonization, we investigated the ability of annexin V labeled with technetium Tc 99m (99mTc-ANX) to provide functional imaging of these vegetations in experimental models of infective endocarditis. This ability was assessed in rabbits and rats because of the different interest of these 2 species in preclinical analysis.
METHODS AND RESULTS: Nonbacterial thrombotic endocarditis was induced with the use of a catheter left indwelling through the aortic or tricuspid valve, and animals were injected with either a bacterial inoculum or saline. Scintigraphic investigations were performed 5 days later and showed a higher 99mTc-ANX uptake by vegetations in infected versus noninfected animals (ratio, 1.3 for in vivo acquisitions and 2 for autoradiography; P<0.0001 for all), whereas no significant uptake was present in controls. Right-sided endocarditis was associated with pulmonary uptake foci corresponding to emboli. Histological analysis of vegetations showed a specific uptake of 99mTc-ANX at the interface between circulating blood and vegetation. In parallel, underlying myocardial tissue showed myocyte apoptosis and mucoid degeneration, without extracellular matrix degradation at this stage.
CONCLUSIONS: 99mTc-ANX is suitable for functional imaging of platelet-fibrin vegetations in endocarditis, as well as embolic events. 99mTc-ANX uptake reflects mainly platelet activation in the luminal layer of vegetations. This uptake is enhanced by bacterial colonization.

Protease nexin-1 interacts with thrombomodulin and modulates its anticoagulant effect
Bouton MC, Venisse L, Richard B, Pouzet C, Arocas V, Jandrot-Perrus M

2007 • Circ Res • [pdf]

The endothelial cell membrane glycoprotein thrombomodulin (TM) plays a critical role in the regulation of coagulation. TM is an essential cofactor in protein C activation by thrombin, and a direct inhibitor of thrombin-induced platelet activation and fibrinogen clotting. Protease nexin-1 (PN-1) is a serpin synthesized and secreted by a variety of cells including endothelial cells. PN-1 bound to the cell surface through interactions with glycosaminoglycans, is an efficient inhibitor of thrombin and controls thrombin-induced cell responses. An investigation of the interaction of PN-1 with TM using purified proteins and cultured human aortic endothelial cells was performed. Purified PN-1 was observed to bind to purified TM in a concentration-dependent manner. Double immunofluorescence studies indicated that PN-1 and TM were colocalized at the endothelial cell surface from which they were coprecipitated. Pretreatment of the cells with chondroitinase ABC greatly decreased the amount of the PN-1 associated to TM at the cell surface demonstrating the involvement of the TM chondroitin-sulfate chain in the formation of complexes. The inhibitory activity of the PN-1/TM complexes on the catalytic activity of thrombin, and on thrombin-induced fibrinogen clotting, was markedly enhanced when compared with the inhibitory activity of each partner. PN-1-overexpressing human aortic endothelial cells and PN-1-underexpressing human aortic endothelial cells exhibited respectively a significantly reduced ability and enhanced capacity to activate protein C. Furthermore, PN-1 decreased the cofactor activity of TM on thrombin activable fibrinolysis inhibitor activation by thrombin. These data show for the first time that PN-1 forms complexes with TM and modulates its anticoagulant activity.

Phosphorylcholine-targeting immunization reduces atherosclerosis
Caligiuri G, Khallou-Laschet J, Vandaele M, Gaston AT, Delignat S, Mandet C, Kohler HV, Kaveri S, Nicoletti A

2007 • J Am Coll Cardiol • [pdf]

Objectives: The present study evaluated the effect of phosphorylcholine (PC) immunization on the extent of experimental atherosclerosis.
Background: Immunization against oxLDL or Streptococcus pneumoconiae reduces atherosclerosis. Phosphorylcholine is the main epitope recognized by both antipneumococcus and anti-oxLDL antibodies. Therefore we reasoned that PC-specific antibodies might play an important role in atherogenesis.
Methods: Apolipoprotein E knockout mice were immunized with PC every second week over 4 months. At the end of the study, serum antibodies directed to either PC or oxLDL were measured. Splenic and peritoneal B cells were analyzed by flow cytometry. Aortic root atherosclerotic lesions were quantified by morphometry and phenotyped by immunohistochemistry. Immune and control sera were also tested for their effect on foam cell formation in macrophage culture in the presence of oxLDL.
Results: The PC-immunized mice showed 3-fold increase in titers of anti-PC and -oxLDL antibodies compared with control mice. The PC-immunized mice also showed a significant increase in the number of splenic mature B cells. The extent of atherosclerotic aorta root lesions was reduced by >40% in the PC-immunized mice. Immunohistochemistry showed reduced expression of MHC II antigens and the presence of B-cell clusters in plaques of PC-immunized mice. Finally, PC-immune serum was able to reduce macrophage-derived foam cell formation in the presence of oxLDL in vitro.
Conclusions: PC immunization drives a specific humoral immune response that reduces foam cell formation in vitro and is atheroprotective in vivo.

A new macromolecular paramagnetic MR contrast agent binds to activated human platelets
Chaubet F, Bertholon I, Serfaty JM, Bazeli R, Alsaid H, Jandrot-Perrus M, Zahir C, Even P, Bachelet L, Touat Z, Lancelot E, Corot C, Canet-Soulas E, Letourneur D

2007 • Contrast Media Mol Imaging • [pdf]

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.

Atheroprotective effect of CD31 receptor globulin through enrichment of circulating regulatory T-cells
Groyer E, Nicoletti A, Ait-Oufella H, Khallou-Laschet J, Varthaman A, Gaston AT, Thaunat O, Kaveri SV, Blatny R, Stockinger H, Mallat Z, Caligiuri G

2007 • J Am Coll Cardiol• [pdf]

Objectives: To evaluate whether replacing CD31 (PECAM-1) signaling can restore the regulation of lymphocyte activation and improve experimental atherosclerosis.
Background: Atherosclerosis is due to the development of a pathogenic immune response within the vascular wall and is aggravated by the reduction of regulatory T-cells. CD31, a transmembrane adhesion molecule with inhibitory signaling functions, is physiologically expressed on blood and vascular resting cells but is lost in pathologic conditions associated with atherosclerosis.
Methods: Replacement therapy with a CD31 receptor globulin (Rg) by in vivo gene transfer in 6-week-old apolipoprotein E knockout mice every 5 weeks for 6 months (Control groups: truncated CD31Rg or with vehicle alone).
Results: Atherosclerotic lesions of CD31Rg-treated mice were smaller and showed less neovascularization and intraplaque hemorrhage compared with control subjects. Furthermore, circulating regulatory T-cells were increased in vivo and showed normal suppressive function on proliferation of conventional T-cells in vitro. Indeed, CD31Rg treatment led to blunted blood T-cell activation and reduced T-cell infiltration within plaques.
Conclusions: CD31 plays a key role in the regulation of the immune response linked to atherosclerosis. CD31-targeting therapeutic approaches may therefore be envisaged for preventing and treating atherosclerotic diseases.

Involvement of intraplaque hemorrhage in atherothrombosis evolution via neutrophil protease enrichment
Leclercq A, Houard X, Philippe M, Ollivier V, Sebbag U, Meilhac O, Michel JB

2007 • J Leukoc Biol • [pdf]

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 (alpha1-antitrypsin/elastase complexes, myeloperoxidase, and alpha-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.

Topological determinants and consequences of adventitial responses to arterial wall injury
Michel JB, Thaunat O, Houard X, Meilhac O, Caligiuri G, Nicoletti A

2007 • Arterioscl Thromb Vasc Biol • [pdf]

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. We propose that adventitial events have an impact not only on the vessel wall biology but also on the surrounding tissue.

Percutaneous intracoronary delivery of SERCA gene increases myocardial function: a tissue Doppler imaging echocardiographic study
Logeart D, Vinet L, Ragot T, Heimburger M, Louedec L, Michel JB, Escoubet B, Mercadier JJ

2006 • Am J Physiol Heart Circ Physiol • [pdf]

The aim of this study was to examine the efficiency of adenovirus-mediated overexpression of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1a) gene in a realistic model based on percutaneous intracoronary delivery and on noninvasive functional monitoring. Catheter-based selective coronary delivery of saline or adenoviruses (Ad.CMV.SERCA1a or Ad.CMV.lacZ, 10(10) plaque-forming units) was performed in the circumflex artery of rabbits. Effects were assessed and compared by using serial Doppler echocardiography, hemodynamics, and measurements of SERCA protein and Ca(2+) uptake activity. On day 3, a 21% increase in SERCA proteins and a 37% increase in the maximal rate of Ca(2+) uptake were observed in the transfected left ventricular (LV) walls of Ad.CMV.SERCA1a rabbits. Baseline hemodynamics and conventional echographic measurements of global LV function were poorly affected. In contrast, tissue Doppler imaging (TDI) was able to assess a strong increase in the baseline function of transfected LV walls, as assessed with maximal wall velocities (+32% and +43%, respectively) and strain rates (+18% and +30%, respectively). TDI parameters were closely related to the maximal rate of Ca(2+) uptake (r(2) = 0.68 for the systolic strain rate). Serial TDI analysis during follow-up showed that the effects lasted for 7 days and were no longer detectable 15 days after adenoviruses injection. In conclusion, LV function can be increased by adenovirus-mediated overexpression of SERCA in a clinically relevant model, and TDI provides an accurate and noninvasive tool for monitoring effects on global as well as regional myocardial function.

Lymphoid Neogenesis in chronic rejection: the murderer is in the house
Thaunat O, Patey N, Morelon E, Michel JB, Nicoletti A

2006 • Curr Opin Immunol • [pdf]

Although chronic rejection is currently the main cause of long-term allograft failure, its pathogenesis remains elusive, hereby preventing the development of effective therapy. Recent advances in the comprehension of the pathophysiology of chronic inflammatory diseases could shed new light on the pathogenesis of chronic rejection. Lymphoid neogenesis is a mechanism responsible for the progressive organization of chronic inflammatory infiltrates into functional ectopic germinal centers, and has been evidenced recently in various pathological situations sharing a common feature: the failure of the immune response to eradicate the targeted antigen(s). Chronic rejection is such a situation as it results from a sustained alloimmune response against the donor's antigens that are constantly replenished by the grafted tissue. Accordingly, functional ectopic germinal centers develop within chronically rejected organs. This implies that, during chronic rejection, graft is at the same time the target and the site of elicitation of the alloimmune response.

Direct and indirect effects of alloantibodies link neointimal and medial remodeling in graft arteriosclerosis
Thaunat O, Louedec L, Dai J, Bellier F, Groyer E, Delignat S, Gaston AT, Caligiuri G, Joly E, Plissonnier D, Michel JB, Nicoletti A

2006 • ATVB • [pdf]

Objective— Chronic vascular rejection, the main cause of allograft failure, is characterized by the destruction of smooth muscle cells (SMCs) in the media concomitantly with the proliferation of SMCs in the adjacent neointima. We hypothesized that alloantibodies might be responsible for these 2 opposite but coordinated events.
Methods and Results— We used the rat aortic interposition model of chronic vascular rejection. During the rejection process, a neointima composed of proliferating SMCs from the recipient developed, whereas the SMCs in the media, all of donor origin, underwent apoptosis. Alloantibody deposition was detected only in the media. Using in vitro cultures experiments, we observed that alloantibody binding to donor SMCs exerts (1) a rapid upregulation of the transcription of growth factors genes, followed by (2) the induction of apoptosis after 24 hours. The transient production of growth factors by donor SMCs in response to the binding of alloantibodies induced the proliferation of recipient SMCs in culture supernatant transfer experiments. Additional data suggest that among the repertoire of alloantibodies, those directed against major histocompatibility complex I might carry the remodeling effect.
Conclusions— Our data suggest that during chronic vascular rejection, alloantibody binding to donor medial SMCs is a crucial event that links neointimal and medial remodeling.

Is defective lymphatic drainage a trigger for lymphoid neogenesis ?
Thaunat O, Kerjaschki D, Nicoletti A

2006 • Trends Immunol • [pdf]

The progressive organization of cellular infiltrates into functional ectopic germinal centers (i.e. lymphoid neogenesis) has been recently evidenced in various chronic inflammatory diseases. Failure of the immune system to eradicate the targeted antigen(s) is a shared feature of all of the pathological situations associated with lymphoid neogenesis. Although necessary, inability of the immune system to eradicate the antigen(s) seems insufficient to trigger lymphoid neogenesis by itself. We propose that both defective lymphatic drainage of the inflamed tissue and enduring local antigenic stimulation are the crucial triggers of the cascade of events leading to lymphoid neogenesis. In turn, ectopic germinal centers prevent the restoration of lymph outflow by diverting inflammation-dependent lymphangiogenesis. Antigens and immune effectors are rerouted towards the neoformed ectopic lymphoid structures. A self-perpetuating feedback loop, which further sustains the development of the local immune response, is now imposed.

The proatherogenic role of T cells requires cell division and is dependent on the stage of the disease
Khallou-Laschet J, Caligiuri G, Groyer E, Tupin E, Gaston A-T, Poirier B, Kronenberg M, Cohen JL, Klatzmann D, Kaveri SV, Nicoletti A

2006 • ATVB • [pdf]

Objective— The mechanism by which T cells exert a proatherogenic potential is unclear. In order to determine whether this potential requires their replication, we crossed atherosclerosis-prone apolipoprotein E knockout mice (ApoE°) with transgenic mice in which exclusive and conditional ablation of dividing T cells relies on their specific expression of the herpes simplex type 1 thymidine kinase (TK) suicide gene.
Methods and Results— We first showed that conalbumin-immunized ApoE°TK mice mounted a significant immune response to the antigen that was fully and specifically blocked by an in vivo ganciclovir (GCV) treatment. Next, ApoE°TK mice and ApoE° mice were treated or not with GCV either during the first 4 weeks (GCV 1 to 4w), the last 4 weeks (GCV 5 to 8w), or during 8 weeks (GCV 1 to 8w). Strikingly, ApoE°TK mice displayed a dramatic decrease in lesion development in the GCV 1 to 8w and GCV 5 to 8w groups, whereas the GCV had no effect when administered during the first 4 weeks. In protected mice, the inflammatory parameters in lesions, the percentage of CD69+CD3+ splenocytes, and the circulating natural killer T cells were reduced.
Conclusions— The present study, therefore, shows that the proatherogenic potential of T cells is crucial in the progression of fatty streaks to mature plaques and requires cell division. Atherosclerosis-prone ApoE°TK mice in which targeted ablation of dividing T cells can be achieved were used to show that the proatherogenic potential of T cells is crucial in the progression from fatty streaks to mature plaques and requires cell division.

Reduced immunoregulatory CD31+ T cells in patients with atherosclerotic abdominal aortic aneurysm
Caligiuri G, Rossignol P, Julia P, Groyer E, Mouradian D, Urbain D, Misra N, Ollivier V, Boutouyrie P, Kaveri SV, Nicoletti A, Lafont A

2006 • ATVB • [pdf]

Background--Cell-mediated immunity is considered to contribute to the pathogenesis of abdominal aortic aneurysms (AAA). In particular, infiltrating macrophages and CD8+ T lymphocytes participate in the destruction of the aortic wall extracellular matrix and smooth muscle cells. We surmise that these pathological events are controlled by circulating regulatory lymphocytes.

Methods and Results--Circulating CD4+/CD31+ cells were reduced in AAA patients (n=80, 8.9±0.6%) as compared with controls (n=69, 13.7±0.8%; P<0.001) and inversely proportional to AAA size. Exclusion of the aneurysm by an endoprothesis did not affect CD31+ T cell values. Reduction of blood CD4+/CD31+ cells was not attributable to their enrichment in AAA tissue. In contrast, CD8+/CD31+ cells were slightly reduced in the blood while increased in the aneurysmal tissue (29.2±0.5 versus 20.2±4.7% in blood, n=6; P<0.05). Remarkably, high percentages of CD4+/CD31+ cells were able to regulate proliferation and cytokine production of CD8+ lymphocytes, as well as CD8+ cell-mediated cytotoxicity of aortic smooth muscle cells (P<0.01). Finally, CD4+/CD31+ cells reduced the production and activity of metalloproteinase-9 by lipopolysaccharide-stimulated macrophages.

Conclusions--Circulating CD4+/CD31+ T cells regulate macrophage and CD8+ T cell activation and effector function in the arterial wall. Their reduction might promote the development of AAA.

Reduced Immunoregulatory CD31+ T Cells in the Blood of Atherosclerotic Mice With Plaque Thrombosis
G Caligiuri, E Groyer, J Khallou-Laschet, A Al Haj Zen, J Sainz, D Urbain, A-T Gaston, M Lemitre, A Nicoletti, A Lafont

2005 • ATVB • [pdf]

Objective—Lymphocyte activation is thought to play a major role in the pathogenesis of atherosclerotic complications such as plaque thrombosis. Circulating CD31+ T cells have been shown to regulate human T cell activation. Aim of this study was to evaluate whether the proportion of circulating immunoregulatory CD31+ T cells is correlated to the occurrence of plaque thrombosis in aged apolipoprotein (apo) E knockout (KO) mice.

Methods and Results—CD31+ T cell depletion of spleen T cells enhanced proliferation (P<0.05) and interferon g-production (P<0.01) while reducing interleukin (IL)-4 (P<0.001) and IL-10 (P<0.001) secretion in response to minimally modified low-density lipoprotein. CD31+ T cells were counted in 65 apoE KO mice (46-week-old) by flow cytometry. Organizing thrombi could be documented in 28 of 195 (14%) lesions and in at least one of the aorta root lesions in 23 of 65 mice (35%). CD31+ T cell count was significantly reduced in mice showing plaque thrombosis (72.3 1.5% versus 84.1 1.2%; P<0.0001), but such reduction did not follow induced plaque rupture or experimentally controlled thrombosis.

Conclusions—Reduced CD31+ T cells in circulating blood is a hallmark of atherosclerotic plaque thrombosis. Our data suggest that CD31+ T cells may play an important regulatory role in the development of plaque thrombosis.

Lymphoid neogenesis in chronic rejection: Evidence for a local humoral alloimmune response
Thaunat O, Field AC, Dai J, Louedec L, Patey N, Bloch MF, Mandet C, Belair MF, Bruneval P, Meilhac O, Bellon B, Joly E, Michel JB, Nicoletti A

2005 • PNAS • [pdf]

Recent advances indicate that, in various chronic inflammatory disorders, the activation of the immune system is triggered locally rather than in lymphoid organs. In this study, we have evaluated whether the humoral alloimmune response involved in chronic rejection is elicited within the graft. We used the rat aortic interposition model and microdissected the adventitia of the graft. Over time, the T cell infiltrate shifted toward a B helper phenotype. B lymphocyte clusters were detected and were the site of intense proliferation and apoptosis. Simultaneously, adventitial vascular endothelium acquired a high endothelial venule phenotype. Similar features were evidenced in the interstitium of chronically allografts (hearts and kidneys). Strikingly, ganocultured graft interstitial tissue was found to be the site of production of antibodies directed against donor MHC-I molecules. These findings, therefore, document the appearance of germinal centers in chronically rejected tissues. This lymphoid neogenesis implies that the graft is not only the target of the alloimmune response but also a site where this response actually develops, so as to optimize the communication between the targeted tissue and the immune effectors.

Role of the intrinsic coagulation pathway in atherogenesis assessed in hemophilic apolipoprotein E knockout mice
J Khallou-Laschet, Caligiuri G, Tupin E, Gaston A-T, Poirier B, Groyer E, Urbain D, Maisnier-Patin S, Sarkar R, Kaveri SV, Lacroix-Desmazes S, Nicoletti A

2005 • ATVB • [pdf]

Objective— The contribution of thrombosis and coagulation in atherogenesis is largely unknown. We investigated the contribution of the coagulation intrinsic factor VIII (FVIII)–dependent pathway in atherogenesis.

Methods and Results— Apolipoprotein E and FVIII double–deficient mice (E°/FVIII°) were generated. Aortic root lesions were analyzed in 14-week-old and 22-week-old female mice maintained for 8 or 16 weeks, respectively, on a normal chow diet or a hypercholesterolemic diet.

Conclusion— Despite a higher plasma total cholesterol concentration compared with E° mice, E°/FVIII° mice developed dramatically less early-stage atherosclerotic lesions. Whereas early lesions in E° mice contained abundant fibrin(ogen) deposits on which few platelets adhered, lesions in E°/FVIII° were almost devoid of fibrin(ogen), and no platelets could be detected. The genotype effect on development and composition of lesions tended to decrease with time. This study demonstrates that the activation of the intrinsic pathway of coagulation is potently proatherogenic at the early stage of atherogenesis.