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AimsIgE type immunoglobulins and their specific effector cells, mast cells (MCs), are associated with abdominal aortic aneurysm (AAA) progression. In parallel, immunoglobulin-producing B cells, organised in tertiary lymphoid organs (TLOs) within the aortic wall, have also been linked to aneurysmal progression. We aimed at investigating the potential role and mechanism linking local MCs, TLO B cells, and IgE production in aneurysmal progression.Methods and ResultsThrough histological assays conducted on human surgical samples from AAA patients, we uncovered that activated MCs were enriched at sites of unhealed haematomas, due to subclinical aortic wall fissuring, in close proximity to adventitial IgE+ TLO B cells. Remarkably,in vitrothe IgEs deriving from these samples enhanced MC production of IL-4, a cytokine which favors IgE class-switching and production by B cells. Finally, the role of MCs in aneurysmal progression was further analysedin vivoin ApoE-/-mice subjected to angiotensin II infusion aneurysm model, through MC-specific depletion after the establishment of dissecting aneurysms. MC-specific depletion improved intramural haematoma healing and reduced aneurysmal progression.ConclusionsOur data suggest that MC located close to aortic wall fissures are activated by adventitial TLO B cell-produced IgEs and participate to their own activation by providing support for further IgE synthesis through IL-4 production. By preventing prompt repair of aortic subclinical fissures, such a runaway MC activation loop could precipitate aneurysmal progression, suggesting that MC-targeting treatments may represent an interesting adjunctive therapy for reducing AAA progression.

BACKGROUND AND AIMS Gemcitabine (GEM) is an anticancer drug, indicated in the treatment of several types of solid cancers such as pancreatic, breast, bladder cancer, etc. This molecule is effective in tumor processes but causes adverse effects on other organs, including the haematopoietic system and the kidney. The renal toxicity of gemcitabine is illustrated by thrombotic microangiopathy (TMA), whose onset is dramatic and associated in more than 97% of cases with acute renal failure. This adverse effect induces discontinuation of treatment with negative consequences on tumor control. It is, therefore, essential to understand the pathophysiological mechanisms of gemcitabine nephrotoxicity. TMA at the glomerular level reflects damage to the endothelial cell (ECs). However, previously, data of our team show that other glomerular cells, parietal epithelial cells (PECs), could be involved in these lesions. Thus, in this project, we want: i) to describe the modifications of the PEC and EC proteome exposed to gemcitabine and ii) to analyse PEC and EC secretome exposed to gemcitabine and to study the cell communication between PECs and ECs. METHOD In our in vitro experiments, we used a murine PEC cell line and a murine EC cell line. The PECs are exposed to 100 μM GEM for 24-h and the culture supernatants are recovered. These PEC supernatants are used in conditioned media experiments to expose ECs for 24-h. On the other hand, ECs were directly exposed to 1 μM GEM for 24-h and culture supernatants were also recovered. Analyses of protein expression are carried out by western blot and immunofluorescence staining. RESULTS When PECs are exposed to GEM (100 µM, 24-h), we observe an increase in apoptosis (caspase 3 cleavage) as well as DNA damage (γH2AX). However, PECs specific markers, such as PAX2 and Claudin-1, do not appear to be affected. Our results also show an increase in the expression of the transcription factor CHOP and the chaperone protein BIP, suggesting the presence of endoplasmic reticulum (ER) stress in PECs exposed to gemcitabine. In order to study cell communication between PECs and ECs, we set up conditioned media experiments. We first studied the expression of different endothelial markers on ECs exposed to PEC supernatants. The expression of the endothelial markers such as E-cadherin, CD31 and PDGFRβ decreases compared with control cells when ECs are exposed to PEC supernatants. Actin cytoskeleton reorganization is also observed with an increase in cortical actin. Moreover, the microtubule network seems to be also affected with a tendency to decrease tubulin expression. Finally, there is an increase in γH2AX suggesting the presence of DNA damage in ECs exposed to PEC supernatant. The next experiments aim at identifying the factors responsible for this cell communication (proteins, lipid mediators, non-coding RNA, extracellular vesicles). CONCLUSION Our results show GEM has a direct effect on PECs (apoptosis, DNA damage, ER stress). In addition, we show the effect of PEC supernatants exposed to GEM on ECs (decrease in endothelial markers, cytoskeleton modifications, DNA damage).

The aim of the study was to evaluate the ability of technetium-99m-fucoidan ([99mTc]fucoidan), a molecular imaging agent specific for selectins, in the assessment of early localized immunity in a rat model of experimental autoimmune myocarditis (EAM). EAM was induced in Lewis rats and troponin T; brain natriuretic peptide (BNP) and anti-myosin antibodies were measured in plasma. Separately, [99mTc]fucoidan single-photon emission computed tomography (SPECT)/x-ray computed tomography (CT) was performed in the very early phase of myocarditis at 10, 15, and 21 days after immunization. Then, hearts were collected and used for autoradiography, well counting, histology, and flow cytometry analysis. The EAM acute phase is characterized by extensive myocardial necrosis, release of troponin and BNP, and pericardial effusion. [99mTc]Fucoidan uptake was significantly increased in EAM compared with controls starting from D15. There was a close relationship between uptake of the tracer and myocardial content in CD45+, CD8+, CD11b+, and CD31+ cells. [99mTc]Fucoidan SPECT/CT accurately diagnosed the autoimmune attack in the early steps of EAM and could be used to monitor disease evolution and therapy efficiency.

Background Intraleaflet hematomas are associated with advanced stages of aortic valve calcification and suspected to be involved in disease progression. However, the mechanism by which the entry of blood cells into the valves affects the biology of aortic valvular interstitial cells (VICs) remains to be elucidated. Objectives This study sought to evaluate the putative link between intraleaflet hematoma and aortic valve calcification and to assess its pathophysiological implications. Methods The spatial relationship between calcium deposits and intraleaflet hematomas was analyzed by whole-mount staining of calcified and noncalcified human aortic valves, obtained in the context of heart transplantation and from patients who underwent surgical valve replacement. Endothelial microfissuring was evaluated by en face immunofluorescence and scanning electron microscopic analyses of the fibrosa surface. Red blood cell (RBC) preparations were used in vitro to assess, by immunofluorescence microscopy and Alizarin red staining, the potential impact of intraleaflet hematomas on phenotypic changes in VICs. Results Intraleaflet hematomas, revealed by iron deposits and RBCs into the fibrosa, secondary to endothelial microfissuring, were consistently found in noncalcified valves. The contact of primary VICs derived from these valves with RBCs resulted in a global inflammatory and osteoblastic phenotype, reflected by the up-regulation of interleukin-6, interleukin-1β, bone sialoprotein, osteoprotegerin, receptor activator of nuclear factor kappa B, bone morphogenic protein 2, and muscle segment homeobox 2, the production of osteocalcin, and the formation of calcium deposits. Conclusions The acquisition of an osteoblastic phenotype in VICs that come into contact with the senescent RBCs of intraleaflet hematomas may play a critical role in the initiation of calcium deposition into the fibrosa of human aortic valves.

Aims Inflammatory mediators, including blood cells and their products, contribute critically to atherogenesis, but the igniting triggers of inflammation remain elusive. Atherosclerosis develops at sites of flow perturbation, where the enhanced haemodynamic stress could initiate the atherogenic inflammatory process due to the occurrence of mechanic injury. We investigated the role of haemodynamic stress-induced breaches, allowing the entry of blood cells in the arterial intima, in triggering inflammation-driven atherogenesis. Methods and results Human coronary samples isolated from explanted hearts, (n = 47) displayed signs of blood entry (detected by the presence of iron, ferritin, and glycophorin A) in the subintimal space (54%) as assessed by histology, immunofluorescence, high resolution episcopic microscopy, and scanning electron microscopy. Computational flow dynamic analysis showed that intimal haemorrhagic events occurred at sites of flow disturbance. Experimental carotid arteries from Apoe deficient mice showed discrete endothelial breaches and intimal haemorrhagic events specifically occurring at the site of flow perturbation, within 3 days after the exacerbation of the local haemodynamic stress. Endothelial tearing was associated with increased VCAM-1 expression and, within 7 days, substantial Ly6G+ leucocytes accumulated at the sites of erythrocyte-derived iron and lipids droplets accumulation, pathological intimal thickening and positive oil red O staining. The formation of fatty streaks at the sites of intimal breaches was prevented by the depletion of Ly6G+ leucocytes, suggesting that the local injury driven by haemodynamic stress-induced breaches triggers atherogenic inflammation. Conclusion Haemodynamic-driven breaches of the arterial intima drive atherogenic inflammation by triggering the recruitment of leucocyte at sites of disturbed arterial flow.

The formation of tertiary lymphoid organs (TLOs) is orchestrated by the stromal cells of tissues chronically submitted to inflammatory stimuli, in order to uphold specific adaptive immune responses. We have recently shown that the smooth muscle cells of the arterial wall orchestrate the formation of the TLOs associated with atherosclerosis in response to the local release of TNF-α. Observational studies have recently documented the presence of structures resembling TLOs the creeping fat that develops in the mesentery of patients with Crohn's disease (CD), an inflammatory condition combining a complex and as yet not elucidated infectious and autoimmune responses. We have performed a comprehensive analysis of the TLO structures in order to decipher the mechanism leading to their formation in the mesentery of CD patients, and assessed the effect of infectious and/or inflammatory inducers on the potential TLO-organizer functions of adipocytes. Quantitative analysis showed that both T and B memory cells, as well as plasma cells, are enriched in the CD-affected mesentery, as compared with tissue from control subjects. Immunohistochemistry revealed that these cells are concentrated within the creeping fat of CD patients, in the vicinity of transmural lesions; that T and B cells are compartmentalized in clearly distinct areas; that they are supplied by post-capillary high endothelial venules and drained by lymphatic vessels indicating that these nodules are fully mature TLOs. Organ culture showed that mesenteric tissue samples from CD patients contained greater amounts of adipocyte-derived chemokines and the use of the conditioned medium from these cultures in functional assays was able to actively recruit T and B lymphocytes. Finally, the production of chemokines involved in TLO formation by 3T3-L1 adipocytes was directly elicited by a combination of TNF-α and LPS in vitro. We therefore propose a mechanism in which mesenteric adipocyte, through their production of key chemokines in response to inflammatory/bacterial stimuli, may orchestrate the formation of functional TLOs developing in CD-affected mesentery.

The aim of the study was to evaluate the ability of technetium-99m-fucoidan ([EAM was induced in Lewis rats and troponin T; brain natriuretic peptide (BNP) and anti-myosin antibodies were measured in plasma. Separately, [The EAM acute phase is characterized by extensive myocardial necrosis, release of troponin and BNP, and pericardial effusion. [[

Background The authors recently found that a CD31 agonist peptide reaches macrophages in injured aortas and exerts beneficial effects on apolipoprotein E-knockout (Apo E−/−) mice subjected to angiotensin (Ang) II infusion, a model of experimental acute aortic dissection and intramural hematoma (ADIM). Objectives The purpose of this study was to evaluate the therapeutic potential of a drug-suitable agonist peptide in experimental ADIM. Methods P8RI, a retro-inverso sequence of the best candidate identified by functional in vitro screening of a peptide library, passed an absorption, distribution, metabolism, excretion and toxicology analysis. Apo E−/− mice (male, 28-week-old) implanted with Ang II-releasing pumps received P8RI (2.5 mg/kg/day) or vehicle from day 14 (n = 10/group). Leukocytes were analyzed by flow cytometry. Healing features of human and mouse dissected aortic segments were assessed by histology and immunofluorescence. The effect of CD31 on macrophages was evaluated using cells from CD31−/− mice and P8RI, in vitro. Results Human and experimental ADIM were characterized by the infiltration of proinflammatory macrophages. The absence of CD31 enhanced the proinflammatory polarization of macrophages, whereas the CD31 agonist P8RI favored reparative macrophages both in vitro and in vivo. The administration of P8RI after the occurrence of ADIM prevented aneurysmal transformation by promoting the resolution of intramural hematoma and the production of collagen in dissected aortas in vivo, associated with enrichment of M2 macrophages at the site of injury. Conclusions CD31 signaling promotes the switching of proinflammatory macrophages to the reparative phenotype and favors the healing of experimental dissected aortas. Treatment with a drug-suitable CD31 agonist may facilitate the clinical management of ADIM.

Background Intraleaflet hematoma has been associated with advanced stages of aortic valve stenosis. Its presence is suspected to be involved in faster disease progression. However, the mechanism whereby the entry and the presence of blood elements into the valves would have an impact on the biology of aortic valvular interstitial cells (VICs) remains to be deciphered. Purpose To evaluate the presence of intraleaflet hematoma, in respect to calcium deposition, at all stages of aortic valve calcification (AC) and to assess its putative pathophysiologic implication. Methods The spatial relationship between calcium deposits and intraleaflet hematomas was analyzed by whole-mount staining of calcified and non-calcified human aortic valves, obtained in the context of heart transplantation and from patients who underwent surgical valve replacement. Endothelial micro-fissuring was evaluated by en face immunofluorescence and scanning electron microscope analyses of the fibrosa surface. Red blood cell preparations (RBCs) were used in vitro in order to assess, by immunofluorescence microscopy and Alizarin red staining, the potential impact of intraleaflet hematomas on the phenotype change of human VICs. Results Intraleaflet hematomas revealed by ferric iron deposits and extensive penetration of RBCs into the fibrosa, secondary to endothelial micro-fissuring, were found both in calcified and non-calcified valves. Primary VICs derived from non-calcified valves and cultured in the presence of senescent RBCs acquired an osteoblastic phenotype, as demonstrated by the production of osteocalcin and the formation of calcium deposits. Conclusions Intraleaflet hematomas occurring through endothelial micro-fissuring may play a critical role in the initiation of calcium deposition into human aortic valves by driving the acquisition of an osteoblastic phenotype by the VICs that enter in contact with the infiltrated RBCs.

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.

Recent evidence suggests that adaptive immunity develops during abdominal aortic aneurysm evolution. Uncertainties remain about the antigens implicated and their role in inducing rupture. Because antigens from the extracellular matrix (ECM) have been suspected, the aim of this experimental study was to characterize the role of adaptive immunity directed against antigens from the aortic ECM.In a first step, an experimental model of abdominal aortic aneurysm rupture based on adaptive immunity against the ECM was developed and characterized. Forty 4-week-old male Lewis rats were divided into two groups. In the ECM group (n = 20), rats were presensitized against the guinea pig aortic ECM before implantation of a decellularized aortic xenograft (DAX). In the control group (n = 20), rats were not presensitized before DAX implantation. In each group, half the rats were sacrificed at day 3 to analyze early mechanisms involved after DAX implantation. In a second step, we aimed to assess which ECM component was most efficient in inducing rupture. For this purpose, the nonfibrillar and fibrillar ECM components were sequentially extracted from the guinea pig aortic wall. Forty Lewis rats were then divided into four groups. Each group was presensitized against one ECM component (structural glycoproteins and proteoglycans, collagen, elastin alone, and elastin-associated glycoproteins) before DAX implantation. Apart from those that experienced rupture, rats were sacrificed at day 21. Xenografts were harvested for histologic, immunofluorescence, and conditioned medium analyses.In total, early aortic rupture occurred in 80% of the ECM group vs 0% of the control group (P .001). In the ECM group, major circumferential immunoglobulin deposits were observed in combination with the C3 complement fraction, without cell infiltration. Conditioned medium analysis revealed that matrix metalloproteinase 9 and myeloperoxidase levels and elastase activities were significantly increased in this group. Immunofluorescence analysis demonstrated that myeloperoxidase co-localized with tissue-free DNA and histone H4, highlighting local neutrophil activation and formation of neutrophil extracellular traps. Following differential presensitization, it appeared that rats presensitized against structural glycoproteins and proteoglycans were significantly more susceptible to rupture after DAX implantation.Stimulating adaptive immunity against the aortic ECM, especially structural glycoproteins and proteoglycans, triggers rupture after DAX implantation. Further studies are needed to assess the precise proteins involved.

Introduction CD31 is a mechanosensory receptor and recent studies in CD31 knockout mice have suggested a protective role for CD31 in condition of altered mechanic stress such as in heart failure. Objective To directly assess the impact of CD31 signaling on the outcome of heart failure (HF) with both reduced and preserved ejection fraction (HFrEF and HFpEF, respectively). Method Permanent ligature of the proximal left coronary in C57Bl6 mice was used for modeling experimental HFrEF. Five days after surgery, mice displaying a myocardial infarction involving > 30% of the left ventricle (LV) at echocardiography were randomized to receive either a CD31 agonist peptide (P8RI, 2.5 mg/kg) or placebo by subcutaneous continuous infusion (osmotic pumps). Low dose angiotensin II infusion was applied to apolipoprotein E knockout (ApoE KO) mice as a model of HEpEF. The osmotic pumps delivered angiotensin II (200 μg/Kg/day) alone or together with P8RI. The Ejection Fraction (EF%) and the E/E’ ratio were evaluated by echocardiography. Results In the HFrEF model, the daily treatment with the CD31 agonist peptide starting from the 5th day after the myocardial infarction, improved both the EF% (68 ± 3% vs. 47 ± 4%, P = 0.05) evaluated 30 days later. Also, the LV filling pressure was improved by the treatment, as reflected by the E/E’ ratio (24.6 ± 2.4% vs. 49.7 ± 7.5, P = 0.05). An even more striking beneficial effect was observed in the model of HEpEF, in which the CD31 agonist treatment prevented the development of the diastolic LV dysfunction induced by the low dose of angiotensin II infusion as determined by the variation of the E/E’ ratio at the end of the treatment period (6 ± 7 in P8RI treated mice vs. 37 ± 8 in controls, P Conclusion CD31 agonist drugs can contribute to improve function of the heart in experimental conditions of myocardial mechanic stress and may represent a novel therapeutic strategy for heart failure with both reduced and preserved ejection fraction.

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 to the control of the T follicular helper–germinal center B-cell axis during atherogenesis. Genetic disruption of CD8 + regulatory T cell function in atherosclerosis-prone apolipoprotein E knockout mice resulted in overactivation of this axis in secondary lymphoid organs, led to the increased development of tertiary lymphoid organs in the aorta, and enhanced disease development. In contrast, restoring control of the T follicular helper–germinal center B-cell axis by blocking the ICOS-ICOSL pathway reduced the development of atherosclerosis and the formation of tertiary lymphoid organs. Moreover, analyses of human atherosclerotic aneurysmal arteries by flow cytometry, gene expression analysis, and immunofluorescence confirmed the presence of T follicular helper cells within tertiary lymphoid organs. Conclusions— This study is the first to demonstrate that the T follicular helper–germinal center B-cell axis is proatherogenic and that CD8 + regulatory T cells control the germinal center reaction in both secondary and tertiary lymphoid organs. Therefore, disrupting this axis represents an innovative therapeutic approach.

International audience; no abstract

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-κB nuclear translocation, expression of costimulatory molecules, and production of immunogenic cytokines (e.g., IL-12, IL-6), whereas the expression of TGF-β 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-γ(+)) 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.

Background Experimental atherosclerosis is characterized by the formation of tertiary lymphoid structures (TLOs) within the adventitial layer, which involves the chemokine-expressing aortic smooth muscle cells (SMCs). TLOs have also been described around human atherothrombotic arteries but the mechanisms of their formation remain poorly investigated. Herein, we tested whether human vascular SMCs play the role of chemokine-expressing cells that would trigger the formation of TLOs in atherothrombotic arteries. Results We first characterized, by flow cytometry and immunofluorescence analysis, the prevalence and cell composition of TLOs in human abdominal aneurysms of the aorta (AAAs), an evolutive form of atherothrombosis. Chemotaxis experiments revealed that the conditioned medium from AAA tissues recruited significantly more B and T lymphocytes than the conditioned medium from control (N-AAA) tissues. This was associated with an increase in the concentration of CXCL13, CXCL16, CCL19, CCL20, and CCL21 chemokines in the conditioned medium from AAA tissues. Immunofluorescence analysis of AAA cryosections revealed that α-SMA-positive SMCs were the main contributors to the chemokine production. These results were confirmed by RT-qPCR assays where we found that primary vascular SMCs from AAA tissues expressed significantly more chemokines than SMCs from N-AAA. Finally, in vitro experiments demonstrated that the inflammatory cytokines found to be increased in the conditioned medium from AAA were able to trigger the production of chemokines by primary SMCs. Conclusion Together, these results suggest that human vascular SMCs in atherothrombotic arteries, in response to inflammatory signals, are converted into chemokine-expressing cells that trigger the recruitment of immune cells and the formation of aortic TLOs.

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 (TLOs). Methods and results Mouse bone marrow-derived M1 macrophages acted as lymphoid tissue inducer cells. Indeed, they expressed high levels of tumour necrosis factor (TNF)-α and membrane-bound lymphotoxin (LT)-α, two inducing cytokines that triggered expression of the chemokines CCL19, CCL20, and CXCL16, as did M1 supernatant. The blockade of LTβR signalling with LTβR-Ig had no effect, whereas that of TNFR1/2 signalling reduced chemokine expression by VSMCs in both wild-type (WT) and LTβR KO mice, demonstrating that LTβR signalling is dispensable for the M1-inducing effect. This effect was corroborated by the development of TLOs observed in LTβR KO->apolipoprotein E knockout (ApoE KO) aortic segments after orthotopic transplantation. Furthermore, treatment of ApoE KO mice with anti-TNF-α 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 LTβR signalling. We propose that the dialogue between macrophages and VSMCs—established across the vascular wall—contributes to the formation of aortic TLOs.

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.

International audience; 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 by 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, sc) 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 leucocyte 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 intended to prevent the inflammatory cell processes, such as those underlying progression of atherosclerosis and development of AAA.

Aims The presence of intravalve hemorrhage close to calcium crystals has recently been described in calcified aortic valves histologic slides. Those hemorrhages can play a pathogenic and initiating role in calcification process but they could also be a mere consequence of the mechanical damage due to calcifications. We aimed at assessing whether iron deposits are present both in calcified valves and non calcified valves. Methods and results Aortic valves deriving from patients operated at the Bichat Hospital were consecutively (February-April 2014) included in our study (Table 1). We have developed a morphologic analysis technique in 3D whole mounts, using Perls’ blue staining for ferric iron and Alizarin red staining for calcium crystals, in situ. The valves are optically claryfied using benzyl benzoate prior to analysis. Results Ferric iron staining was positive in 18/20 (90%) non-calcified valves. Ferric iron deposits were present in 93% of calcified valves (78% in the group « severe aortic stenosis » and 100% in the group « moderate aortic stenosis »). Ferric iron deposits displayed two types of pattern: either in « close contact » with calcium crystals (74%), or located within the healthy tissue, « distant » from the calcium crystals (42,5%). Both types of pattern were concomitanly present in 30% of all calcified valves. Conclusion Our study shows that ferric iron deposits can be found in non-calcified valves, suggesting that hemorrhage are not necessarily a consequence of valve calcification. The iron deposits that are not spatially associated with calcium crystals may precede the development of calcifications, possibly playing an active role. Abstract 0428 – Table: Baseline characteristics Type of procedure Nb (men) Mean age, years Mean gradient, mmHg Macroscopically calcified valves Aortic valve replacement 45 (22) 72,5 46,7 45 Severe aortic stenosis 40 (18) 73 49,5 40 Moderate aortic stenosis 5(4) 71,5 25 5 Bentall procedure 16 (14) 53 – 2 Explanted heart 4 (4) 62 – 0

Tertiary lymphoid organs (TLOs), composed of B cell follicles, develop in the adventitia of atherosclerotic arteries. However, it is not known whether TLOs play an effective role in atherogenesis. In order to address this issue it is necessary to modulate the formation/function of these ectopic B cell follicles within the adventitia. Recent studies have shown that the function of germinal center B cells in secondary lymphoid organs (SLO) is tightly controlled by the Qa-1-restricted CD8+ T cells (CD8+ Treg) which regulate T follicular helper (TfH) cells function. We therefore evaluated whether regulation of TfH by CD8+ Treg can control the development of TLOs and, in turn, of atherogenesis in hypercholesterolemic (ApoE°) mice. We firstly found that the frequency of CD8+ Treg in ApoE° mice significantly decreases with age in the spleen and peripheral lymph nodes of this model. We therefore crossed ApoE° mice with the D227K-Tg mice in which, due to a Qa-1 point mutation, the CD8+ Treg are unable to control the development/function of TfH cells. ApoE° were compared with ApoE°/D227K-Tg littermates (n=5-12/group) at 8, 15 and 35 weeks of age. Qa-1 mutation increased the TfH cell population in SLO of ApoE°/D227K-Tg as compared to ApoE° mice. This effect was accompanied by an increase in the percentage of activated/memory CD4+ and CD8+ T cells and of B cells in lymph nodes of ApoE°/D227K-Tg, at 15 and 35 weeks of age. In parallel, lesion development was accelerated: atherosclerotic plaques in the aortic root of ApoE°/D227K-Tg mice were 2-fold greater than those of ApoE° mice. Immunofluorescent analysis of adventitial whole mounts revealed a greater incidence of TLOs (organized B cell follicles) in ApoE°/D227K-Tg than in ApoE° mice. Our data therefore suggest that CD8+ Treg control the development of adventitial pro-atherogenic TLOs in ApoE° mice through their regulatory action on TfH cells.

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

Myocarditis, an inflammatory disease of the myocardium, is a major cause of death and dilated cardiomyopathy. However its work-up is not standardized due to the lack of reliable marker of progression. The disease is characterized by clusters of lymphoid and myeloid cell infiltrates, and endothelial activation which hallmark is the expression of adhesion molecules such as selectins. The aim of the study was to evaluate the ability of 99mTc-fucoidan, an imaging agent specific of selectins, in the assessment of inflammation in a model of experimental autoimmune myocarditis (EAM).MethodsEAM has been induced in male Lewis rats with purified rat cardiomyosin and compared with controls. The experiments were performed in the very early phase of myocarditis (10, 15 and 21 days after immunization) and included (1) quantification of 99mTc-fucoidan myocardial/lung ratio using SPECT/CT; (2) qualitatively: comparative analysis on adjacent slices of tracer uptake on autoradiography and selectins on immuno-histochemistry (CD62p, CD62e); and (3) quantitatively: relationship between 99mTc-fucoidan uptake by well counting (expressed as %ID/g of myocardium) and myocardial content of inflammatory cells (CD45; CD11b) using flow cytometry.Resultsmyocardial uptake of 99mTc-fucoidan was significantly increased in EAM compared to controls (SPECT uptake ratio: 1.6±0.1 vs. 1.2±0.4, p=0.04 at D10; 2.3±0.2 vs. 1.3±0.8, p