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Objective: Many early-onset epilepsies present as developmental and epileptic encephalopathy associated with refractory seizures, altered psychomotor development, and disorganized interictal cortical activity. Abnormal upregulation of specific N-methyl-d-aspartate receptor (NMDA-R) subunits is being disentangled as one of the mechanisms of severe early-onset epilepsies. In tuberous sclerosis complex (TSC), upregulation of the GluN2C subunit of the NMDA-R with slow deactivation kinetic results in increased neuronal excitation and synchronization., Methods: Starting from an available GluN2C/D antagonist, NMDA-R-modulating compounds were developed and screened using a patch clamp on neuronal culture to select those with the strongest inhibitory effect on glutamatergic NMDA currents. For these selected compounds, blood pharmacokinetics and passage through the blood-brain barrier were studied. We tested the effect of the most promising compounds on epileptic activity in Tsc1 +/- mice brain slices with multielectrode array, and then in vivo at postnatal ages P14-P17, comparable with the usual age at epilepsy onset in human TSC., Results: Using a double-electrode voltage clamp on isolated NMDA currents, we identified the most prominent antagonists of the GluN2C subunit with no effect on GluN2A as a means of preventing side effects. The best compound passing through the blood-brain barrier was selected. Applied in vivo in six Tsc1 +/- mice at P14-P17, this compound reduced or completely stopped spontaneous seizures in four of them, and decreased the background activity disorganization. Furthermore, ictal-like discharges stopped on a human brain sample from an infant with epilepsy due to TSC., Interpretation: Subunit-selective inhibition is a valuable target for developing drugs for severe epilepsies resulting from an upregulation of NMDA-R subunit-mediated transmission., (© 2022 International League Against Epilepsy.)

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., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2018. For permissions, please email: journals.permissions@oup.com.)

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., (Copyright © 2019 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Objective: We aimed to assess a cohort of young patients with Dravet syndrome (DS) for intellectual disability (ID) and autism spectrum disorder (ASD) using standardized tools and parental questionnaires to delineate their specific profiles., Methods: We included 35 patients with DS aged 24 months to 7 years, excluding patients with a developmental age (DA) <18 months (n = 5). We performed specific tests adapted for ID (Psychoeducational Profile, Third Edition [PEP-3]), in addition to the Child Development Inventory (CDI) and Vineland Adaptive Behavior Scales, Second Edition (VABS-II) questionnaires. We used 2 standardized tools for ASD: the Autism Diagnostic Observation Schedule, Second Edition (ADOS-2) and the Autism Diagnostic Interview-Revised (ADI-R). We compared the with parental questionnaires and the VABS-II, and with ASD characteristics., Results: PEP-3 subscales showed pathologic development in all but one patient (97%): ID in 23 of 30 (77%), and borderline cognitive functioning in 6 of 30 (22%). Eleven patients (39%) had ASD and 2 (7%) had a Social Communication Disorder (SCD) diagnosis. We found no difference between PEP-3 and CDI categorization except for fine motor skills. We found significant negative correlations between ADOS-2 and PEP-3 for the majority of scores. For patients aged older than 50 months, 2 groups emerged (ASD/no ASD) with significant difference in DA. The logistic regression for ASD diagnosis explained by VABS-II showed a significant effect for Socialization, Motor Skills, and Adaptive Behavior., Significance: We found a high prevalence of ID in patients with DS. ID is characterized by expressive and comprehensive communication deficits in addition to visuospatial difficulties. ASD showed a specific profile with a relative preservation of social skills, emphasizing a possible underdiagnosis. Parental questionnaires can provide a good assessment of cognitive profile and might allow the difficulty of addressing cognitive scales in DS to be overcome. The profile of ID and ASD should help to establish early adapted rehabilitation programs and emphasizes the global need for care beyond seizures in DS and other developmental epileptic encephalopathies., Competing Interests: None of the authors has any conflict of interest to disclose. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

Background: CD31 is a key transmembrane neutrophil immunoregulatory receptor. Mesenteric ischemia/reperfusion-induced neutrophil activation leads to a massive cleavage and shedding of the most extracellular domains of CD31 into plasma, enhancing the deleterious effect of neutrophil activation. We have evaluated the preventive therapeutic potential of an engineered synthetic octapeptide (P8RI), which restores the inhibitory intracellular signaling of cleaved CD31, in an experimental model of acute mesenteric ischemia/reperfusion., Methods: In a randomized, controlled, and experimenter-blinded preclinical study, mesenteric ischemia/reperfusion (I/R) was induced in Wistar rats by superior mesenteric artery occlusion for 30 min followed by 4 h of reperfusion. Three groups of rats were compared: I/R + saline perfusion (I/R controls group, n = 7), I/R + preventive P8RI perfusion (P8RI group, n = 7), and sham-operated rats + saline perfusion (sham group, n = 7)., Results: Compared with I/R controls, P8RI perfusion significantly decreased intestinal ischemia/reperfusion injury (Chiu's score, P = 0.01; epithelial area, P = 0.001) and bacterial translocation (plasma Escherichia coli DNA, P = 0.04) and could limit intestinal bleeding (P = 0.09). P8RI decreased neutrophil activation assessed by matrix metalloproteinase-9 release in plasma (P < 0.001) and in the intestinal wall, albeit without statistical significance (P = 0.06 and P = 0.058 for myeloperoxydase). Inhibition of CD31 cleavage from neutrophils could play a major role in the protective effects of P8RI (P < 0.0001)., Conclusions: Preventive administration of P8RI, a CD31-agonist peptide, could decrease I/R-induced intestinal injury by potentially limiting neutrophil activation.

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., (Copyright © 2018 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Objective: Experimental studies suggest that maternal hypercholesterolemia may be relevant for the early onset of cardiovascular disease in offspring. We investigated the effect of perinatal hypercholesterolemia on the atherosclerosis development in the offspring of apolipoprotein E-deficient mice and the underlying mechanism., Approach and Results: Atherosclerosis and related parameters were studied in adult male or female apolipoprotein E-deficient mice offspring from either normocholesterolemic or hypercholesterolemic mothers and normocholesterolemic fathers. Female born to hypercholesterolemic mothers had more aortic root lesions than female born to normocholesterolemic mothers. Lesions in whole aorta did not differ between groups. Higher trimethylamine-N-oxide levels and Fmo3 hepatic gene expression were higher in female born to hypercholesterolemic mothers offspring compared with female born to normocholesterolemic mothers and male. Trimethylamine-N-oxide levels were correlated with the size of atherosclerotic root lesions. Levels of hepatic cholesterol and gallbladder bile acid were greater in male born to hypercholesterolemic mothers compared with male born to normocholesterolemic mothers. At 18 weeks of age, female born to hypercholesterolemic mothers showed lower hepatic Scarb1 and Cyp7a1 but higher Nr1h4 gene expression compared with female born to normocholesterolemic mothers. Male born to hypercholesterolemic mothers showed an increase in Scarb1 and Ldlr gene expression compared with male born to normocholesterolemic mothers. At 25 weeks of age, female born to hypercholesterolemic mothers had lower Cyp7a1 gene expression compared with female born to normocholesterolemic mothers. DNA methylation of Fmo3, Scarb1 , and Ldlr promoter regions was slightly modified and may explain the mRNA expression modulation., Conclusions: Our findings suggest that maternal hypercholesterolemia may exacerbate the development of atherosclerosis in female offspring by affecting metabolism of trimethylamine-N-oxide and bile acids. These data could be explained by epigenetic alterations., (© 2017 American Heart Association, Inc.)

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

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., (© 2014 American Heart Association, Inc.)

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.

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.

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.

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.

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.

We have shown that the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is the kinase involved in the endogenous phosphorylation of the alpha1 subunit of the gamma-aminobutyric acid (GABA)(A) receptor (GABA(A)R), maintaining GABA(A)-R function. GABA(A)R endogenous phosphorylation is opposed by one or several atypical phosphatases. We have shown in addition, using cerebral tissue obtained during epilepsy surgery and control tissue from patients undergoing brain tumor surgery, that both endogenous phosphorylation and GABA(A)R function are significantly reduced in the "epileptogenic" cerebral cortex when compared to control. This dysfunction likely contributes to seizure generation and/or transition from the interictal to the ictal state. The therapeutic challenge is to alleviate the endogenous phosphorylation deficiency of GABA(A)R in the epileptogenic cortical tissue, either through activating the endogenous kinase activity, or inhibiting dephosphorylation of the alpha1 subunit. Following the first trail, we have shown that spermine (the most effective polyamine) increases the GAPDH kinase activity on GABA(A)R and that subsequently such modulation potentiates its function as assessed by rundown studies on isolated neurons. Following the second trail, we have developed methods to identify these atypical membrane-bound phosphatases. Their activities were detected using two synthetic phosphopeptides corresponding to the alpha1 regions of phosphorylation by GAPDH. After purification, the active fractions are submitted to proteomic analysis by nanoLC-Maldi-TOF/TOF for protein identification. Two candidate proteins have been identified, which will be used as targets for high-throughput screening in order to develop original antiepileptic molecules.

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.

The function of the gamma-aminobutyric acid type A receptor (GABA(A)R) is maintained by endogenous phosphorylation. We have shown that the corresponding kinase is the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), using the locally produced glycolytic ATP. In addition, using cerebral tissue obtained during curative surgery for epilepsy, we showed that both the endogenous phosphorylation and the GABA(A)R function are significantly reduced in the "epileptogenic" cerebral cortex when compared to "control" tissue. This dysfunction likely contributes to seizure generation and/or transition from the interictal to the ictal state. Glucose utilization is decreased in the epileptogenic cortex of patients with partial epilepsy in the interictal state, but the relationship to the disorder remains unclear. We propose that this hypometabolism is related to the deficiency in the endogenous phosphorylation of GABA(A)R and the resulting greater lability of GABAergic inhibition. Several lines of evidences indeed suggest that GABAergic inhibition is costly in terms of metabolic consumption. The deficiency of this glycolysis-dependent mechanism may thus link epileptogenicity to glucose hypometabolism. The antiepileptic effect of ketogenic diets may be mediated by the subsequent rise in the NADH/NAD(+) index, which favors GABA(A)R endogenous phosphorylation and should contribute to restoration of GABAergic inhibition in the epileptogenic zone.

The immuno-inflammatory response is central to the development of atherosclerosis. The important players of the adaptive immune system are all involved in this pathologic process. Several antigens have been identified these last years and they are mostly self-molecules that have been modified due to the complex microenvironment that is generated within the diseased artery. Pro-atherogenic autoreactive T cells have been characterized. The presence of auto-reactive natural antibodies has also been confirmed in the lesions. All these, together with the data showing that adoptive transfer of lymphocytes is able to modulate the disease, fulfill the criteria put forth by Witebsky and Rose to define a disease as being autoimmune. However, the complexity of the disease process extends to the immune system. Although T cells are known to be pro-atherogenic, B cells have been clearly shown to be athero-protective. The fine balance between the two extensions of the adaptive immune response is the key to a successful therapeutic approach towards atherosclerosis.

Background: Macrophages, key regulators of healing/regeneration processes, strongly infiltrate ischemic tissues from patients suffering from critical limb ischemia (CLI). However pro-inflammatory markers correlate with disease progression and risk of amputation, suggesting that modulating macrophage activation state might be beneficial. We previously reported that thrombospondin-1 (TSP-1) is highly expressed in ischemic tissues during CLI in humans. TSP-1 is a matricellular protein that displays well-known angiostatic properties in cancer, and regulates inflammation in vivo and macrophages properties in vitro. We therefore sought to investigate its function in a mouse model of CLI., Methods and Findings: Using a genetic model of tsp-1(-/-) mice subjected to femoral artery excision, we report that tsp-1(-/-) mice were clinically and histologically protected from necrosis compared to controls. Tissue protection was associated with increased postischemic angiogenesis and muscle regeneration. We next showed that macrophages present in ischemic tissues exhibited distinct phenotypes in tsp-1(-/-) and wt mice. A strong reduction of necrotic myofibers phagocytosis was observed in tsp-1(-/-) mice. We next demonstrated that phagocytosis of muscle cell debris is a potent pro-inflammatory signal for macrophages in vitro. Consistently with these findings, macrophages that infiltrated ischemic tissues exhibited a reduced postischemic pro-inflammatory activation state in tsp-1(-/-) mice, characterized by a reduced Ly-6C expression and a less pro-inflammatory cytokine expression profile. Finally, we showed that monocyte depletion reversed clinical and histological protection from necrosis observed in tsp-1(-/-) mice, thereby demonstrating that macrophages mediated tissue protection in these mice., Conclusion: This study defines targeting postischemic macrophage activation state as a new potential therapeutic approach to protect tissues from necrosis and promote tissue repair during CLI. Furthermore, our data suggest that phagocytosis plays a crucial role in promoting a deleterious intra-tissular pro-inflammatory macrophage activation state during critical injuries. Finally, our results describe TSP-1 as a new relevant physiological target during critical leg ischemia.

Background: Recent studies clearly suggest that regulatory T cells play a critical role in the control of the immunoinflammatory response in atherosclerosis and substantially limit lesion development. Measles virus infection or vaccination is associated with immune depression, in part through the induction of an antiinflammatory response by measles virus nucleoprotein. We hypothesized that the antiinflammatory properties of measles virus nucleoprotein may limit the development atherosclerosis., Methods and Results: Here, we show for the first time that repetitive administration of measles virus nucleoprotein to apolipoprotein E-deficient mice promotes an antiinflammatory T-regulatory-cell type 1-like response and inhibits macrophage and T-cell accumulation within the lesions. Treatment with measles virus nucleoprotein significantly reduces the development of new atherosclerotic plaques and markedly inhibits the progression of established lesions. The antiatherosclerotic potential of nucleoprotein is retained in its short N-terminal segment. The protective effects on lesion size are lost in mice with lymphocyte deficiency., Conclusions: Our findings identify a novel mechanism of immune modulation by measles virus nucleoprotein through the promotion of a regulatory T-cell response and suggest that this property may be harnessed for treating atherosclerosis, the first cause of heart disease and stroke.

Objectives: The present study evaluated the effect of phosphorylcholine (PC) immunization on the extent of experimental atherosclerosis., Background: Immunization against oxidized lipoprotein (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 (p < 0.01). 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 (p < 0.01). Immunohistochemistry showed reduced expression of major histocompatibility complex class II antigens (p < 0.05) 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: Phosphorylcholine immunization drives a specific humoral immune response that reduces foam cell formation in vitro and is atheroprotective in vivo.

Objectives: This study was designed to evaluate whether replacing CD31 (PECAM-1) signaling can restore the regulation of lymphocyte activation and improve experimental atherosclerosis., Background: Atherosclerosis, the principal cause of myocardial infarction and stroke, 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) was delivered by in vivo gene transfer in 6-week-old apolipoprotein E knockout mice (n = 14 per group) every 5 weeks for 6 months. Control groups were treated with a truncated CD31Rg or with vehicle alone. At the end of the study, plaque size and morphology and blood T-cell compartment were analyzed in all mice., Results: Atherosclerotic lesions of CD31Rg-treated mice were smaller (p < 0.01) and showed less neovascularization and intraplaque hemorrhage (p < 0.05) compared with control subjects. Furthermore, circulating regulatory T-cells were increased in vivo (p < 0.01) and showed normal suppressive function on proliferation of conventional T-cells in vitro. Indeed, CD31Rg treatment led to blunted blood T-cell activation (p < 0.05) and reduced T-cell infiltration within plaques (p < 0.01)., Conclusions: Our data suggest that 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.

A reduction in GABAergic neurotransmission has been put forward as a pathophysiological mechanism for human epilepsy. However, in slices of human epileptogenic neocortex, GABAergic inhibition can be clearly demonstrated. In this article we present data showing an increase in the functional lability of GABAergic inhibition in epileptogenic tissue compared with nonepileptogenic human tissue. We have previously shown that the glycolytic enzyme GAPDH is the kinase involved in the glycolysis-dependent endogenous phosphorylation of the alpha1-subunit of GABA(A) receptor, a mechanism necessary for maintaining GABA(A) function. In human epileptogenic cortex obtained during curative surgery of patients with partial seizures, we demonstrate an intrinsic deficiency of GABA(A) receptor endogenous phosphorylation resulting in an increased lability of GABAergic currents in neurons isolated from this tissue when compared with neurons from nonepileptogenic human tissue. This feature was not related to a reduction in the number of GABA(A) receptor alpha1-subunits in the epileptogenic tissue as measured by [(3)H]flunitrazepam photoaffinity labeling. Maintaining the receptor in a phosphorylated state either by favoring the endogenous phosphorylation or by inhibiting a membrane-associated phosphatase is needed to sustain GABA(A) receptor responses in epileptogenic cortex. The increased functional lability induced by the deficiency in phosphorylation can account for transient GABAergic disinhibition favoring seizure initiation and propagation. These findings imply new therapeutic approaches and suggest a functional link to the regional cerebral glucose hypometabolism observed in patients with partial epilepsy, because the dysfunctional GABAergic mechanism depends on the locally produced glycolytic ATP.

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 degrees) 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 degrees 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 degrees TK mice and ApoE degrees 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 degrees 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.

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-gamma 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.