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.chronic rejection ͉ transplantation ͉ B cells ͉ germinal centers ͉ adventitia D espite recent advances in transplantation, the long-term outcome of transplanted organs remains impeded by chronic rejection (1). Accumulating evidence suggests that humoral immunity (2-4) and, particularly, alloantibodies directed against donor MHC I molecules (5-8) are critical in the pathogenesis of chronic vascular rejection.Clinically, chronic rejection is responsible for a slow deterioration of graft function, which correlates with typical histological changes. Excluding organ-specific manifestations, the most common histopathological feature is chronic vascular rejection, also known as allograft arteriosclerosis, characterized by widespread and diffuse narrowing of the vascular lumen as a result of intimal proliferation of smooth muscle cells and fibroblasts and destruction of smooth muscle cells from the media (9-12). Chronic vascular rejection is also typified by an abundant adventitial inflammatory infiltrate (9). We therefore evaluated whether the humoral alloimmune response was elicited within the adventitia of the graft. In such case, chronic vascular rejection would be similar to other chronic inflammatory disorders in which tissue destruction results from a vicious circle maintained by an uncontrolled local immune response.We demonstrate the involvement of intragraft lymphoid neogenesis in the development of chronic rejection in an animal model, based on aortic transplantation between histoincompatible strains of rats (13-16). We show that the adventitial inflammatory infiltrate harbors a secondary lymphoid organ structure and that anti-donor MHC I antibodies are produced within these structures. Additionally, we provide insights into the clinical relevance of these observations because similar lymphoid str...

Background-We hypothesized that normal and pathological vessel walls display a differential pattern of secreted proteins. We have recently set up the conditions for comparing secretomes from carotid atherosclerotic plaques and control arteries using a proteomic approach to assess whether differentially secreted proteins could represent markers for atherosclerosis. Methods and Results-Normal endartery segments and different regions of endarterectomy pieces (noncomplicated/ complicated plaques) were incubated in protein-free medium, and the released proteins were analyzed by 2D electrophoresis (2-DE). Among the differently secreted proteins, we have identified heat shock protein-27 (HSP27 Beyond the classic risk factors (dyslipidemias, diabetes, and hypertension), humoral markers of plaque vulnerability related primarily to inflammation (eg, high-sensitivity C-reactive protein, interleukin-6, -10, and -18, CD40L) or reflecting pathological vascular remodeling (eg, immune activation, apoptosis, extracellular matrix degradation) have recently been highlighted. 1 Emerging noninvasive imaging techniques for assessment of subclinical atherosclerosis permit measurement of intima-media thickness or peripheral flow-mediated dilatation, which are inversely correlated with coronary artery diseases. 2 Despite these achievements, intermediate phenotypes between risk factors and clinical complications are needed to target vulnerable patients. 3 We hypothesized that the patterns of protein secretion are different between atherosclerotic plaques and normal endarteries. Whereas the existing markers were found by monitoring the variations of a candidate protein related to the pathology, our strategy is to compare the secretome from normal and pathological arteries using a differential proteomic approach to identify new biological markers potentially released by the arterial wall within the plasma. 4 The incubation of complicated and noncomplicated endarterectomy samples or control endarteries in a serum-free culture medium allowed us to harvest separately the proteins released from lesioned and healthy areas. Two-dimensional electrophoresis (2-DE) enabled us to analyze these secretomes globally and to identify, among the differentially secreted proteins, heat shock protein 27 (HSP27) as a potential marker of atherosclerosis. Confirming these results, plasma HSP27 was markedly decreased in atherosclerotic patients relative to healthy subjects. Methods Tissue SamplingTwenty-eight patients (carotid stenosis Ͼ70%, 21 men/7 women; age, 68Ϯ9 years; 86% hypertensive, 39% diabetic, 54% hyperlipidemic) undergoing carotid endarterectomy at our institutions were included. Informed consent was obtained before enrollment. Blood samples were collected from these patients the day of endarterecto- Tissue CultureCarotid endarterectomy samples were dissected as described previously, 4 separating the stenosing complicated zone (origin of the internal carotid artery) from the adjacent plaque (common and external carotid endartery). Histological ana...

BackgroundAbdominal 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 FindingsOur 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.ConclusionsThus, the control of periodontal disease may represent a therapeutic target to limit human AAA progression.

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

Abstract-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. Physiologically, soluble vascular mediators are centrifugally conveyed by mass transport toward the adventitia. Moreover, in pathological conditions, neomediators and antigens can be generated within the arterial wall, whose outward conveyance triggers different patterns of local adventitial response. Adventitial angiogenesis, immunoinflammation, and fibrosis sequentially interact and their net balance defines the participation of the adventitial response in arterial pathology. In the present review we discuss 4 pathological entities in which the adventitial response to arterial wall injury participates in arterial wall remodeling. Hence, the adventitial adaptive immune response predominates in chronic rejection. Inflammatory phagocytic cell recruitment and initiation of a shift from innate to adaptive immunity characterize the adventitial response to products of proteolysis in abdominal aortic aneurysm. Adventitial sprouting of neovessels, leading to intraplaque hemorrhages, predominates in atherothrombosis. Adventitial fibrosis characterizes the response to mechanical stress and is responsible for the constrictive remodeling of arterial segments and initiating interstitial fibrosis in perivascular tissues. These adventitial events, therefore, have an impact not only on the vessel wall biology but also on the surrounding tissue.

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AimsNeutrophils/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 resultsConditioned media from luminal, intermediate, and abluminal layers of 29 human ILTs were analysed for neutrophil markers [elastase/α1-antitrypsin and MMP9/NGAL complexes, myeloperoxidase (MPO), and α-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.ConclusionTaken 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.

Oxidized low density lipoproteins (oxLDL) are thought to play a major role in atherosclerosis. OxLDL exhibit a wide variety of biological effects resulting from their ability to interfere with intracellular signaling. The cellular targets and primary signaling events of oxLDL are unknown. We report that oxLDL elicit, in intact cells, tyrosine phosphorylation of the epithelial growth factor receptor (EGFR) and activation of its signaling pathway. This activation triggered by oxLDL was associated with derivatization of reactive amino groups of EGFR and was mimicked by 4-hydroxynonenal (4-HNE, a major lipid peroxidation product of oxLDL). Immunopurified EGFR was derivatized and activated in vitro by oxLDL lipid extracts and 4-HNE, thus indicating that 1) EGFR may be a primary target of oxidized lipids and 2) EGFR derivatization may be associated with activation. The reported data suggest that EGFR acts as a sensor for oxidized lipids. We therefore propose a novel concept of the mechanism by which oxidized lipids (contained in oxLDL or more generally produced during oxidative stress) are able to activate receptor tyrosine kinase and subsequent signaling pathways, resulting finally in a gain of function.