Ex vivo expansion of regulatory T cells from abdominal aortic aneurysm patients inhibits aneurysm in humanized murine model.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is a chronic inflammatory disease. Studies of human aneurysm tissue demonstrate dense inflammatory cell infiltrates with CD4+ T cells predominating. Regulatory T cells (Tregs) play an important role in inhibiting pro-inflammatory T cell proliferation, therefore, limiting collateral tissue destruction. The aim of this study was to investigate whether ex vivo augmentation of human Tregs attenuates aneurysm formation in humanized murine model of AAA. METHODS: Circulating Treg population in AAA patients and age- and gender-matched controls were determined by real-time polymerase chain reaction and flow cytometry. To create humanized murine model of AAA, irradiated Rag1-deficient (Rag1-/-) mice, without mature T lymphocytes, at 7 weeks of age were given 5 × 106 of human CD4+ T cells intraperitoneally. Then the mice underwent CaCl2 aneurysm induction. Aortic diameters were measured before and at 6 weeks after aneurysm induction. Aortic tissue was collected for histology and protein extraction. Verhoeff-Van Gieson stain was used for staining elastic fiber. CD4+ T cells in the aortic tissue were detected by immunohistochemical staining. RESULTS: In human peripheral blood mononuclear cells, the proportion of Tregs are decreased in AAA patients compared with matched control patients with significant vascular disease. We first validated the role of Tregs in the CaCl2 model of AAA. To determine the role of human T cells in AAA formation, Rag1-/- mice, resistant to CaCl2-aneurysm induction, were transplanted with human CD4+ T cells. Human CD4+ T cells were able to drive aneurysm formation in Rag1-/- mice. We show that ex vivo augmentation of human Tregs by interleukin-2 resulted in decreased aneurysm progression. CONCLUSIONS: These data suggest that the ex vivo expansion of human Tregs may be a potential therapeutic strategy for inhibiting progression of AAA.

IL-1ß (Interleukin-1ß) and TNF-α (Tumor Necrosis Factor-α) Impact Abdominal Aortic Aneurysm Formation by Differential Effects on Macrophage Polarization.

OBJECTIVE: Abdominal aortic aneurysms are inflammatory in nature and are associated with some risk factors that also lead to atherosclerotic occlusive disease, most notably smoking. The purpose of our study was to identify differential cytokine expression in patients with abdominal aortic aneurysm and those with atherosclerotic occlusive disease. Based on this analysis, we further explored and compared the mechanism of action of IL (interleukin)-1ß versus TNF-α (tumor necrosis factor-α) in abdominal aortic aneurysm formation. APPROACH AND RESULTS: IL-1ß was differentially expressed in human plasma with lower levels detected in patients with abdominal aortic aneurysm compared with matched atherosclerotic controls. We further explored its mechanism of action using a murine model and cell culture. Genetic deletion of IL-1ß and IL-1R did not inhibit aneurysm formation or decrease MMP (matrix metalloproteinase) expression. The effects of IL-1ß deletion on M1 macrophage polarization were compared with another proinflammatory cytokine, TNF-α. Bone marrow-derived macrophages from IL-1ß-/- and TNF-α-/- mice were polarized to an M1 phenotype. TNF-α deletion, but not IL-1ß deletion, inhibited M1 macrophage polarization. Infusion of M1 polarized TNF-α-/- macrophages inhibited aortic diameter growth; no inhibitory effect was seen in mice infused with M1 polarized IL-1ß-/- macrophages. CONCLUSIONS: Although IL-1ß is a proinflammatory cytokine, its effects on aneurysm formation and macrophage polarization differ from TNF-α. The differential effects of IL-1ß and TNF-α inhibition are related to M1/M2 macrophage polarization and this may account for the differences in clinical efficacy of IL-1ß and TNF-α antibody therapies in management of inflammatory diseases.

Elastin-Derived Peptides Promote Abdominal Aortic Aneurysm Formation by Modulating M1/M2 Macrophage Polarization.

Abdominal aortic aneurysm is a dynamic vascular disease characterized by inflammatory cell invasion and extracellular matrix degradation. Damage to elastin in the extracellular matrix results in release of elastin-derived peptides (EDPs), which are chemotactic for inflammatory cells such as monocytes. Their effect on macrophage polarization is less well known. Proinflammatory M1 macrophages initially are recruited to sites of injury, but, if their effects are prolonged, they can lead to chronic inflammation that prevents normal tissue repair. Conversely, anti-inflammatory M2 macrophages reduce inflammation and aid in wound healing. Thus, a proper M1/M2 ratio is vital for tissue homeostasis. Abdominal aortic aneurysm tissue reveals a high M1/M2 ratio in which proinflammatory cells and their associated markers dominate. In the current study, in vitro treatment of bone marrow-derived macrophages with EDPs induced M1 macrophage polarization. By using C57BL/6 mice, Ab-mediated neutralization of EDPs reduced aortic dilation, matrix metalloproteinase activity, and proinflammatory cytokine expression at early and late time points after aneurysm induction. Furthermore, direct manipulation of the M1/M2 balance altered aortic dilation. Injection of M2-polarized macrophages reduced aortic dilation after aneurysm induction. EDPs promoted a proinflammatory environment in aortic tissue by inducing M1 polarization, and neutralization of EDPs attenuated aortic dilation. The M1/M2 imbalance is vital to aneurysm formation.

Background differences in baseline and stimulated MMP levels influence abdominal aortic aneurysm susceptibility.

OBJECTIVE: Evidence has demonstrated profound influence of genetic background on cardiovascular phenotypes. Murine models in Marfan syndrome (MFS) have shown that genetic background-related variations affect thoracic aortic aneurysm formation, rupture, and lifespan of mice. MFS mice with C57Bl/6 genetic background are less susceptible to aneurysm formation compared to the 129/SvEv genetic background. In this study, we hypothesize that susceptibility to abdominal aortic aneurysm (AAA) will be increased in 129/SvEv mice versus C57Bl/6 mice. We tested this hypothesis by assessing differences in aneurysm size, tissue properties, immune response, and MMP expression. METHODS: Mice of C57Bl/6 or 129/SvEv background underwent AAA induction by periaortic application of CaCl2. Baseline aortic diameters, tissue properties and MMP levels were measured. After aneurysm induction, diameters, MMP expression, and immune response (macrophage infiltration and bone marrow transplantation) were measured. RESULTS: Aneurysms were larger in 129/SvEv mice than C57Bl/6 mice (83.0% ± 13.6 increase compared to 57.8% ± 6.4). The aorta was stiffer in the 129/SvEv mice compared to C57Bl/6 mice (952.5 kPa ± 93.6 versus 621.4 kPa ± 84.2). Baseline MMP-2 and post-aneurysm MMP-2 and -9 levels were higher in 129/SvEv aortas compared to C57Bl/6 aortas. Elastic lamella disruption/fragmentation and macrophage infiltration were increased in 129/SvEv mice. Myelogenous cell reversal by bone marrow transplantation did not affect aneurysm size. CONCLUSIONS: These data demonstrate that 129/SvEv mice are more susceptible to AAA compared to C57Bl/6 mice. Intrinsic properties of the aorta between the two strains of mice, including baseline expression of MMP-2, influence susceptibility to AAA.

Inflammatory cell phenotypes in AAAs: their role and potential as targets for therapy.

Abdominal aortic aneurysms (AAAs) are characterized by chronic inflammatory cell infiltration. AAA is typically an asymptomatic disease and caused ≈15 000 deaths annually in the United States. Previous studies have examined both human and murine aortic tissue for the presence of various inflammatory cell types. Studies show that in both human and experimental AAAs, prominent inflammatory cell infiltration, such as CD4(+) T cells and macrophages, occurs in the damaged aortic wall. These cells have the ability to undergo phenotypic modulation based on microenvironmental cues, potentially influencing disease progression. Proinflammatory CD4(+) T cells and classically activated macrophages dominate the landscape of aortic infiltrates. The skew to proinflammatory phenotypes alters disease progression and plays a role in causing chronic inflammation. The local cytokine production and presence of inflammatory mediators, such as extracellular matrix breakdown products, influence the uneven balance of the inflammatory infiltrate phenotypes. Understanding and developing new strategies that target the proinflammatory phenotype could provide useful therapeutic targets for a disease with no current pharmacological intervention.

A bimodular oxidoreductase mediates the specific reduction of phylloquinone (vitamin K1) in chloroplasts.

Plants and certain species of cyanobacteria are the only organisms capable of synthesizing phylloquinone (vitamin K1 for vertebrates), which they use as an electron carrier during photosynthesis. Recent studies, however, have identified a plastidial pool of non-photoactive phylloquinone that could be involved in additional cellular functions. Here, we characterized an Arabidopsis bimodular enzyme--the At4g35760 gene product--comprising an integral domain homologous to the catalytic subunit of mammalian vitamin K1 epoxide reductase (VKORC1, EC 1.1.4.1) that is fused to a soluble thioredoxin-like moiety. GFP-fusion experiments in tobacco mesophyll cells established that the plant protein is targeted to plastids, and analyses of transcript and protein levels showed that expression is maximal in leaf tissues. The fused and individual VKORC1 domains were separately expressed in yeast, removing their chloroplast targeting pre-sequence and adding a C-terminal consensus signal for retention in the endoplasmic reticulum. The corresponding microsomal preparations were equally effective at mediating the dithiotreitol-dependent reduction of phylloquinone and menaquinone into their respective quinol forms. Strikingly, unlike mammalian VKORC1, the Arabidopsis enzyme did not reduce phylloquinone epoxide, and was resistant to inhibition by warfarin. The isoprenoid benzoquinone conjugates plastoquinone and ubiquinone were not substrates, establishing that the plant enzyme evolved strict specificity for the quinone form of naphthalenoid conjugates. In vitro reconstitution experiments established that the soluble thioredoxin-like domain can function as an electron donor for its integral VKORC1 partner.