RESUMO
Abdominal aortic aneurysm (AAA) formation is characterized by inflammation, leukocyte infiltration, and vascular remodeling. This study investigates the role of TRPV4 channels, which are transmembrane calcium channels that can regulate vascular tone, in modulating AAA formation. The elastase-treatment model of AAA in C57BL6 (WT) mice and Angiotensin II treatment model in ApoE-/- mice were used to confirm our hypotheses. The administration of a specific TRPV4 antagonist, GSK2193874, in elastase-treated WT mice and in AngII-treated ApoE-/- mice caused a significant attenuation of aortic diameter, decrease in pro-inflammatory cytokines (IL-1ß, IL-6, IL-17, MCP-1, MIP-1α, MIP-2, RANTES, and TNF-α), inflammatory cell infiltration (CD3 + T cells, macrophages, and neutrophils), elastic fiber disruption, and an increase in smooth muscle cell α-actin expression compared to untreated mice. Similarly, elastase-treated TRPV4-/- mice had a significant decrease in AAA formation, aortic inflammation, and vascular remodeling compared to elastase-treated WT mice on Day 14. In vitro studies demonstrated that the inhibition of TRPV4 channels mitigates aortic smooth muscle cell-dependent inflammatory cytokine production as well as decreases neutrophil transmigration through aortic endothelial cells. Therefore, our results suggest that TRPV4 antagonism can attenuate aortic inflammation and remodeling via decreased smooth muscle cell activation and neutrophil transendothelial migration during AAA formation.
Assuntos
Aneurisma da Aorta Abdominal/prevenção & controle , Inflamação/prevenção & controle , Macrófagos/efeitos dos fármacos , Piperidinas/farmacologia , Quinolinas/farmacologia , Canais de Cátion TRPV/antagonistas & inibidores , Animais , Aneurisma da Aorta Abdominal/etiologia , Aneurisma da Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/patologia , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Elastase Pancreática/metabolismoRESUMO
The formation of an abdominal aortic aneurysm (AAA) is characterized by inflammation, macrophage infiltration, and vascular remodeling. In this study, we tested the hypothesis that mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) immunomodulate aortic inflammation, to mitigate AAA formation via modulation of microRNA-147. An elastase-treatment model of AAA was used in male C57BL/6 wild-type (WT) mice. Administration of EVs in elastase-treated WT mice caused a significant attenuation of aortic diameter and mitigated proinflammatory cytokines, inflammatory cell infiltration, an increase in smooth muscle cell α-actin expression, and a decrease in elastic fiber disruption, compared with untreated mice. A 10-fold up-regulation of microRNA (miR)-147, a key mediator of macrophage inflammatory responses, was observed in murine aortic tissue in elastase-treated mice compared with controls on d 14. EVs derived from MSCs transfected with miR-147 mimic, but not with miR-147 inhibitor, attenuated aortic diameter, inflammation, and leukocyte infiltration in elastase-treated mice. In vitro studies of human aortic tissue explants and murine-derived CD11b+ macrophages induced proinflammatory cytokines after elastase treatment, and the expression was attenuated by cocultures with EVs transfected with miR-147 mimic, but not with miR-147 inhibitor. Thus, our findings define a critical role of MSC-derived EVs in attenuation of aortic inflammation and macrophage activation via miR-147 during AAA formation.-Spinosa, M., Lu, G., Su, G., Bontha, S. V., Gehrau, R., Salmon, M. D., Smith, J. R., Weiss, M. L., Mas, V. R., Upchurch, G. R., Sharma, A. K. Human mesenchymal stromal cell-derived extracellular vesicles attenuate aortic aneurysm formation and macrophage activation via microRNA-147.
RESUMO
OBJECTIVE: Resolvins have been shown to attenuate inflammation, whereas NETosis, the process of neutrophils releasing neutrophil extracellular traps (NETs), produces increased inflammation. It is hypothesized that treatment of animals with resolvin D1 (RvD1) would reduce abdominal aortic aneurysm (AAA) formation by inhibiting NETosis. METHODS: Wild-type 8- to 12-week-old C57BL/6 male mice (n = 47) and apolipoprotein E-deficient (ApoE-/-) mice (n = 20) were used in two models to demonstrate the effects of RvD1 on AAA growth. In the topical elastase AAA model, wild-type mice were divided into three groups: a deactivated elastase control group, in which sham surgery was performed using deactivated elastase and mice were intravenously injected with phosphate-buffered saline (PBS) once a day until harvest; an elastase group, in which active elastase was used to induce AAA and mice were injected with PBS daily until harvest; and an RvD1-treated group, in which AAA was induced and mice were injected with RvD1 daily until harvest. In the angiotensin II (Ang II)-induced AAA model, ApoE-/- mice were fed a high-fat diet and implanted with osmotic infusion pumps containing Ang II (1000 ng/kg/min). The Ang II model was divided into two groups: an Ang II control group, in which Ang II was delivered and mice were injected with PBS daily until harvest; and an RvD1-treated group, in which Ang II was delivered and mice were injected with RvD1 daily until harvest. On postoperative day 3, day 14, or day 28, aortic and blood samples were collected for Western blot, histology, cytokine array, enzyme-linked immunosorbent assay, and gelatin zymography after aortic diameter measurement. RESULTS: The day 14 RvD1-treated group demonstrated 42% reduced AAA diameter compared with the elastase group (P < .001). On postoperative day 3, the RvD1-treated group showed decreased levels of NETosis markers citrullinated histone H3 (P = .04) and neutrophil elastase (P = .002) compared with the elastase group. Among important cytokines involved in AAA formation, interleukin (IL) 1ß was downregulated (P = .02) whereas IL-10, a protective cytokine, was upregulated (P = .01) in the RvD1-treated group. Active matrix metalloproteinase 2 also decreased in the RvD1-treated group (P = .03). The RvD1-treated group in the Ang II AAA model, a second model, demonstrated reduced AAA diameter compared with the Ang II control group on day 28 (P < .046). The RvD1-treated group showed decreased levels of citrullinated histone H3 on day 3 (P = .002). Cytokines interferon γ, IL-1ß, C-X-C motif chemokine ligand 10, monocyte chemotactic protein 1, and regulated on activation, normal T cell expressed and secreted (RANTES) were all decreased on day 28 (P < .05). CONCLUSIONS: RvD1-mediated inhibition of NETosis may represent a future medical treatment for the attenuation of AAA growth.
Assuntos
Anti-Inflamatórios/farmacologia , Aorta Abdominal/efeitos dos fármacos , Aneurisma da Aorta Abdominal/prevenção & controle , Ácidos Docosa-Hexaenoicos/farmacologia , Armadilhas Extracelulares/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Animais , Aorta Abdominal/metabolismo , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/patologia , Citrulinação , Citocinas/metabolismo , Modelos Animais de Doenças , Armadilhas Extracelulares/metabolismo , Histonas/metabolismo , Mediadores da Inflamação/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Neutrófilos/metabolismo , Neutrófilos/patologia , Elastase Pancreática , Remodelação Vascular/efeitos dos fármacosRESUMO
OBJECTIVE: Abdominal aortic aneurysm (AAA) formation is characterized by inflammation, smooth muscle activation, and matrix degradation. This study tests the hypothesis that macrophage-produced high mobility group box 1 (HMGB1) production is dependent on nicotinamide adenine dinucleotide phosphate oxidase (Nox2), which leads to increase in interleukin (IL)-17 production resulting in AAA formation and that treatment with human mesenchymal stem cells (MSCs) can attenuate this process thereby inhibiting AAA formation. APPROACH AND RESULTS: Human aortic tissue demonstrated a significant increase in HMGB1 expression in AAA patients when compared with controls. An elastase-perfusion model of AAA demonstrated a significant increase in HMGB1 production in C57BL/6 (wild-type [WT]) mice, which was attenuated by MSC treatment. Furthermore, anti-HMGB1 antibody treatment of WT mice attenuated AAA formation, IL-17 production, and immune cell infiltration when compared with elastase-perfused WT mice on day 14. Elastase-perfused Nox2(-/y) mice demonstrated a significant attenuation of HMGB1 and IL-17 production, cellular infiltration, matrix metalloproteinase activity, and AAA formation when compared with WT mice on day 14. In vitro studies showed that elastase-treated macrophages from WT mice, but not from Nox2(-/y) mice, produced HMGB1, which was attenuated by MSC treatment. The production of macrophage-dependent HMGB1 involved Nox2 activation and superoxide anion production, which was mitigated by MSC treatment. CONCLUSIONS: These results demonstrate that macrophage-produced HMGB1 leads to aortic inflammation and acts as a trigger for CD4(+) T-cell-produced IL-17 during AAA formation. HMGB1 release is dependent on Nox2 activation, which can be inhibited by MSCs leading to attenuation of proinflammatory cytokines, especially IL-17, and protection against AAA formation.
Assuntos
Aorta Abdominal/enzimologia , Aneurisma da Aorta Abdominal/prevenção & controle , Proteína HMGB1/metabolismo , Macrófagos/enzimologia , Glicoproteínas de Membrana/metabolismo , Transplante de Células-Tronco Mesenquimais , NADPH Oxidases/metabolismo , Animais , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/enzimologia , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/patologia , Linfócitos T CD4-Positivos/metabolismo , Estudos de Casos e Controles , Dilatação Patológica , Modelos Animais de Doenças , Predisposição Genética para Doença , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-17/metabolismo , Ativação Linfocitária , Ativação de Macrófagos , Masculino , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidase 2 , NADPH Oxidases/deficiência , NADPH Oxidases/genética , Elastase Pancreática , Fenótipo , Transdução de Sinais , Fatores de Tempo , Técnicas de Cultura de TecidosRESUMO
Pannexin-1 (Panx1) channels have been shown to regulate leukocyte trafficking and tissue inflammation but the mechanism of Panx1 in chronic vascular diseases like abdominal aortic aneurysms (AAA) is unknown. Here we demonstrate that Panx1 on endothelial cells, but not smooth muscle cells, orchestrate a cascade of signaling events to mediate vascular inflammation and remodeling. Mechanistically, Panx1 on endothelial cells acts as a conduit for ATP release that stimulates macrophage activation via P2X7 receptors and mitochondrial DNA release to increase IL-1ß and HMGB1 secretion. Secondly, Panx1 signaling regulates smooth muscle cell-dependent intracellular Ca2+ release and vascular remodeling via P2Y2 receptors. Panx1 blockade using probenecid markedly inhibits leukocyte transmigration, aortic inflammation and remodeling to mitigate AAA formation. Panx1 expression is upregulated in human AAAs and retrospective clinical data demonstrated reduced mortality in aortic aneurysm patients treated with Panx1 inhibitors. Collectively, these data identify Panx1 signaling as a contributory mechanism of AAA formation.
Assuntos
Aneurisma da Aorta Abdominal , Células Endoteliais , Trifosfato de Adenosina/metabolismo , Aneurisma da Aorta Abdominal/genética , Conexinas/genética , Conexinas/metabolismo , Células Endoteliais/metabolismo , Humanos , Inflamação/metabolismo , Macrófagos/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Estudos RetrospectivosRESUMO
INTRODUCTION: Given the unknown biologic antecedents before aortic aneurysm rupture, the purpose of this study was to establish a reproducible model of aortic aneurysm rupture. METHODS: We fed 7-week-old apolipoprotein E deficient mice a high-fat diet for 4 weeks and osmotic infusion pumps containing Angiotensin II were implanted. Angiotensin II was delivered continuously for 4 weeks at either 1,000 ng/kg/min (n = 25) or 2,000 ng/kg/min (n = 29). A third group (n = 14) were given Angiotensin II at 2,000 ng/kg/min and 0.2% ß-aminopropionitrile dissolved in drinking water. Surviving mice were killed 28 days after pump placement, aortic diameters were measured, and molecular analyses were performed. RESULTS: Survival at 28 days was significantly different among groups with 80% survival in the 1,000 ng/kg/min group, 52% in the 2,000 ng/kg/min group, and only 14% in the Angiotensin II/ß-aminopropionitrile group (P = .0001). Concordantly, rupture rates were statistically different among groups (8% versus 38% versus 79%, P < .0001). Rates of abdominal aortic aneurysm were 48%, 55%, and 93%, respectively, with statistically higher rates in the Angiotensin II/ß-aminopropionitrile group compared with both the 1,000 ng and 2,000 ng Angiotensin II groups (P = .006 and P = .0165, respectively). Rates of thoracic aortic aneurysm formation were 12%, 52%, and 79% in the 3 groups with a statistically higher rate in the Angiotensin II/ß-aminopropionitrile group compared with 1,000 ng group (P < .0001). CONCLUSIONS: A reproducible model of aortic aneurysm rupture was developed with a high incidence of abdominal and thoracic aortic aneurysm. This model should enable further studies investigating the pathogenesis of aortic rupture, as well as allow for targeted strategies to prevent human aortic aneurysm rupture.
Assuntos
Ruptura Aórtica , Modelos Animais de Doenças , Aminopropionitrilo , Angiotensina II , Animais , Aorta/metabolismo , Citocinas/metabolismo , Masculino , Camundongos , Camundongos Knockout para ApoERESUMO
BACKGROUND: Tamsulosin, an α1A-adrenergic receptor inhibitor, is prescribed to treat benign prostatic hyperplasia in men >60 years of age, the same demographic most susceptible to abdominal aortic aneurysm. The goal of this study was to investigate the effect of tamsulosin on abdominal aortic aneurysm pathogenesis. METHODS: Abdominal aortic aneurysms were induced in WT C57BL/6 male mice (nâ¯=â¯9-18/group), using an established topical elastase abdominal aortic aneurysm model. Osmotic pumps were implanted in mice 5 days before operation to create the model, administering either low dose (0.125 µg/day tamsulosin), high dose (0.250µg/day tamsulosin), or vehicle treatments with and without topical application of elastase. Blood pressures were measured preoperatively and on postoperative days 0, 3, 7, and 14. On postoperative day 14, aortic diameter was measured before harvest. Sample aortas were prepared for histology and cytokine analysis. RESULTS: Measurements of systolic blood pressure did not differ between groups. Mice treated with the low dose of tamsulosin and with the high dose of tamsulosin showed decreased aortic diameter compared with vehicle-treated control (93% ± 24 versus 94% ± 30 versus 132% ± 24, respectively; Pâ¯=â¯.0003, Pâ¯=â¯.0003). Cytokine analysis demonstrated downregulation of pro-inflammatory cytokines in both treatment groups compared with the control (P < .05). Histology exhibited preservation of elastin in both low- and high-dose tamsulosin-treated groups (Pâ¯=â¯.0041 and Pâ¯=â¯.0018, respectively). CONCLUSION: Tamsulosin attenuates abdominal aortic aneurysm formation with increased preservation of elastin and decreased production of pro-inflammatory cytokines. Further studies are necessary to elucidate the mechanism by which tamsulosin attenuates abdominal aortic aneurysm pathogenesis.
Assuntos
Antagonistas de Receptores Adrenérgicos alfa 1/farmacologia , Aorta Abdominal/efeitos dos fármacos , Aneurisma da Aorta Abdominal/prevenção & controle , Tansulosina/farmacologia , Antagonistas de Receptores Adrenérgicos alfa 1/uso terapêutico , Animais , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/patologia , Pressão Sanguínea/efeitos dos fármacos , Citocinas/metabolismo , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Elastina/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Elastase Pancreática/toxicidade , Tansulosina/uso terapêutico , Resultado do TratamentoRESUMO
OBJECTIVE: Ex vivo lung perfusion has been successful in the assessment of marginal donor lungs, including donation after cardiac death (DCD) donor lungs. Ex vivo lung perfusion also represents a unique platform for targeted drug delivery. We sought to determine whether ischemia-reperfusion injury would be decreased after transplantation of DCD donor lungs subjected to prolonged cold preservation and treated with an adenosine A2A receptor agonist during ex vivo lung perfusion. METHODS: Porcine DCD donor lungs were preserved at 4°C for 12 hours and underwent ex vivo lung perfusion for 4 hours. Left lungs were then transplanted and reperfused for 4 hours. Three groups (n = 4/group) were randomized according to treatment with the adenosine A2A receptor agonist ATL-1223 or the dimethyl sulfoxide vehicle: Infusion of dimethyl sulfoxide during ex vivo lung perfusion and reperfusion (DMSO), infusion of ATL-1223 during ex vivo lung perfusion and dimethyl sulfoxide during reperfusion (ATL-E), and infusion of ATL-1223 during ex vivo lung perfusion and reperfusion (ATL-E/R). Final Pao2/Fio2 ratios (arterial oxygen partial pressure/fraction of inspired oxygen) were determined from samples obtained from the left superior and inferior pulmonary veins. RESULTS: Final Pao2/Fio2 ratios in the ATL-E/R group (430.1 ± 26.4 mm Hg) were similar to final Pao2/Fio2 ratios in the ATL-E group (413.6 ± 18.8 mm Hg), but both treated groups had significantly higher final Pao2/Fio2 ratios compared with the dimethyl sulfoxide group (84.8 ± 17.7 mm Hg). Low oxygenation gradients during ex vivo lung perfusion did not preclude superior oxygenation capacity during reperfusion. CONCLUSIONS: After prolonged cold preservation, treatment of DCD donor lungs with an adenosine A2A receptor agonist during ex vivo lung perfusion enabled Pao2/Fio2 ratios greater than 400 mm Hg after transplantation in a preclinical porcine model. Pulmonary function during ex vivo lung perfusion was not predictive of outcomes after transplantation.