Juliette Raffort1, Fabien Lareyre2, Marc Clément3, Claudine Moratal4, Elixène Jean-Baptiste5, Réda Hassen-Khodja5, Fanny Burel-Vandenbos6, Patrick Bruneval7, Giulia Chinetti8, Ziad Mallat9. 1. Division of Cardiovascular Medicine, Department of Medicine, University of Cambridge, Cambridge, United Kingdom; Institut National de la Santé et de la Recherche Médicale (Inserm), Unit 970, Paris Cardiovascular Research Center, Paris, France; Department of Clinical Biochemistry, University Hospital of Nice, Nice, France; Université Côte d'Azur, CHU, Inserm U1065, C3M, Nice, France. Electronic address: juliette.raffort@hotmail.fr. 2. Division of Cardiovascular Medicine, Department of Medicine, University of Cambridge, Cambridge, United Kingdom; Université Côte d'Azur, CHU, Inserm U1065, C3M, Nice, France; Department of Vascular Surgery, University Hospital of Nice, Nice, France. 3. Division of Cardiovascular Medicine, Department of Medicine, University of Cambridge, Cambridge, United Kingdom. 4. Université Côte d'Azur, CHU, Inserm U1065, C3M, Nice, France. 5. Université Côte d'Azur, CHU, Inserm U1065, C3M, Nice, France; Department of Vascular Surgery, University Hospital of Nice, Nice, France. 6. Department of Pathology, University Hospital of Nice, Nice, France. 7. Department of Pathology, Hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux de Paris, Paris, France. 8. Department of Clinical Biochemistry, University Hospital of Nice, Nice, France; Université Côte d'Azur, CHU, Inserm U1065, C3M, Nice, France. 9. Division of Cardiovascular Medicine, Department of Medicine, University of Cambridge, Cambridge, United Kingdom; Institut National de la Santé et de la Recherche Médicale (Inserm), Unit 970, Paris Cardiovascular Research Center, Paris, France.
Abstract
OBJECTIVE: Macrophages play a critical role in the initiation and progression of abdominal aortic aneurysm (AAA) and are classically distinguished into M1 "proinflammatory" and M2 "anti-inflammatory" macrophages. Topical application of elastase associated with transforming growth factor β (TGF-β) systemic neutralization reproduces the main pathologic features of human AAA, offering a new model to investigate their role. The aim of this study was to investigate whether macrophages contribute to the expression of canonical M1/M2 markers in the aorta in the AAA model induced by elastase and systemic blockade of TGF-β and whether blocking of TGF-β activity affects macrophage phenotype and the expression of the M2 marker arginase 1 (ARG1). METHODS: C57Bl/6J male mice (6-8 weeks old) were randomly assigned to three experimental groups: mice that had local application of heat-inactivated elastase or elastase and mice that had elastase application and received injection of anti-TGF-β (elastase + anti-TGF-β group). Monocyte-macrophage depletion was achieved in the elastase + anti-TGF-β group using liposome clodronate. Macrophage phenotype was characterized by quantitative polymerase chain reaction, flow cytometry, and immunohistochemistry. Human infrarenal AAA tissues (n = 10) were obtained to analyze ARG1 expression. RESULTS: Analysis of gene expression in the infrarenal aortic wall revealed that after 14 days, no significant difference for the expression of CCL2, NOS2, and Ym1/2 was observed in the elastase group compared with the elastase + anti-TGF-β group, whereas the expression of ARG1, interleukin (IL) 1β, and IL-6 was significantly increased. Macrophage depletion in the elastase + anti-TGF-β group led to a significant decrease of IL-1β, IL-6, ARG1, and Ym1/2 gene expression. Immunofluorescent staining confirmed that TGF-β neutralization significantly enhanced ARG1 protein expression in the aneurysmal tissue. Flow cytometry analysis revealed an increase of macrophages expressing ARG1 in the aorta of mice treated with elastase + anti-TGF-β compared with the elastase group, and their proportion increased with aneurysmal dilation. In humans, ARG1 protein expression was increased in aneurysmal tissues compared with controls, and positive cells were mainly found in the adventitia. CONCLUSIONS: TGF-β neutralization finely tunes macrophage phenotype in elastase-induced AAA and leads to an increase in ARG1 gene and protein expression in the aortic wall. Even if further studies are required to elucidate its role in AAA development, ARG1 could represent a new prognostic or therapeutic target in aneurysmal disease.
OBJECTIVE: Macrophages play a critical role in the initiation and progression of abdominal aortic aneurysm (AAA) and are classically distinguished into M1 "proinflammatory" and M2 "anti-inflammatory" macrophages. Topical application of elastase associated with transforming growth factor β (TGF-β) systemic neutralization reproduces the main pathologic features of human AAA, offering a new model to investigate their role. The aim of this study was to investigate whether macrophages contribute to the expression of canonical M1/M2 markers in the aorta in the AAA model induced by elastase and systemic blockade of TGF-β and whether blocking of TGF-β activity affects macrophage phenotype and the expression of the M2 marker arginase 1 (ARG1). METHODS: C57Bl/6J male mice (6-8 weeks old) were randomly assigned to three experimental groups: mice that had local application of heat-inactivated elastase or elastase and mice that had elastase application and received injection of anti-TGF-β (elastase + anti-TGF-β group). Monocyte-macrophage depletion was achieved in the elastase + anti-TGF-β group using liposome clodronate. Macrophage phenotype was characterized by quantitative polymerase chain reaction, flow cytometry, and immunohistochemistry. Human infrarenal AAA tissues (n = 10) were obtained to analyze ARG1 expression. RESULTS: Analysis of gene expression in the infrarenal aortic wall revealed that after 14 days, no significant difference for the expression of CCL2, NOS2, and Ym1/2 was observed in the elastase group compared with the elastase + anti-TGF-β group, whereas the expression of ARG1, interleukin (IL) 1β, and IL-6 was significantly increased. Macrophage depletion in the elastase + anti-TGF-β group led to a significant decrease of IL-1β, IL-6, ARG1, and Ym1/2 gene expression. Immunofluorescent staining confirmed that TGF-β neutralization significantly enhanced ARG1 protein expression in the aneurysmal tissue. Flow cytometry analysis revealed an increase of macrophages expressing ARG1 in the aorta of mice treated with elastase + anti-TGF-β compared with the elastase group, and their proportion increased with aneurysmal dilation. In humans, ARG1 protein expression was increased in aneurysmal tissues compared with controls, and positive cells were mainly found in the adventitia. CONCLUSIONS: TGF-β neutralization finely tunes macrophage phenotype in elastase-induced AAA and leads to an increase in ARG1 gene and protein expression in the aortic wall. Even if further studies are required to elucidate its role in AAA development, ARG1 could represent a new prognostic or therapeutic target in aneurysmal disease.
Authors: Laura Lopez-Sanz; Susana Bernal; Luna Jimenez-Castilla; Ignacio Prieto; Sara La Manna; Sergio Gomez-Lopez; Luis Miguel Blanco-Colio; Jesus Egido; Jose Luis Martin-Ventura; Carmen Gomez-Guerrero Journal: Clin Transl Med Date: 2021-07