TGFβ (transforming growth factor-β) blockade induces a human-like disease in a nondissecting mouse model of abdominal aortic aneurysm

Objective-Current experimental models of abdominal aortic aneurysm (AAA) do not accurately reproduce the major features of human AAA. We hypothesized that blockade of TGF beta (transforming growth factor-beta) activity-a guardian of vascular integrity and immune homeostasis-would impair vascular healing in models of nondissecting AAA and would lead to sustained aneurysmal growth until rupture. Approach and Results-Here, we test this hypothesis in the elastase-induced AAA model in mice. We analyze AAA development and progression using ultrasound in vivo, synchrotron-based ultrahigh resolution imaging ex vivo, and a combination of biological, histological, and flow cytometry-based cellular and molecular approaches in vitro. Systemic blockade of TGF beta using a monoclonal antibody induces a transition from a self-contained aortic dilatation to a model of sustained aneurysmal growth, associated with the formation of an intraluminal thrombus. AAA growth is associated with wall disruption but no medial dissection and culminates in fatal transmural aortic wall rupture. TGF beta blockade enhances leukocyte infiltration both in the aortic wall and the intraluminal thrombus and aggravates extracellular matrix degradation. Early blockade of IL-1 beta or monocyte-dependent responses substantially limits AAA severity. However, blockade of IL-1 beta after disease initiation has no effect on AAA progression to rupture. Conclusions-Endogenous TGF beta activity is required for the healing of AAA. TGF beta blockade may be harnessed to generate new models of AAA with better relevance to the human disease. We expect that the new models will improve our understanding of the pathophysiology of AAA and will be useful in the identification of new therapeutic targets

Indoleamine 2 3-dioxygenase knockout limits angiotensin II-induced aneurysm in low density lipoprotein receptor-deficient mice fed with high fat diet.

AIMS: Abdominal aortic aneurysm (AAA) is an age-associated disease characterized by chronic inflammation, vascular cell apoptosis and metalloproteinase-mediated extracellular matrix degradation. Despite considerable progress in identifying targets involved in these processes, therapeutic approaches aiming to reduce aneurysm growth and rupture are still scarce. Indoleamine 2-3 dioxygenase 1 (IDO) is the first and rate-limiting enzyme involved in the conversion of tryptophan (Trp) into kynurenine (Kyn) pathway. In this study, we investigated the role of IDO in two different models of AAA in mice. METHODS AND RESULTS: Mice with deficiencies in both low density receptor-deficient (Ldlr-/-) and IDO (Ldlr-/-Ido1-/-) were generated by cross-breeding Ido1-/- mice with Ldlr-/-mice. To induce aneurysm, these mice were infused with angiotensin II (Ang II) (1000 ng/min/kg) and fed with high fat diet (HFD) during 28 days. AAAs were present in almost all Ldlr-/- infused with AngII, but only in 50% of Ldlr-/-Ido1-/- mice. Immunohistochemistry at an early time point (day 7) revealed no changes in macrophage and T lymphocyte infiltration within the vessel wall, but showed reduced apoptosis, as assessed by TUNEL assay, and increased α-actin staining within the media of Ldlr-/-Ido1-/- mice, suggesting enhanced survival of vascular smooth muscle cells (VSMCs) in the absence of IDO. In another model of elastase-induced AAA in C57Bl/6 mice, IDO deficiency had no effect on aneurysm formation. CONCLUSION: Our study showed that the knockout of IDO prevented VSMC apoptosis in AngII -treated Ldlr-/- mice fed with HFD, suggesting a detrimental role of IDO in AAA formation and thus would be an important target for the treatment of aneurysm

Vascular Smooth Muscle Cell Plasticity and Autophagy in Dissecting Aortic Aneurysms.

Objective- Recent studies suggested the occurrence of phenotypic switching of vascular smooth muscle cells (VSMCs) during the development of aortic aneurysm (AA). However, lineage-tracing studies are still lacking, and the behavior of VSMCs during the formation of dissecting AA is poorly understood. Approach and Results- We used multicolor lineage tracing of VSMCs to track their fate after injury in murine models of Ang II (angiotensin II)-induced dissecting AA. We also addressed the direct impact of autophagy on the response of VSMCs to AA dissection. Finally, we studied the relevance of these processes to human AAs. Here, we show that a subset of medial VSMCs undergoes clonal expansion and that VSMC outgrowths are observed in the adventitia and borders of the false channel during Ang II-induced development of dissecting AA. The clonally expanded VSMCs undergo phenotypic switching with downregulation of VSMC differentiation markers and upregulation of phagocytic markers, indicative of functional changes. In particular, autophagy and endoplasmic reticulum stress responses are activated in the injured VSMCs. Loss of autophagy in VSMCs through deletion of autophagy protein 5 gene ( Atg5) increases the susceptibility of VSMCs to death, enhances endoplasmic reticulum stress activation, and promotes IRE (inositol-requiring enzyme) 1α-dependent VSMC inflammation. These alterations culminate in increased severity of aortic disease and higher incidence of fatal AA dissection in mice with VSMC-restricted deletion of Atg5. We also report increased expression of autophagy and endoplasmic reticulum stress markers in VSMCs of human dissecting AAs. Conclusions- VSMCs undergo clonal expansion and phenotypic switching in Ang II-induced dissecting AAs in mice. We also identify a critical role for autophagy in regulating VSMC death and endoplasmic reticulum stress-dependent inflammation with important consequences for aortic wall homeostasis and repair

The Dendritic Cell Receptor DNGR-1 Promotes the Development of Atherosclerosis in Mice.

RATIONALE: Necrotic core formation during the development of atherosclerosis is associated with a chronic inflammatory response and promotes accelerated plaque development and instability. However, the molecular links between necrosis and the development of atherosclerosis are not completely understood. Clec9a (C-type lectin receptor) or DNGR-1 (dendritic cell NK lectin group receptor-1) is preferentially expressed by the CD8α+ subset of dendritic cells (CD8α+ DCs) and is involved in sensing necrotic cells. We hypothesized that sensing of necrotic cells by DNGR-1 plays a determinant role in the inflammatory response of atherosclerosis. OBJECTIVE: We sought to address the impact of total, bone marrow-restricted, or CD8α+ DC-restricted deletion of DNGR-1 on atherosclerosis development. METHODS AND RESULTS: We show that total absence of DNGR-1 in Apoe (apolipoprotein e)-deficient mice (Apoe-/-) and bone marrow-restricted deletion of DNGR-1 in Ldlr (low-density lipoprotein receptor)-deficient mice (Ldlr-/-) significantly reduce inflammatory cell content within arterial plaques and limit atherosclerosis development in a context of moderate hypercholesterolemia. This is associated with a significant increase of the expression of interleukin-10 (IL-10). The atheroprotective effect of DNGR-1 deletion is completely abrogated in the absence of bone marrow-derived IL-10. Furthermore, a specific deletion of DNGR-1 in CD8α+ DCs significantly increases IL-10 expression, reduces macrophage and T-cell contents within the lesions, and limits the development of atherosclerosis. CONCLUSIONS: Our results unravel a new role of DNGR-1 in regulating vascular inflammation and atherosclerosis and potentially identify a new target for disease modulation

Genetic and Pharmacological Inhibition of TREM-1 Limits the Development of Experimental Atherosclerosis.

BACKGROUND: Innate immune responses activated through myeloid cells contribute to the initiation, progression, and complications of atherosclerosis in experimental models. However, the critical upstream pathways that link innate immune activation to foam cell formation are still poorly identified. OBJECTIVES: This study sought to investigate the hypothesis that activation of the triggering receptor expressed on myeloid cells (TREM-1) plays a determinant role in macrophage atherogenic responses. METHODS: After genetically invalidating Trem-1 in chimeric Ldlr-/-Trem-1-/- mice and double knockout ApoE-/-Trem-1-/- mice, we pharmacologically inhibited Trem-1 using LR12 peptide. RESULTS: Ldlr-/- mice reconstituted with bone marrow deficient for Trem-1 (Trem-1-/-) showed a strong reduction of atherosclerotic plaque size in both the aortic sinus and the thoracoabdominal aorta, and were less inflammatory compared to plaques of Trem-1+/+ chimeric mice. Genetic invalidation of Trem-1 led to alteration of monocyte recruitment into atherosclerotic lesions and inhibited toll-like receptor 4 (TLR 4)-initiated proinflammatory macrophage responses. We identified a critical role for Trem-1 in the upregulation of cluster of differentiation 36 (CD36), thereby promoting the formation of inflammatory foam cells. Genetic invalidation of Trem-1 in ApoE-/-/Trem-1-/- mice or pharmacological blockade of Trem-1 in ApoE-/- mice using LR-12 peptide also significantly reduced the development of atherosclerosis throughout the vascular tree, and lessened plaque inflammation. TREM-1 was expressed in human atherosclerotic lesions, mainly in lipid-rich areas with significantly higher levels of expression in atheromatous than in fibrous plaques. CONCLUSIONS: We identified TREM-1 as a major upstream proatherogenic receptor. We propose that TREM-1 activation orchestrates monocyte/macrophage proinflammatory responses and foam cell formation through coordinated and combined activation of CD36 and TLR4. Blockade of TREM-1 signaling may constitute an attractive novel and double-hit approach for the treatment of atherosclerosis

Exploring the role of TREM-1 in experimental abdominal aortic aneurysm

L'anévrisme de l'aorte abdominale (AAA) représente une des principales causes de décès cardiovasculaires à travers le monde, principalement parmi les hommes de plus de 65 ans. L'AAA se manifeste par une dilatation focale de l'aorte, qui est la conséquence d'un remodelage délétère de la paroi aortique, associant une infiltration inflammatoire importante, une perte des cellules musculaires lisses et un remaniement de la matrice extracellulaire, et dont le risque majeur est la rupture, potentiellement létale. De nombreuses études expérimentales et cliniques suggèrent que la dérégulation du système rénine-angiotensine serait impliquée dans le développement de l'AAA. TREM-1 (Triggering Receptor Expressed on Myeloid Cells-1) est un récepteur membranaire exprimé principalement par les cellules myéloïdes, dont l'activation amplifie la réponse immune innée, en association avec la voie des TLRs. L'objectif de ce travail de thèse est d'étudier le rôle du récepteur TREM-1 dans le développement de l'anévrisme de l'aorte abdominale expérimental, en utilisant différentes approches « perte et gain de fonction » dans un modèle murin d'AAA induit par infusion continue d'angiotensine II (AngII). Nous avons montré que l'infusion d'Ang II augmente le pool de monocytes classiques Ly6Chi exprimant TREM-1, via une activation du récepteur AT1R de façon indépendante du récepteur TLR4 chez la souris. La délétion génique de Trem-1 chez la souris hypercholestérolémique Apoe-/-Trem-1-/- 1 limite l'infiltration macrophagique, la réponse inflammatoire locale, l'activité métalloprotéinase, la dégradation du réseau élastique dans la paroi, et in fine réduit l'incidence et la sévérité de la dilatation anévrismale aortique. A contrario, la stimulation pharmacologique de TREM-1 par un anticorps agoniste augmente le recrutement des monocytes classiques Ly6Chi dans la paroi aortique, la réponse pro-inflammatoire locale, la dégradation du réseau élastique et finalement aggrave la maladie anévrismale aortique. Enfin, le traitement par LR-12, un peptide inhibant l'activation de TREM-1, chez la souris Apoe-/- sous angiotensine II limite l'inflammation aortique, la dégradation matricielle et protège contre la dilatation aortique. Chez l'homme, l'expression génique et protéique de TREM-1 est augmentée dans les aortes anévrismales par rapport aux aortes saines et colocalise localement avec la présence de macrophages. Pour conclure, nous avons montré que le récepteur TREM-1 est exprimé en réponse à l'angiotensine II et que TREM-1 est impliqué dans la physiopathologie de la maladie anévrismale aortique, en régulant le trafic et l'activation des monocytes dans la paroi aortique. L'inhibition de TREM-1 pourrait constituer une nouvelle stratégie thérapeutique pour limiter le développement et les complications de l'anévrisme de l'aorte abdominale.Abdominal Aortic Aneurysm (AAA) is one of the leading causes of cardiovascular death globally, especially among men over 65 years of age. AAA is characterized by a focal dilation of the aorta, which is the consequence of an adverse remodeling occuring in the aortic wall that entails a massive infiltration of inflammatory cells, a loss of smooth muscle cells and a structural and functional reorganization of the extracellular matrix resulting in life-threatening aneurysmal rupture. Clinical and experimental studies suggest that an inappropriate renin-angiotensin system is involved in AAA pathophysiology. TREM-1 (Triggering Receptor Expressed on Myeloid Cells-1) is a cell surface receptor mainly expressed by myeloid cells. TREM-1 engagement leads to the amplification of the innate immune response, in synergy with TLR pathways activation. The aim of this PhD project was to decipher the role of TREM-1 in AAA development and complications, using loss- or gain-of-function experimental strategies in a murine AngII-induced AAA model. Here, we showed that AngII increases the pool of Ly6Chi classical monocytes expressing TREM-1 via AT1R stimulation, in a TLR4-independent manner, suggesting an interaction between renin-angiotensin system and TREM-1 signaling pathway. Genetic deletion of Trem-1 in dyslipidemic Apoe-/-Trem-1-/- limits macrophage infiltration, inflammatory response, metalloproteinase activity and extracellular matrix remodeling, hampering AAA development. Conversely, pharmacologic stimulation of TREM-1 with an agonist antibody increases Ly6Chi monocyte recruitment in the aortic wall, local pro-inflammatory response and elastin degradation, which promotes AAA development. Pharmacologic inhibition of TREM-1 with LR12, an inhibitory peptide, was demonstrated to reduce AAA development by limiting early aortic inflammation and extracellular matrix degradation. In addition, TREM-1 was found to be localized with macrophage infiltration and that the gene and protein expression of TREM-1 were also found to be increased in human aneurysmal aortic tissue compared to control counterparts. To conclude, we showed that TREM-1 receptor engagement is induced in response to AngII stimulation and is implicated in AAA development through regulation of monocyte trafficking and activation into the aortic wall. Inhibition of TREM-1 may thus constitute a promising novel therapeutic strategy to limit AAA development

Exploration et modulation du récepteur TREM-1 dans le développement de l'anévrisme de l'aorte abdominale expérimental

Abdominal Aortic Aneurysm (AAA) is one of the leading causes of cardiovascular death globally, especially among men over 65 years of age. AAA is characterized by a focal dilation of the aorta, which is the consequence of an adverse remodeling occuring in the aortic wall that entails a massive infiltration of inflammatory cells, a loss of smooth muscle cells and a structural and functional reorganization of the extracellular matrix resulting in life-threatening aneurysmal rupture. Clinical and experimental studies suggest that an inappropriate renin-angiotensin system is involved in AAA pathophysiology. TREM-1 (Triggering Receptor Expressed on Myeloid Cells-1) is a cell surface receptor mainly expressed by myeloid cells. TREM-1 engagement leads to the amplification of the innate immune response, in synergy with TLR pathways activation. The aim of this PhD project was to decipher the role of TREM-1 in AAA development and complications, using loss- or gain-of-function experimental strategies in a murine AngII-induced AAA model. Here, we showed that AngII increases the pool of Ly6Chi classical monocytes expressing TREM-1 via AT1R stimulation, in a TLR4-independent manner, suggesting an interaction between renin-angiotensin system and TREM-1 signaling pathway. Genetic deletion of Trem-1 in dyslipidemic Apoe-/-Trem-1-/- limits macrophage infiltration, inflammatory response, metalloproteinase activity and extracellular matrix remodeling, hampering AAA development. Conversely, pharmacologic stimulation of TREM-1 with an agonist antibody increases Ly6Chi monocyte recruitment in the aortic wall, local pro-inflammatory response and elastin degradation, which promotes AAA development. Pharmacologic inhibition of TREM-1 with LR12, an inhibitory peptide, was demonstrated to reduce AAA development by limiting early aortic inflammation and extracellular matrix degradation. In addition, TREM-1 was found to be localized with macrophage infiltration and that the gene and protein expression of TREM-1 were also found to be increased in human aneurysmal aortic tissue compared to control counterparts. To conclude, we showed that TREM-1 receptor engagement is induced in response to AngII stimulation and is implicated in AAA development through regulation of monocyte trafficking and activation into the aortic wall. Inhibition of TREM-1 may thus constitute a promising novel therapeutic strategy to limit AAA development.L'anévrisme de l'aorte abdominale (AAA) représente une des principales causes de décès cardiovasculaires à travers le monde, principalement parmi les hommes de plus de 65 ans. L'AAA se manifeste par une dilatation focale de l'aorte, qui est la conséquence d'un remodelage délétère de la paroi aortique, associant une infiltration inflammatoire importante, une perte des cellules musculaires lisses et un remaniement de la matrice extracellulaire, et dont le risque majeur est la rupture, potentiellement létale. De nombreuses études expérimentales et cliniques suggèrent que la dérégulation du système rénine-angiotensine serait impliquée dans le développement de l'AAA. TREM-1 (Triggering Receptor Expressed on Myeloid Cells-1) est un récepteur membranaire exprimé principalement par les cellules myéloïdes, dont l'activation amplifie la réponse immune innée, en association avec la voie des TLRs. L'objectif de ce travail de thèse est d'étudier le rôle du récepteur TREM-1 dans le développement de l'anévrisme de l'aorte abdominale expérimental, en utilisant différentes approches « perte et gain de fonction » dans un modèle murin d'AAA induit par infusion continue d'angiotensine II (AngII). Nous avons montré que l'infusion d'Ang II augmente le pool de monocytes classiques Ly6Chi exprimant TREM-1, via une activation du récepteur AT1R de façon indépendante du récepteur TLR4 chez la souris. La délétion génique de Trem-1 chez la souris hypercholestérolémique Apoe-/-Trem-1-/- 1 limite l'infiltration macrophagique, la réponse inflammatoire locale, l'activité métalloprotéinase, la dégradation du réseau élastique dans la paroi, et in fine réduit l'incidence et la sévérité de la dilatation anévrismale aortique. A contrario, la stimulation pharmacologique de TREM-1 par un anticorps agoniste augmente le recrutement des monocytes classiques Ly6Chi dans la paroi aortique, la réponse pro-inflammatoire locale, la dégradation du réseau élastique et finalement aggrave la maladie anévrismale aortique. Enfin, le traitement par LR-12, un peptide inhibant l'activation de TREM-1, chez la souris Apoe-/- sous angiotensine II limite l'inflammation aortique, la dégradation matricielle et protège contre la dilatation aortique. Chez l'homme, l'expression génique et protéique de TREM-1 est augmentée dans les aortes anévrismales par rapport aux aortes saines et colocalise localement avec la présence de macrophages. Pour conclure, nous avons montré que le récepteur TREM-1 est exprimé en réponse à l'angiotensine II et que TREM-1 est impliqué dans la physiopathologie de la maladie anévrismale aortique, en régulant le trafic et l'activation des monocytes dans la paroi aortique. L'inhibition de TREM-1 pourrait constituer une nouvelle stratégie thérapeutique pour limiter le développement et les complications de l'anévrisme de l'aorte abdominale

Une mutation « gain de fonction » de JAK2 favorise la formation d’anévrismes aortiques disséquants en induisant un phénotype pro-inflammatoire des macrophages vasculaires résidents

International audienceNo abstract availabl

Rôle du récepteur TREM-1 dans les maladies cardiovasculaires

La réponse immunitaire innée joue un rôle important dans le déclenchement et la progression des maladies cardiovasculaires ainsi que dans leurs complications, potentiellement mortelles. TREM-1, un récepteur membranaire principalement exprimé par les cellules myéloïdes, agit comme un chef d’orchestre de l’inflammation amplifiant la production de cytokines et de chimiokines. De récentes études expérimentales montrent que l’inhibition de TREM-1 limite le développement de l’athérosclérose, la dilatation aortique anévrismale, ainsi que les complications cardiaques et cérébrales lors de l’ischémie aiguë. Chez l’homme, la forme soluble de TREM-1, libérée après son activation, est un biomarqueur intéressant, qui permet d’identifier les patients à haut risque cardiovasculaire, et qui pourrait ouvrir la voie vers une approche immuno-modulatrice personnalisée des maladies cardiovasculaires

Indoleamine 2 3-dioxygenase knockout limits angiotensin II-induced aneurysm in low density lipoprotein receptor-deficient mice fed with high fat diet.

AIMS: Abdominal aortic aneurysm (AAA) is an age-associated disease characterized by chronic inflammation, vascular cell apoptosis and metalloproteinase-mediated extracellular matrix degradation. Despite considerable progress in identifying targets involved in these processes, therapeutic approaches aiming to reduce aneurysm growth and rupture are still scarce. Indoleamine 2-3 dioxygenase 1 (IDO) is the first and rate-limiting enzyme involved in the conversion of tryptophan (Trp) into kynurenine (Kyn) pathway. In this study, we investigated the role of IDO in two different models of AAA in mice. METHODS AND RESULTS: Mice with deficiencies in both low density receptor-deficient (Ldlr-/-) and IDO (Ldlr-/-Ido1-/-) were generated by cross-breeding Ido1-/- mice with Ldlr-/-mice. To induce aneurysm, these mice were infused with angiotensin II (Ang II) (1000 ng/min/kg) and fed with high fat diet (HFD) during 28 days. AAAs were present in almost all Ldlr-/- infused with AngII, but only in 50% of Ldlr-/-Ido1-/- mice. Immunohistochemistry at an early time point (day 7) revealed no changes in macrophage and T lymphocyte infiltration within the vessel wall, but showed reduced apoptosis, as assessed by TUNEL assay, and increased α-actin staining within the media of Ldlr-/-Ido1-/- mice, suggesting enhanced survival of vascular smooth muscle cells (VSMCs) in the absence of IDO. In another model of elastase-induced AAA in C57Bl/6 mice, IDO deficiency had no effect on aneurysm formation. CONCLUSION: Our study showed that the knockout of IDO prevented VSMC apoptosis in AngII -treated Ldlr-/- mice fed with HFD, suggesting a detrimental role of IDO in AAA formation and thus would be an important target for the treatment of aneurysm