ABSTRACT
BACKGROUND: Exposure to ionizing radiation has been linked to cardiovascular diseases. However, the impact of moderate doses of radiation on abdominal aortic aneurysm (AAA) remains unknown. METHODS: Angiotensin II-infused Apoe-/- mice were irradiated (acute, 1 Gray) either 3 days before (Day-3) or 1 day after (Day+1) pomp implantation. Isolated primary aortic vascular smooth muscle cells (VSMCs) were irradiated (acute 1 Gray) for mechanistic studies and functional testing in vitro. RESULTS: Day-3 and Day+1 irradiation resulted in a significant reduction in aorta dilation (Control: 1.39+/-0.12; Day-3: 1.12+/-0.11; Day+1: 1.15+/-0.08 mm, P<0.001) and AAA incidence (Control: 81.0%; Day-3: 33.3%, Day+1: 53.3%) compared to the non-irradiated group. Day-3 and Day+1 irradiation led to an increase in collagen content in the adventitia (Thickness control: 23.64+/-2.9; Day-3: 54.39+/-15.5; Day+1 37.55+/-10.8 mm, P = 0.006). However, the underlying protective mechanisms were different between Day-3 and Day+1 groups. Irradiation before Angiotensin II (AngII) infusion mainly modulated vascular smooth muscle cell (VSMC) phenotype with a decrease in contractile profile and enhanced proliferative and migratory activity. Irradiation after AngII infusion led to an increase in macrophage content with a local anti-inflammatory phenotype characterized by the upregulation of M2-like gene and IL-10 expression. CONCLUSION: Moderate doses of ionizing radiation mitigate AAA either through VSCM phenotype or inflammation modulation, depending on the time of irradiation.
Subject(s)
Angiotensin II , Aortic Aneurysm, Abdominal , Muscle, Smooth, Vascular , Myocytes, Smooth Muscle , Radiation, Ionizing , Animals , Aortic Aneurysm, Abdominal/pathology , Aortic Aneurysm, Abdominal/metabolism , Aortic Aneurysm, Abdominal/etiology , Mice , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/radiation effects , Muscle, Smooth, Vascular/pathology , Angiotensin II/pharmacology , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/radiation effects , Myocytes, Smooth Muscle/pathology , Male , Disease Models, Animal , Interleukin-10/metabolism , Interleukin-10/genetics , Collagen/metabolism , Cell Proliferation/radiation effectsABSTRACT
BACKGROUND: The long isoform of the Wnk1 (with-no-lysine [K] kinase 1) is a ubiquitous serine/threonine kinase, but its role in vascular smooth muscle cells (VSMCs) pathophysiology remains unknown. METHODS: AngII (angiotensin II) was infused in Apoe-/- to induce experimental aortic aneurysm. Mice carrying an Sm22-Cre allele were cross-bred with mice carrying a floxed Wnk1 allele to specifically investigate the functional role of Wnk1 in VSMCs. RESULTS: Single-cell RNA-sequencing of the aneurysmal abdominal aorta from AngII-infused Apoe-/- mice revealed that VSMCs that did not express Wnk1 showed lower expression of contractile phenotype markers and increased inflammatory activity. Interestingly, WNK1 gene expression in VSMCs was decreased in human abdominal aortic aneurysm. Wnk1-deficient VSMCs lost their contractile function and exhibited a proinflammatory phenotype, characterized by the production of matrix metalloproteases, as well as cytokines and chemokines, which contributed to local accumulation of inflammatory macrophages, Ly6Chi monocytes, and γδ T cells. Sm22Cre+Wnk1lox/lox mice spontaneously developed aortitis in the infrarenal abdominal aorta, which extended to the thoracic area over time without any negative effect on long-term survival. AngII infusion in Sm22Cre+Wnk1lox/lox mice aggravated the aortic disease, with the formation of lethal abdominal aortic aneurysms. Pharmacological blockade of γδ T-cell recruitment using neutralizing anti-CXCL9 (anti-CXC motif chemokine ligand 9) antibody treatment, or of monocyte/macrophage using Ki20227, a selective inhibitor of CSF1 receptor, attenuated aortitis. Wnk1 deletion in VSMCs led to aortic wall remodeling with destruction of elastin layers, increased collagen content, and enhanced local TGF-ß (transforming growth factor-beta) 1 expression. Finally, in vivo TGF-ß blockade using neutralizing anti-TGF-ß antibody promoted saccular aneurysm formation and aorta rupture in Sm22 Cre+ Wnk1lox/lox mice but not in control animals. CONCLUSION: Wnk1 is a key regulator of VSMC function. Wnk1 deletion promotes VSMC phenotype switch toward a pathogenic proinflammatory phenotype, orchestrating deleterious vascular remodeling and spontaneous severe aortitis in mice.
Subject(s)
Angiotensin II , Aortic Aneurysm, Abdominal , Aortitis , Muscle, Smooth, Vascular , Myocytes, Smooth Muscle , WNK Lysine-Deficient Protein Kinase 1 , Animals , Aortitis/genetics , Aortitis/metabolism , Aortitis/pathology , Mice , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/pathology , Aortic Aneurysm, Abdominal/genetics , Aortic Aneurysm, Abdominal/metabolism , Aortic Aneurysm, Abdominal/pathology , Humans , WNK Lysine-Deficient Protein Kinase 1/genetics , WNK Lysine-Deficient Protein Kinase 1/metabolism , Mice, Inbred C57BL , Male , Cells, Cultured , Mice, Knockout, ApoE , Disease Models, Animal , Inflammation/metabolism , Inflammation/genetics , Inflammation/pathology , Aorta, Abdominal/metabolism , Aorta, Abdominal/pathologyABSTRACT
Tryptophan (Trp) is an essential amino acid, whose metabolism is a key gatekeeper of intestinal homeostasis. Yet, its systemic effects, particularly on atherosclerosis, remain unknown. Here we show that high-fat diet (HFD) increases the activity of intestinal indoleamine 2, 3-dioxygenase 1 (IDO), which shifts Trp metabolism from the production of microbiota-derived indole metabolites towards kynurenine production. Under HFD, the specific deletion of IDO in intestinal epithelial cells leads to intestinal inflammation, impaired intestinal barrier, augmented lesional T lymphocytes and atherosclerosis. This is associated with an increase in serotonin production and a decrease in indole metabolites, thus hijacking Trp for the serotonin pathway. Inhibition of intestinal serotonin production or supplementation with indole derivatives alleviates plaque inflammation and atherosclerosis. In summary, we uncover a pivotal role of intestinal IDO in the fine-tuning of Trp metabolism with systemic effects on atherosclerosis, paving the way for new therapeutic strategies to relieve gut-associated inflammatory diseases.
Subject(s)
Atherosclerosis , Diet, High-Fat , Indoleamine-Pyrrole 2,3,-Dioxygenase , Intestinal Mucosa , Mice, Inbred C57BL , Serotonin , Tryptophan , Animals , Tryptophan/metabolism , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Indoleamine-Pyrrole 2,3,-Dioxygenase/genetics , Atherosclerosis/metabolism , Atherosclerosis/pathology , Atherosclerosis/genetics , Atherosclerosis/drug therapy , Diet, High-Fat/adverse effects , Mice , Serotonin/metabolism , Intestinal Mucosa/metabolism , Kynurenine/metabolism , Male , Gastrointestinal Microbiome , Indoles/pharmacology , Inflammation/metabolism , Mice, Knockout , Intestines/pathology , T-Lymphocytes/metabolism , T-Lymphocytes/immunology , Disease Models, AnimalABSTRACT
Caspase recruitment-domain containing protein 9 (CARD9) is a key signaling pathway in macrophages but its role in atherosclerosis is still poorly understood. Global deletion of Card9 in Apoe-/- mice as well as hematopoietic deletion in Ldlr-/- mice increases atherosclerosis. The acceleration of atherosclerosis is also observed in Apoe-/-Rag2-/-Card9-/- mice, ruling out a role for the adaptive immune system in the vascular phenotype of Card9 deficient mice. Card9 deficiency alters macrophage phenotype through CD36 overexpression with increased IL-1ß production, increased lipid uptake, higher cell death susceptibility and defective autophagy. Rapamycin or metformin, two autophagy inducers, abolish intracellular lipid overload, restore macrophage survival and autophagy flux in vitro and finally abolish the pro-atherogenic effects of Card9 deficiency in vivo. Transcriptomic analysis of human CARD9-deficient monocytes confirms the pathogenic signature identified in murine models. In summary, CARD9 is a key protective pathway in atherosclerosis, modulating macrophage CD36-dependent inflammatory responses, lipid uptake and autophagy.
Subject(s)
Atherosclerosis , Humans , Animals , Mice , Atherosclerosis/metabolism , Autophagy/genetics , Apolipoproteins E/genetics , Lipids , CARD Signaling Adaptor Proteins/metabolism , Mice, Knockout , Mice, Inbred C57BLSubject(s)
Bone Marrow , Macrophage Colony-Stimulating Factor , Monocytes , Humans , Bone Marrow/metabolism , Bone Marrow Cells/metabolism , Cells, Cultured , Endothelial Cells , Mesenchymal Stem Cells , Monocytes/metabolism , Stem Cell Niche , Stromal Cells , Macrophage Colony-Stimulating Factor/metabolismABSTRACT
JAK2V617F mutation is associated with an increased risk for athero-thrombotic cardiovascular disease, but its role in aortic disease development and complications remains unknown. In a cohort of patients with myeloproliferative neoplasm, JAK2V617F mutation was identified as an independent risk factor for dilation of both the ascending and descending thoracic aorta. Using single-cell RNA-seq, complementary genetically-modified mouse models, as well as pharmacological approaches, we found that JAK2V617F mutation was associated with a pathogenic pro-inflammatory phenotype of perivascular tissue-resident macrophages, which promoted deleterious aortic wall remodeling at early stages, and dissecting aneurysm through the recruitment of circulating monocytes at later stages. Finally, genetic manipulation of tissue-resident macrophages, or treatment with a Jak2 inhibitor, ruxolitinib, mitigated aortic wall inflammation and reduced aortic dilation and rupture. Overall, JAK2V617F mutation drives vascular resident macrophages toward a pathogenic phenotype and promotes dissecting aortic aneurysm.
Subject(s)
Aortic Aneurysm , Aortic Dissection , Mice , Animals , Aortic Dissection/pathology , Phenotype , Mutation , Macrophages/pathology , Aortic Aneurysm/genetics , Aortic Aneurysm/complicationsABSTRACT
Peripheral arterial disease occurs more frequently and has a worse prognosis in patients with chronic kidney disease (CKD). The receptor for advanced glycation end products (RAGE) is involved in multiple aspects of uremia-associated vasculopathy. Previous data suggest that the RAGE pathway may promote soluble fms-like tyrosine kinase 1 (sFlt1) production, an anti-angiogenic molecule. Thus, we tested the hypothesis that the deletion of AgeR would decrease sFlt1 production and improve post-ischemic revascularization in uremic condition. We used a well-established CKD model (5/6 nephrectomy) in WT and AgeR-/- C57/Bl6 mice. Hindlimb ischemia was induced by femoral artery ligation. Revascularization was evaluated by complementary approaches: ischemic limb retraction, LASCA imagery, and capillary density. The production of sFlt1 was assessed at both RNA and protein levels. After hindlimb ischemia, uremic mice showed slower functional recovery (p < 0.01), decreased reperfusion (p < 0.01), lower capillary density (p = 0.02), and increased circulating sFlt1 levels (p = 0.03). AgeR deletion restored post-ischemic angiogenesis and was protective from sFlt1 increase in uremic mice. These findings show the main role of RAGE in post-ischemic angiogenesis impairment associated with CKD. RAGE may represent a key target for building new therapeutic approaches to improve the outcome of CKD patients with PAD.
Subject(s)
Gene Deletion , Ischemia/complications , Neovascularization, Physiologic , Receptor for Advanced Glycation End Products/deficiency , Uremia/complications , Vascular Endothelial Growth Factor Receptor-1/biosynthesis , Animals , Biomarkers/blood , Cell Line , Humans , Ligands , Male , Mice, Inbred C57BL , RNA/metabolism , Receptor for Advanced Glycation End Products/metabolism , Solubility , Up-RegulationABSTRACT
The triggering receptor expressed on myeloid cells 1 (TREM-1) drives inflammatory responses in several cardiovascular diseases but its role in abdominal aortic aneurysm (AAA) remains unknown. Our objective was to explore the role of TREM-1 in a mouse model of angiotensin II-induced (AngII-induced) AAA. TREM-1 expression was detected in mouse aortic aneurysm and colocalized with macrophages. Trem1 gene deletion (Apoe-/-Trem1-/-), as well as TREM-1 pharmacological blockade with LR-12 peptide, limited both AAA development and severity. Trem1 gene deletion attenuated the inflammatory response in the aorta, with a reduction of Il1b, Tnfa, Mmp2, and Mmp9 mRNA expression, and led to a decreased macrophage content due to a reduction of Ly6Chi classical monocyte trafficking. Conversely, antibody-mediated TREM-1 stimulation exacerbated Ly6Chi monocyte aorta infiltration after AngII infusion through CD62L upregulation and promoted proinflammatory signature in the aorta, resulting in worsening AAA severity. AngII infusion stimulated TREM-1 expression and activation on Ly6Chi monocytes through AngII receptor type I (AT1R). In human AAA, TREM-1 was detected and TREM1 mRNA expression correlated with SELL mRNA expression. Finally, circulating levels of sTREM-1 were increased in patients with AAA when compared with patients without AAA. In conclusion, TREM-1 is involved in AAA pathophysiology and may represent a promising therapeutic target in humans.
Subject(s)
Angiotensin II/adverse effects , Aortic Aneurysm, Abdominal/metabolism , Cell Movement/drug effects , Monocytes/metabolism , Triggering Receptor Expressed on Myeloid Cells-1/metabolism , Angiotensin II/pharmacology , Animals , Aortic Aneurysm, Abdominal/chemically induced , Aortic Aneurysm, Abdominal/genetics , Aortic Aneurysm, Abdominal/pathology , Cell Movement/genetics , Gene Deletion , Humans , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 2/metabolism , Mice , Mice, Knockout, ApoE , Monocytes/pathology , Triggering Receptor Expressed on Myeloid Cells-1/genetics , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolismABSTRACT
BACKGROUND: Cell therapy has been proposed for patients with critical limb ischemia (CLI). Autologous bone marrow derived cells (BMCs) have been mostly used, mesenchymal stem cells (MSCs) being an alternative. The aim of this study was to characterize two types of MSCs and evaluate their efficacy. METHODS: MSCs were obtained from CLI-patients BMCs. Stimulated- (S-) MSCs were cultured in endothelial growth medium. Cells were characterized by the expression of cell surface markers, the relative expression of 6 genes, the secretion of 10 cytokines and the ability to form vessel-like structures. The cell proangiogenic properties was analysed in vivo, in a hindlimb ischemia model. Perfusion of lower limbs and functional tests were assessed for 28 days after cell infusion. Muscle histological analysis (neoangiogenesis, arteriogenesis and muscle repair) was performed. RESULTS: S-MSCs can be obtained from CLI-patients BMCs. They do not express endothelial specific markers but can be distinguished from MSCs by their secretome. S-MSCs have the ability to form tube-like structures and, in vivo, to induce blood flow recovery. No amputation was observed in S-MSCs treated mice. Functional tests showed improvement in treated groups with a superiority of MSCs and S-MSCs. In muscles, CD31+ and αSMA+ labelling were the highest in S-MSCs treated mice. S-MSCs induced the highest muscle repair. CONCLUSIONS: S-MSCs exert angiogenic potential probably mediated by a paracrine mechanism. Their administration is associated with flow recovery, limb salvage and muscle repair. The secretome from S-MSCs or secretome-derived products may have a strong potential in vessel regeneration and muscle repair. Trial registration NCT00533104.
Subject(s)
Culture Media/pharmacology , Endothelial Cells/cytology , Extremities/blood supply , Ischemia/therapy , Mesenchymal Stem Cells/cytology , Adult , Aged , Animals , Arteries/growth & development , Cells, Cultured , Endothelial Cells/drug effects , Extremities/pathology , Female , Hindlimb/blood supply , Humans , Ischemia/pathology , Male , Mesenchymal Stem Cell Transplantation , Mice, Inbred BALB C , Mice, Nude , Middle Aged , Muscles/blood supply , Muscles/pathology , Neovascularization, Physiologic , Organogenesis , Regional Blood FlowABSTRACT
We have investigated the development of murine hindlimb ischemia from day 1 to day 55 after femoral artery ligation (FAL) using blood flow analysis, functional tests, histopathological staining, and in vivo Raman spectroscopy. FAL resulted in hindlimb blood deprivation and the loss of functionality as attested by the blood flow analysis and functional tests, respectively. The limbs recovered a normal circulation progressively without recovering complete functionality. Histological analysis showed changes in the morphology of muscle fibers with intense inflammation. From day 22 to day 55 post-ischemia, regeneration of the myofibers was observed. Raman spectroscopic results related to subcutaneous analysis made the identification of modification in the biochemical constituents of hindlimb muscles possible during disease progression. Ischemia was characterized by a quantitative increase in the lipid content and a decrease in the protein content. The lipid to protein ratio can be used as a spectroscopic marker to score the severity of ischemia. Multivariate statistical analysis PC-LDA (Principal Component-Linear Discriminant Analysis) was used to classify all the data measured for the normal and ischemic tissues. This classification illustrated an excellent separation between the control and ischemic tissues at any time during the course of ischemic development. In vivo Raman spectroscopy was then applied to assess the potential of this technique as a screening tool to explore an ischemic disease non-invasively (transcutaneously). For this purpose, the influence of skin on the diagnostic accuracy was evaluated; transcutaneous analysis revealed the accuracy of this technique, indicating its potential in the in situ monitoring of muscle structural changes during ischemia.
Subject(s)
Hindlimb/metabolism , Ischemia/diagnosis , Ischemia/metabolism , Muscle Fibers, Skeletal/metabolism , Skin/metabolism , Animals , Blood Circulation/physiology , Discriminant Analysis , Hindlimb/blood supply , Male , Mice, Inbred BALB C , Multivariate Analysis , Muscle Fibers, Skeletal/pathology , Spectrum Analysis, Raman/methodsABSTRACT
BACKGROUND: Cell therapy has been proposed as a salvage limb procedure in critical limb ischemia (CLI). In spite of the fact that clinical trials found some efficacy, the mechanism of action remains elusive. The objective of this study was to characterize two autologous cell therapy products (CTPs) obtained from patients with advanced peripheral arterial disease. METHODS: Bone marrow (BM-CTPs) (n = 20) and CTPs obtained by non-mobilized cytapheresis (peripheral blood [PB]-CTPs) (n = 20) were compared. CTPs were characterized by their cell composition, by the quantification of endothelial progenitor cells (EPCs) and mesenchymal stromal cells (MSCs) and by transcriptomic profiling. The angiogenic profile and the 6-month outcome of CLI patients are described. RESULTS: Patients presented inflammation syndrome and high levels of CXCL12, soluble stem cell factor and granulocyte colony-stimulating factor, whereas granulocyte macrophage colony-stimulating factor was low. Circulating CD34+ cells represented rare events. BM and PB-CTPs were heterogeneous. Mature cells and colony-forming unit-endothelial cells were in higher concentration in PB-CTPs, whereas CD34+ stem cells and EPCs were more abundant in BM-CTPs. MSCs were identified in both CTPs. Transcriptomic profiling revealed the strong angiogenic potential of BM-CTPs. Transcutaneous partial pressure of oxygen, C-reative protein and neutrophil content in CTPs are predictive of the clinical outcome at 6 months. DISCUSSION: Transcriptomic allows an accurate characterization of CTPs. BM-CTPs have the richest content in terms of stem cells and transcriptome. The high content of mature cells in PB-CTPs means that they work via a paracrine mechanism. The clinical outcome indicates the deleterious influence the patients' status and the limits of an autologous approach. In this respect, MSCs may allow an allogenic strategy.