ABSTRACT
BACKGROUND: Atherosclerosis is driven by the infiltration of the arterial intima by diverse immune cells and smooth muscle cells (SMCs). CD8+ T cells promote lesion growth during atherosclerotic lesion development, but their role in advanced atherosclerosis is less clear. Here, we studied the role of CD8+ T cells and their effects on SMCs in established atherosclerosis. METHODS: CD8+ T cells were depleted in (SMC reporter) low-density lipoprotein receptor-deficient (Ldlr-/-) mice with established atherosclerotic lesions. Atherosclerotic lesion formation was examined, and single-cell RNA sequencing of aortic SMCs and their progeny was performed. Additionally, coculture experiments with primary aortic SMCs and CD8+ T cells were conducted. RESULTS: Although we could not detect differences in atherosclerotic lesion size, an increased plaque SMC content was noted in mice after CD8+ T-cell depletion. Single-cell RNA sequencing of aortic lineage-traced SMCs revealed contractile SMCs and a modulated SMC cluster, expressing macrophage- and osteoblast-related genes. CD8+ T-cell depletion was associated with an increased contractile but decreased macrophage and osteoblast-like gene signature in this modulated aortic SMC cluster. Conversely, exposure of isolated aortic SMCs to activated CD8+ T cells decreased the expression of genes indicative of a contractile SMC phenotype and induced a macrophage and osteoblast-like cell state. Notably, CD8+ T cells triggered calcium deposits in SMCs under osteogenic conditions. Mechanistically, we identified transcription factors highly expressed in modulated SMCs, including Runx1, to be induced by CD8+ T cells in cultured SMCs in an IFNγ (interferon-γ)-dependent manner. CONCLUSIONS: We here uncovered CD8+ T cells to control the SMC phenotype in atherosclerosis. CD8+ T cells promote SMC dedifferentiation and drive SMCs to adopt features of macrophage-like and osteoblast-like, procalcifying cell phenotypes. Given the critical role of SMCs in atherosclerotic plaque stability, CD8+ T cells could thus be explored as therapeutic target cells during lesion progression.
Subject(s)
Atherosclerosis , CD8-Positive T-Lymphocytes , Cell Dedifferentiation , Disease Models, Animal , Muscle, Smooth, Vascular , Myocytes, Smooth Muscle , Plaque, Atherosclerotic , Animals , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Myocytes, Smooth Muscle/pathology , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/immunology , Mice , Atherosclerosis/pathology , Atherosclerosis/metabolism , Atherosclerosis/genetics , Atherosclerosis/immunology , Muscle, Smooth, Vascular/pathology , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/immunology , Mice, Inbred C57BL , Mice, Knockout , Cells, Cultured , Male , Receptors, LDL/genetics , Receptors, LDL/deficiency , Phenotype , Core Binding Factor Alpha 2 Subunit/genetics , Core Binding Factor Alpha 2 Subunit/metabolism , Aorta/pathology , Aorta/immunology , Aorta/metabolism , Coculture Techniques , Aortic Diseases/pathology , Aortic Diseases/genetics , Aortic Diseases/immunology , Aortic Diseases/metabolismABSTRACT
Endothelial dysfunction is cause and consequence of cardiovascular diseases. The endothelial hormone C-type natriuretic peptide (CNP) regulates vascular tone and the vascular barrier. Its cGMP-synthesizing guanylyl cyclase-B (GC-B) receptor is expressed in endothelial cells themselves. To characterize the role of endothelial CNP/cGMP signaling, we studied mice with endothelial-selective GC-B deletion. Endothelial EC GC-B KO mice had thicker, stiffer aortae and isolated systolic hypertension. This was associated with increased proinflammatory E-selectin and VCAM-1 expression and impaired nitric oxide bioavailability. Atherosclerosis susceptibility was evaluated in such KO and control littermates on Ldlr (low-density lipoprotein receptor)-deficient background fed a Western diet for 10 weeks. Notably, the plaque areas and heights within the aortic roots were markedly increased in the double EC GC-B/Ldlr KO mice. This was accompanied by enhanced macrophage infiltration and greater necrotic cores, indicating unstable plaques. Finally, we found that EC GC-B KO mice had diminished vascular regeneration after critical hind-limb ischemia. Remarkably, all these genotype-dependent changes were only observed in female and not in male mice. Auto/paracrine endothelial CNP/GC-B/cGMP signaling protects from arterial stiffness, systolic hypertension, and atherosclerosis and improves reparative angiogenesis. Interestingly, our data indicate a sex disparity in the connection of diminished CNP/GC-B activity to endothelial dysfunction.
Subject(s)
Cyclic GMP , Mice, Knockout , Natriuretic Peptide, C-Type , Signal Transduction , Animals , Natriuretic Peptide, C-Type/metabolism , Natriuretic Peptide, C-Type/genetics , Cyclic GMP/metabolism , Mice , Male , Female , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , Atherosclerosis/metabolism , Atherosclerosis/genetics , Atherosclerosis/pathology , Receptors, Atrial Natriuretic Factor/metabolism , Receptors, Atrial Natriuretic Factor/genetics , Endothelial Cells/metabolism , Receptors, LDL/metabolism , Receptors, LDL/genetics , Paracrine Communication , Hypertension/metabolism , Hypertension/genetics , Mice, Inbred C57BL , Aorta/metabolism , Aorta/pathologyABSTRACT
OBJECTIVE: Activated perivascular mast cells (MCs) participate in different cardiovascular diseases. Many factors provoking MC degranulation have been described, while physiological counterregulators are barely known. Endothelial CNP (C-type natriuretic peptide) participates in the maintenance of vascular barrier integrity, but the target cells and mechanisms are unclear. Here, we studied whether MCs are regulated by CNP. Approach and Results: In cultured human and murine MCs, CNP activated its specific GC (guanylyl cyclase)-B receptor and cyclic GMP signaling. This enhanced cyclic GMP-dependent phosphorylation of the cytoskeleton-associated VASP (vasodilator-stimulated phosphoprotein) and inhibited ATP-evoked degranulation. To elucidate the relevance in vivo, mice with a floxed GC-B (Npr2) gene were interbred with a Mcpt5-CreTG line to generate mice lacking GC-B in connective tissue MCs (MC GC-B knockout). In anesthetized mice, acute ischemia-reperfusion of the cremaster muscle microcirculation provoked extensive MC degranulation and macromolecule extravasation. Superfusion of CNP markedly prevented MC activation and endothelial barrier disruption in control but not in MC GC-B knockout mice. Notably, already under resting conditions, such knockout mice had increased numbers of degranulated MCs in different tissues, together with elevated plasma chymase levels. After transient coronary occlusion, their myocardial areas at risk and with infarction were enlarged. Moreover, MC GC-B knockout mice showed augmented perivascular neutrophil infiltration and deep vein thrombosis in a model of inferior vena cava ligation. CONCLUSIONS: CNP, via GC-B/cyclic GMP signaling, stabilizes resident perivascular MCs at baseline and prevents their excessive activation under pathological conditions. Thereby CNP contributes to the maintenance of vascular integrity in physiology and disease.
Subject(s)
Cell Degranulation , Endothelial Cells/metabolism , Mast Cells/metabolism , Myocardial Reperfusion Injury/metabolism , Natriuretic Peptide, C-Type/metabolism , Paracrine Communication , Receptors, Atrial Natriuretic Factor/metabolism , Thrombosis/metabolism , Adenosine Triphosphate/metabolism , Animals , Capillary Permeability , Cell Adhesion Molecules/metabolism , Cell Degranulation/drug effects , Cell Line , Cyclic GMP/metabolism , Disease Models, Animal , Endothelial Cells/drug effects , Endothelial Cells/pathology , Mast Cells/drug effects , Mast Cells/pathology , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Microfilament Proteins/metabolism , Myocardial Reperfusion Injury/genetics , Myocardial Reperfusion Injury/pathology , Natriuretic Peptide, C-Type/pharmacology , Neutrophil Infiltration , Phosphoproteins/metabolism , Phosphorylation , Receptors, Atrial Natriuretic Factor/agonists , Receptors, Atrial Natriuretic Factor/genetics , Signal Transduction , Thrombosis/genetics , Thrombosis/pathologyABSTRACT
AIMS: Atherosclerosis is a chronic inflammatory disease of the vessel wall controlled by local and systemic immune responses. The role of interleukin-23 receptor (IL-23R), expressed in adaptive immune cells (mainly T-helper 17 cells) and γδ T cells, in atherosclerosis is only incompletely understood. Here, we investigated the vascular cell types expressing IL-23R and addressed the function of IL-23R and γδ T cells in atherosclerosis. METHODS AND RESULTS: IL-23R+ cells were frequently found in the aortic root in contrast to the aorta in low-density lipoprotein receptor deficient IL-23R reporter mice (Ldlr-/-Il23rgfp/+), and mostly identified as γδ T cells that express IL-17 and GM-CSF. scRNA-seq confirmed γδ T cells as the main cell type expressing Il23r and Il17a in the aorta. Ldlr-/-Il23rgfp/gfp mice deficient in IL-23R showed a loss of IL-23R+ cells in the vasculature, and had reduced atherosclerotic lesion formation in the aortic root compared to Ldlr-/- controls after 6 weeks of high-fat diet feeding. In contrast, Ldlr-/-Tcrδ-/- mice lacking all γδ T cells displayed unaltered early atherosclerotic lesion formation compared to Ldlr-/- mice. In both HFD-fed Ldlr-/-Il23rgfp/gfp and Ldlr-/-Tcrδ-/- mice a reduction in the plaque necrotic core area was noted as well as an expansion of splenic regulatory T cells. In vitro, exposure of bone marrow-derived macrophages to both IL-17A and GM-CSF induced cell necrosis, and necroptotic RIP3K and MLKL expression, as well as inflammatory mediators. CONCLUSIONS: IL-23R+ γδ T cells are predominantly found in the aortic root rather than the aorta and promote early atherosclerotic lesion formation, plaque necrosis, and inflammation at this site. Targeting IL-23R may thus be explored as a therapeutic approach to mitigate atherosclerotic lesion development.