ABSTRACT
Cardiovascular disease is the leading cause of death worldwide. Advanced insights into disease mechanisms and therapeutic strategies require a deeper understanding of the molecular processes involved in the healthy heart. Knowledge of the full repertoire of cardiac cells and their gene expression profiles is a fundamental first step in this endeavour. Here, using state-of-the-art analyses of large-scale single-cell and single-nucleus transcriptomes, we characterize six anatomical adult heart regions. Our results highlight the cellular heterogeneity of cardiomyocytes, pericytes and fibroblasts, and reveal distinct atrial and ventricular subsets of cells with diverse developmental origins and specialized properties. We define the complexity of the cardiac vasculature and its changes along the arterio-venous axis. In the immune compartment, we identify cardiac-resident macrophages with inflammatory and protective transcriptional signatures. Furthermore, analyses of cell-to-cell interactions highlight different networks of macrophages, fibroblasts and cardiomyocytes between atria and ventricles that are distinct from those of skeletal muscle. Our human cardiac cell atlas improves our understanding of the human heart and provides a valuable reference for future studies.
Subject(s)
Myocardium/cytology , Single-Cell Analysis , Transcriptome , Adipocytes/classification , Adipocytes/metabolism , Adult , Angiotensin-Converting Enzyme 2/analysis , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Epithelial Cells/classification , Epithelial Cells/metabolism , Epithelium , Female , Fibroblasts/classification , Fibroblasts/metabolism , Gene Expression Profiling , Genome-Wide Association Study , Heart Atria/anatomy & histology , Heart Atria/cytology , Heart Atria/innervation , Heart Ventricles/anatomy & histology , Heart Ventricles/cytology , Heart Ventricles/innervation , Homeostasis/immunology , Humans , Macrophages/immunology , Macrophages/metabolism , Male , Muscle, Skeletal/cytology , Muscle, Skeletal/metabolism , Myocytes, Cardiac/classification , Myocytes, Cardiac/metabolism , Neurons/classification , Neurons/metabolism , Pericytes/classification , Pericytes/metabolism , Receptors, Coronavirus/analysis , Receptors, Coronavirus/genetics , Receptors, Coronavirus/metabolism , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Stromal Cells/classification , Stromal Cells/metabolismABSTRACT
RATIONALE: The efficient resolution of tissue hemorrhage is an important homeostatic function. In human macrophages in vitro, heme activates an AMPK (AMP-activated protein kinase)/ATF1 (activating transcription factor-1) pathway that directs Mhem macrophages through coregulation of HO-1 (heme oxygenase-1; HMOX1) and lipid homeostasis genes. OBJECTIVE: We asked whether this pathway had an in vivo role in mice. METHODS AND RESULTS: Perifemoral hematomas were used as a model of hematoma resolution. In mouse bone marrow-derived macrophages, heme induced HO-1, lipid regulatory genes including LXR (lipid X receptor), the growth factor IGF1 (insulin-like growth factor-1), and the splenic red pulp macrophage gene Spic. This response was lost in bone marrow-derived macrophages from mice deficient in AMPK (Prkab1-/-) or ATF1 (Atf1-/-). In vivo, femoral hematomas resolved completely between days 8 and 9 in littermate control mice (n=12), but were still present at day 9 in mice deficient in either AMPK (Prkab1-/-) or ATF1 (Atf1-/-; n=6 each). Residual hematomas were accompanied by increased macrophage infiltration, inflammatory activation and oxidative stress. We also found that fluorescent lipids and a fluorescent iron-analog were trafficked to lipid-laden and iron-laden macrophages respectively. Moreover erythrocyte iron and lipid abnormally colocalized in the same macrophages in Atf1-/- mice. Therefore, iron-lipid separation was Atf1-dependent. CONCLUSIONS: Taken together, these data demonstrate that both AMPK and ATF1 are required for normal hematoma resolution. Graphic Abstract: An online graphic abstract is available for this article.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Activating Transcription Factor 1/metabolism , Hematoma/metabolism , Macrophages/metabolism , AMP-Activated Protein Kinases/genetics , Activating Transcription Factor 1/genetics , Animals , Cells, Cultured , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Disease Models, Animal , Erythrocytes/metabolism , Female , Hematoma/genetics , Heme Oxygenase-1/genetics , Heme Oxygenase-1/metabolism , Insulin-Like Growth Factor I/genetics , Insulin-Like Growth Factor I/metabolism , Iron/metabolism , Lipid Metabolism , Liver X Receptors/genetics , Liver X Receptors/metabolism , Male , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice, Inbred C57BL , Mice, Knockout , Oxidative Stress , Time FactorsABSTRACT
Heart failure is the common final pathway of several cardiovascular conditions and a major cause of morbidity and mortality worldwide. Aberrant activation of the adaptive immune system in response to myocardial necrosis has recently been implicated in the development of heart failure. The ß-adrenergic agonist isoproterenol hydrochloride is used for its cardiac effects in a variety of different dosing regimens with high doses causing acute cardiomyocyte necrosis. To assess whether isoproterenol-induced cardiomyocyte necrosis triggers an adaptive immune response against the heart, we treated C57BL/6J mice with a single intraperitoneal injection of isoproterenol. We confirmed tissue damage reminiscent of human type 2 myocardial infarction. This is followed by an adaptive immune response targeting the heart as demonstrated by the activation of T cells, the presence of anti-heart auto-antibodies in the serum as late as 12 weeks after initial challenge and IgG deposition in the myocardium. All of these are hallmark signs of an established autoimmune response. Adoptive transfer of splenocytes from isoproterenol-treated mice induces left ventricular dilation and impairs cardiac function in healthy recipients. In summary, a single administration of a high dose of isoproterenol is a suitable high-throughput model for future studies of the pathological mechanisms of anti-heart autoimmunity and to test potential immunomodulatory therapeutic approaches.
Subject(s)
Adaptive Immunity , Myocardial Infarction/immunology , Myocardium/pathology , Adoptive Transfer , Animals , Dendritic Cells/immunology , Disease Models, Animal , Female , Fibrosis , Heart Ventricles/pathology , Heart Ventricles/physiopathology , Isoproterenol , Leukocyte Common Antigens/metabolism , Male , Mice, Inbred C57BL , Myocardial Infarction/physiopathology , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Necrosis , Organ Specificity , Spleen/immunology , Systole , T-Lymphocytes, Helper-Inducer/immunology , VasodilationABSTRACT
Heme oxygenase-1 (HO-1) is a vital enzyme in humans that primarily regulates free heme concentrations. The overexpression of HO-1 is commonly associated with cardiovascular and neurodegenerative diseases including atherosclerosis and ischemic stroke. Currently, there are no known chemical probes to detect HO-1 activity, limiting its potential as an early diagnostic/prognostic marker in these serious diseases. Reported here are the design, synthesis, and photophysical and biological characterization of a coumarin-porphyrin FRET break-apart probe to detect HO-1 activity, Fe-L1. We designed Fe-L1 to "break-apart" upon HO-1-catalyzed porphyrin degradation, perturbing the efficient FRET mechanism from a coumarin donor to a porphyrin acceptor fluorophore. Analysis of HO-1 activity using Escherichia coli lysates overexpressing hHO-1 found that a 6-fold increase in emission intensity at 383 nm was observed following incubation with NADPH. The identities of the degradation products following catabolism were confirmed by MALDI-MS and LC-MS, showing that porphyrin catabolism was regioselective at the α-position. Finally, through the analysis of Fe-L2, we have shown that close structural analogues of heme are required to maintain HO-1 activity. It is anticipated that this work will act as a foundation to design and develop new probes for HO-1 activity in the future, moving toward applications of live fluorescent imaging.
Subject(s)
Coumarins/chemistry , Fluorescence Resonance Energy Transfer/methods , Heme Oxygenase-1/chemistry , Protoporphyrins/chemistry , Escherichia coli/enzymology , Escherichia coli/genetics , Heme Oxygenase-1/analysis , Heme Oxygenase-1/genetics , Heme Oxygenase-1/metabolism , Humans , Photochemical Processes , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Spectrophotometry, UltravioletABSTRACT
RATIONALE: The ETS (E-26 transformation-specific) transcription factor ERG (ETS-related gene) is essential for endothelial homeostasis, driving expression of lineage genes and repressing proinflammatory genes. Loss of ERG expression is associated with diseases including atherosclerosis. ERG's homeostatic function is lineage-specific, because aberrant ERG expression in cancer is oncogenic. The molecular basis for ERG lineage-specific activity is unknown. Transcriptional regulation of lineage specificity is linked to enhancer clusters (super-enhancers). OBJECTIVE: To investigate whether ERG regulates endothelial-specific gene expression via super-enhancers. METHODS AND RESULTS: Chromatin immunoprecipitation with high-throughput sequencing in human umbilical vein endothelial cells showed that ERG binds 93% of super-enhancers ranked according to H3K27ac, a mark of active chromatin. These were associated with endothelial genes such as DLL4 (Delta-like protein 4), CLDN5 (claudin-5), VWF (von Willebrand factor), and CDH5 (VE-cadherin). Comparison between human umbilical vein endothelial cell and prostate cancer TMPRSS2 (transmembrane protease, serine-2):ERG fusion-positive human prostate epithelial cancer cell line (VCaP) cells revealed distinctive lineage-specific transcriptome and super-enhancer profiles. At a subset of endothelial super-enhancers (including DLL4 and CLDN5), loss of ERG results in significant reduction in gene expression which correlates with decreased enrichment of H3K27ac and MED (Mediator complex subunit)-1, and reduced recruitment of acetyltransferase p300. At these super-enhancers, co-occupancy of GATA2 (GATA-binding protein 2) and AP-1 (activator protein 1) is significantly lower compared with super-enhancers that remained constant following ERG inhibition. These data suggest distinct mechanisms of super-enhancer regulation in endothelial cells and highlight the unique role of ERG in controlling a core subset of super-enhancers. Most disease-associated single nucleotide polymorphisms from genome-wide association studies lie within noncoding regions and perturb transcription factor recognition sequences in relevant cell types. Analysis of genome-wide association studies data shows significant enrichment of risk variants for cardiovascular disease and other diseases, at ERG endothelial enhancers and super-enhancers. CONCLUSIONS: The transcription factor ERG promotes endothelial homeostasis via regulation of lineage-specific enhancers and super-enhancers. Enrichment of cardiovascular disease-associated single nucleotide polymorphisms at ERG super-enhancers suggests that ERG-dependent transcription modulates disease risk.
Subject(s)
Enhancer Elements, Genetic/genetics , Gene Expression Profiling , Gene Expression Regulation , Cell Line, Tumor , Cells, Cultured , Claudin-5/genetics , Claudin-5/metabolism , GATA2 Transcription Factor/genetics , GATA2 Transcription Factor/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Male , Membrane Proteins/genetics , Membrane Proteins/metabolism , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Transcription Factor AP-1/genetics , Transcription Factor AP-1/metabolism , Transcriptional Regulator ERG/geneticsABSTRACT
Background and Purpose- Extracellular matrix proteins are important in atherosclerotic disease by influencing plaque stability and cellular behavior but also by regulating inflammation. COMP (cartilage oligomeric matrix protein) is present in healthy human arteries and expressed by smooth muscle cells. A recent study showed that transplantation of COMP-deficient bone marrow to apoE-/- mice increased atherosclerotic plaque formation, indicating a role for COMP also in bone marrow-derived cells. Despite the evidence of a role for COMP in murine atherosclerosis, knowledge is lacking about the role of COMP in human atherosclerotic disease. Methods- In the present study, we investigated if COMP was associated with a stable or a vulnerable human atherosclerotic plaque phenotype by analyzing 211 carotid plaques for COMP expression using immunohistochemistry. Results- Plaque area that stained positive for COMP was significantly larger in atherosclerotic plaques associated with symptoms (n=110) compared with asymptomatic plaques (n=101; 9.7% [4.7-14.3] versus 5.6% [2.8-9.8]; P=0.0002). COMP was positively associated with plaque lipids (r=0.32; P=0.000002) and CD68 cells (r=0.15; P=0.036) but was negatively associated with collagen (r=-0.16; P=0.024), elastin (r=-0.14; P=0.041), and smooth muscle cells (r=-0.25; P=0.0002). COMP was positively associated with CD163 (r=0.37; P=0.00000006), a scavenger receptor for hemoglobin/haptoglobin and a marker of Mhem macrophages, and with intraplaque hemorrhage, measured as glycophorin A staining (r=0.28; P=0.00006). Conclusions- The present study shows that COMP is associated to symptomatic carotid atherosclerosis, CD163-expressing cells, and a vulnerable atherosclerotic plaque phenotype in humans.
Subject(s)
Carotid Artery Diseases/metabolism , Cartilage Oligomeric Matrix Protein/metabolism , Macrophages/metabolism , Myocytes, Smooth Muscle/metabolism , Plaque, Atherosclerotic/metabolism , Animals , Antigens, CD/genetics , Antigens, CD/metabolism , Antigens, Differentiation, Myelomonocytic/genetics , Antigens, Differentiation, Myelomonocytic/metabolism , Bone Marrow Transplantation , Carotid Artery Diseases/genetics , Carotid Artery Diseases/pathology , Cartilage Oligomeric Matrix Protein/genetics , Female , Heterografts , Humans , Immunohistochemistry , Macrophages/pathology , Male , Mice , Mice, Knockout, ApoE , Myocytes, Smooth Muscle/pathology , Plaque, Atherosclerotic/genetics , Plaque, Atherosclerotic/pathology , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolismABSTRACT
Endothelial injury and dysfunction precede accelerated arterial disease in allograft vasculopathy and systemic autoimmune diseases and involve pathogenic Abs and complement. Recent reports suggest that switching to rapamycin from calcineurin antagonists reduces posttransplant vasculopathy and prolongs survival following cardiac transplantion. The majority of these patients also receive statin therapy. We examined potential mechanisms underlying this protective response in human endothelial cells and identified synergy between rapamycin and atorvastatin. Mechanistically, atorvastatin and rapamycin activated a protein kinase Cα, AMP-activated kinase, and CREB-dependent vasculoprotective pathway, which induced decay-accelerating factor (DAF) promoter activity via binding to the cAMP response element, mutation of which attenuated promoter activity. This response significantly increased endothelial cell surface DAF and enhanced protection against complement-mediated injury. Synergy with rapamycin was reproduced by simvastatin, whereas combining atorvastatin with cyclosporine or mycophenolate in place of rapamycin was ineffective. Importantly, synergy was reproduced in vivo, in which only atorvastatin and rapamycin therapy in combination was sufficient to induce DAF on murine aortic endothelium. We believe this pathway represents an important therapeutically inducible vasculoprotective mechanism for diseases mediated by pathogenic Abs and complement, including posttransplant vasculopathy and systemic lupus erythematosus. Although our study focuses on the vascular endothelium, the findings are likely to be broadly applicable, given the diverse cellular expression of DAF.
Subject(s)
Cytoprotection/drug effects , Endothelium, Vascular/drug effects , Heptanoic Acids/administration & dosage , Pyrroles/administration & dosage , Signal Transduction/drug effects , Sirolimus/administration & dosage , AMP-Activated Protein Kinases/metabolism , Animals , Atorvastatin , CD55 Antigens/metabolism , Complement Activation/drug effects , Complement Activation/physiology , Complement System Proteins/metabolism , Cyclic AMP Response Element-Binding Protein/metabolism , Cytoprotection/physiology , Drug Synergism , Endothelium, Vascular/metabolism , Humans , Hydroxymethylglutaryl-CoA Reductase Inhibitors/administration & dosage , Immunosuppressive Agents/administration & dosage , Mice , Protein Kinase C/metabolism , Signal Transduction/physiologyABSTRACT
RATIONALE: Intraplaque hemorrhage (IPH) drives atherosclerosis through the dual metabolic stresses of cholesterol-enriched erythrocyte membranes and pro-oxidant heme/iron. When clearing tissue hemorrhage, macrophages are typically seen storing either iron or lipid. We have recently defined hemorrhage-associated macrophages (HA-mac) as a plaque macrophage population that responds adaptively to IPH. OBJECTIVE: This study aimed to define the key transcription factor(s) involved in HO-1 induction by heme. METHODS AND RESULTS: To address this question, we used microarray analysis and transfection with siRNA and plasmids. To maintain physiological relevance, we focused on human blood-derived monocytes. We found that heme stimulates monocytes through induction of activating transcription factor 1 (ATF-1). ATF-1 coinduces heme oxygenase-1 (HO-1) and Liver X receptor beta (LXR-ß). Heme-induced HO-1 and LXR-ß were suppressed by knockdown of ATF-1, and HO-1 and LXR-ß were induced by ATF-1 transfection. ATF-1 required phosphorylation for full functional activity. Expression of LXR-ß in turn led to induction of other genes central to cholesterol efflux, such as LXR-α and ABCA1. This heme-directed state was distinct from known macrophage states (M1, M2, Mox) and, following the same format, we have designated them Mhem. CONCLUSIONS: These results show that ATF-1 mediates HO-1 induction by heme and drives macrophage adaptation to intraplaque hemorrhage. Our definition of an ATF-1-mediated pathway for linked protection from foam cell formation and oxidant stress may have therapeutic potential.
Subject(s)
Activating Transcription Factor 1/metabolism , Foam Cells/pathology , Hemorrhage/pathology , Iron/metabolism , Macrophages/metabolism , Macrophages/pathology , Plaque, Atherosclerotic/pathology , ATP Binding Cassette Transporter 1 , ATP-Binding Cassette Transporters/metabolism , Cell Communication , Cells, Cultured , Heme/pharmacology , Heme Oxygenase-1/metabolism , Humans , Lipid Metabolism/physiology , Liver X Receptors , Macrophages/drug effects , Orphan Nuclear Receptors/metabolism , Oxidative Stress/physiology , Plaque, Atherosclerotic/physiopathology , Plaque, Atherosclerotic/prevention & control , Signal Transduction/physiologyABSTRACT
OBJECTIVE: Intraplaque hemorrhage (IPH) is an important driver of the progression of atherosclerotic plaques. Recently, we characterized Mhem as a novel macrophage phenotype that limits the atherogenicity of IPH. Mhem are directed by activating transcription factor 1 (ATF1), which is activated by phosphorylation. A better understanding of the counteratherogenic ATF1-Mhem pathway may facilitate antiatherosclerotic therapies. APPROACH AND RESULTS: We tested the hypothesis that heme in pathologically relevant concentrations activates the ATF1-Mhem pathway via 5'-AMP-activated protein kinase (AMPK) in primary human monocyte-derived macrophages and mouse bone marrow macrophages. We found that heme (10 µmol/L) activates AMPK, and downstream ATF1-mediated coinduction of heme oxygenase and liver X receptor that characterize Mhem. Heme increased macrophage phospho-AMPK, phospho-ATF1, and its target genes, and these effects were inhibited by the AMPK antagonist dorsomorphin, or by AMPK-knockdown with small inhibitory ribonucleic acid. The AMPK-activating oral hypoglycemic agent metformin also induced and phosphorylated ATF1 at a clinically relevant concentration (10 µmol/L). Functional effects of heme and metformin were inhibited by AMPK-knockdown and included suppression of macrophage oxidative stress; increased cholesterol export; protection from foam-cell formation; and suppression of macrophage inflammatory activation (human leukocyte antigen type DR expression). CONCLUSIONS: Our data indicate that heme activates the ATF1 pathway in human macrophages via AMPK, and that a similar response occurs after treatment of cells with metformin. Our results suggest an in vitro mechanism that may explain the clinical evidence that metformin has vascular protective effects beyond its role in treating hyperglycemia.
Subject(s)
Activating Transcription Factor 1/metabolism , Atherosclerosis/metabolism , Heme/pharmacology , Macrophages/drug effects , Macrophages/metabolism , Metformin/pharmacology , AMP-Activated Protein Kinases/genetics , AMP-Activated Protein Kinases/metabolism , Activating Transcription Factor 1/genetics , Heme Oxygenase-1/genetics , Heme Oxygenase-1/metabolism , Humans , Hypoglycemic Agents/pharmacology , Liver X Receptors , Macrophages/cytology , Orphan Nuclear Receptors/genetics , Orphan Nuclear Receptors/metabolism , Oxidative Stress/drug effects , Oxidative Stress/physiology , Primary Cell Culture , RNA, Small Interfering/pharmacology , Signal Transduction/drug effects , Signal Transduction/physiologyABSTRACT
Atherosclerosis is a chronic inflammatory disease of the medium and large arteries driven in large part by the accumulation of oxidized low-density lipoproteins and other debris at sites rendered susceptible because of the geometry of the arterial tree. As lesions develop, they acquire a pathologic microcirculation that perpetuates lesion progression, both by providing a means for further monocyte and T-lymphocyte recruitment into the arterial wall and by the physical and chemical stresses caused by micro-hemorrhage. This review summarizes work performed in our department investigating the roles of signaling pathways, alone and in combination, that lead to specific programs of gene expression in the atherosclerotic environment. Focusing particularly on cytoprotective responses that might be enhanced therapeutically, the work has encompassed the anti-inflammatory effects of arterial laminar shear stress, mechanisms of induction of membrane inhibitors that prevent complement-mediated injury, homeostatic macrophage responses to hemorrhage, and the transcriptional mechanisms that control the stability, survival, and quiescence of endothelial monolayers. Lastly, while the field has been dominated by investigation into the mechanisms of DNA transcription, we consider the importance of parallel post-transcriptional regulatory mechanisms for fine-tuning functional gene expression repertoires.
Subject(s)
Cell Communication , Endothelial Cells/metabolism , Gene Expression Regulation , Macrophages/metabolism , Plaque, Atherosclerotic/metabolism , Signal Transduction , Animals , Endothelial Cells/pathology , Humans , Macrophages/pathology , Plaque, Atherosclerotic/pathologyABSTRACT
IgG may accelerate atherosclerosis via ligation of proinflammatory Fcγ receptors; however, IgM is unable to ligate FcγR and is often considered vasculoprotective. IgM aggravates ischemia-reperfusion injury, and solid-phase deposits of pure IgM, as seen with IgM-secreting neoplasms, are well known clinically to provoke vascular inflammation. We therefore examined the molecular mechanisms by which immunoglobulins can aggravate vascular inflammation, such as in atherosclerosis. We compared the ability of fluid- and solid-phase immunoglobulins to activate macrophages. Solid-phase immunoglobulins initiated prothrombotic and proinflammatory functions in human macrophages, including NF-κB p65 activation, H(2)O(2) secretion, macrophage-induced apoptosis, and tissue factor expression. Responses to solid-phase IgG (but not to IgM) were blocked by neutralizing antibodies to CD16 (FcγRIII), consistent with its known role. Macrophages from mice deficient in macrophage scavenger receptor A (SR-A; CD204) had absent IgM binding and no activation by solid-phase IgM. RNA interference-mediated knockdown of SR-A in human macrophages suppressed activation by solid-phase IgM. IgM binding to SR-A was demonstrated by both co-immunoprecipitation studies and the binding of fluorescently labeled IgM to SR-A-transfected cells. Immunoglobulins on solid-phase particles around macrophages were found in human plaques, increased in ruptured plaques compared with stable ones. These observations indicate that solid-phase IgM and IgG can activate macrophages and destabilize vulnerable plaques. Solid-phase IgM activates macrophages via a novel SR-A pathway.
Subject(s)
Immunoglobulin G/immunology , Immunoglobulin M/immunology , Macrophage Activation/immunology , Plaque, Atherosclerotic/immunology , Scavenger Receptors, Class A/immunology , Animals , Blood Coagulation/physiology , Cells, Cultured , Complement System Proteins/immunology , Coronary Artery Disease/immunology , Cytotoxicity, Immunologic , GPI-Linked Proteins/immunology , Humans , Hydrogen Peroxide/metabolism , Lipoproteins, LDL/metabolism , Mice , Muscle, Smooth, Vascular/immunology , NF-kappa B/physiology , Protein Denaturation , Receptors, Fc/immunology , Receptors, IgG/immunology , Signal Transduction/immunology , Thromboplastin/physiologyABSTRACT
Vascular endothelial injury predisposes to endothelial dysfunction and atherogenesis. We have investigated the hypothesis that PKCε (protein kinase Cε) is an important upstream regulator of cytoprotective pathways in vascular ECs (endothelial cells). Depletion of PKCε in human ECs reduced expression of the cytoprotective genes A1, A20 and Bcl-2. Conversely, constitutively active PKCε expressed in human ECs increased mRNA and protein levels of these cytoprotective genes, with up-regulation dependent upon ERK1/2 (extracellular-signal-regulated kinase 1/2) activation. Furthermore, inhibition of NF-κB (nuclear factor κB) by the pharmacological antagonist BAY 11-7085 or an IκB (inhibitor of NF-κB) SuperRepressor prevented cytoprotective gene induction. Activation of PKCε enhanced p65 NF-κB DNA binding and elevated NF-κB transcriptional activity. Importantly, although NF-κB activation by PKCε induced cytoprotective genes, it did not up-regulate pro-inflammatory NF-κB targets [E-selectin, VCAM-1 (vascular cell adhesion molecule 1) and ICAM-1 (intercellular adhesion molecule 1)]. Indeed, PKCε exhibited cytoprotective and anti-inflammatory actions, including inhibition of TNFα (tumour necrosis factor α)-induced JNK (c-Jun N-terminal kinase) phosphorylation and ICAM-1 up-regulation, a response attenuated by depletion of A20. Thus we conclude that PKCε plays an essential role in endothelial homoeostasis, acting as an upstream co-ordinator of gene expression through activation of ERK1/2, inhibition of JNK and diversion of the NF-κB pathway to cytoprotective gene induction, and propose that PKCε represents a novel therapeutic target for endothelial dysfunction.
Subject(s)
Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , NF-kappa B/metabolism , Protein Kinase C-epsilon/metabolism , Animals , DNA-Binding Proteins/biosynthesis , Enzyme Activation , Human Umbilical Vein Endothelial Cells , Humans , Intercellular Adhesion Molecule-1/biosynthesis , Intracellular Signaling Peptides and Proteins/biosynthesis , MAP Kinase Kinase 4/metabolism , MAP Kinase Signaling System/physiology , Mice , Minor Histocompatibility Antigens , Nuclear Proteins/biosynthesis , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Tumor Necrosis Factor alpha-Induced Protein 3 , Vascular Cell Adhesion Molecule-1/biosynthesisABSTRACT
PURPOSE OF REVIEW: Several studies have recently shown that haemoglobin drives a novel macrophage subset that is protected from foam cell formation. RECENT FINDINGS: In a previously overlooked area, two centres have independently shown that heme and haemoglobin drive an atheroprotective macrophage subset. We compare and contrast the approaches and findings of the laboratories and discuss some of the underlying biology and implications, concentrating on the aspects of lipidological relevance. SUMMARY: Treatments based on direct heme-mimetics or other agonists of this pathway have enormous potential for linked antioxidant protection via heme oxygenase 1 and reduced foam cell formation via liver X receptor, a potent combination for treating atherosclerosis.
Subject(s)
Foam Cells/pathology , Heme/metabolism , Hemoglobins/metabolism , Macrophages/pathology , Plaque, Atherosclerotic/physiopathology , ATP Binding Cassette Transporter 1 , ATP-Binding Cassette Transporters/metabolism , Activating Transcription Factor 1/metabolism , Animals , Cytoprotection , Foam Cells/metabolism , Heme Oxygenase-1/metabolism , Hemorrhage/physiopathology , Humans , Interleukins/metabolism , Liver X Receptors , Macrophages/metabolism , NF-E2-Related Factor 2/metabolism , Orphan Nuclear Receptors/metabolism , Phenotype , Plaque, Atherosclerotic/therapyABSTRACT
PURPOSE: Targeted tyrosine kinase inhibitors (TKIs) and immune-checkpoint inhibitors (ICIs) revolutionized the treatment of metastatic renal cell carcinoma (RCC). Efforts to translate these therapies into the adjuvant setting for local and locoregional RCC have been pursued over the past decade. We sought to provide an updated review of the literature regarding adjuvant therapy in RCC, as well as an analysis of patient characteristics that may portend the most favorable responses. MATERIALS AND METHODS: Using PubMed, Google Scholar, and Wiley Online Library, we reviewed articles between 2000 and 2022. Search terms included "tyrosine kinase inhibitors," "adjuvant," "immunotherapy," and "renal cell carcinoma." The articles included were original and published in English. Information on clinical trials was collected from ClinicalTrials.gov, accessed in June 2022. RESULTS: Landmark trials investigating adjuvant vascular endothelial growth factor (VEGF) inhibitors produced conflicting results, with only a single trial of sunitinib (S-TRAC) resulting in US Food and Drug Administration-approval on the basis of a slightly prolonged progression-free survival (PFS). Subsequent meta-analyses failed to show a benefit for adjuvant VEGF inhibitors. Several trials evaluating ICIs are currently ongoing, with pembrolizumab (KEYNOTE-564) earning US Food and Drug Administration-approval for a prolonged PFS, although overall survival data are not yet mature. Preliminary results from other adjuvant ICI trials have been conflicting. CONCLUSION: There remains a lack of clear benefit for the use of adjuvant VEGF inhibitors in local and locoregional RCC. Adjuvant ICI investigations are ongoing, with promising results from KEYNOTE-564. It remains to be seen if PFS is an adequate surrogate end point for overall survival. Selection of patients at greatest risk for recurrence, and identification of those at greatest risk of rare but serious adverse events, may improve outcomes.
Subject(s)
Carcinoma, Renal Cell , Kidney Neoplasms , United States , Humans , Carcinoma, Renal Cell/drug therapy , Kidney Neoplasms/drug therapy , Kidney Neoplasms/pathology , Vascular Endothelial Growth Factor A/therapeutic use , Progression-Free Survival , Risk AssessmentABSTRACT
OBJECTIVE: Intraplaque hemorrhage (IPH) is an important progression event in advanced atherosclerosis, in large part because of the delivery of prooxidant hemoglobin in erythrocytes. We have previously defined a novel macrophage phenotype (hemorrhage-associated-mac) in human advanced plaques with IPH. These may be atheroprotective in view of raised heme oxygenase 1 (HO-1), CD163, and interleukin-10 expression and suppressed oxidative stress. METHODS AND RESULTS: We have used a combination of small interfering RNA and pharmacological reagents, protein analysis, and oxidative stress measurements to dissect the pathway leading to the development of this phenotype. We found that erythrocytes, hemoglobin, or purified heme similarly induced CD163 and suppressed human leukocyte antigen and reactive oxygen species. HO-1 was required for the development of each of these features. Challenge of macrophages with purified heme provoked nuclear translocation of Nrf2, and Nrf2 small interfering RNA resulted in significant inhibition of the ability of heme to induce HO-1 protein. Furthermore, tert-butyl-hydroquinone, which activates Nrf2, upregulated CD163, suppressed human leukocyte antigen, and induced interleukin-10, further supporting a role for Nrf2-mediated signaling. However, an inducible protein transactivator is also probably necessary, as heme-induced HO-1 mRNA expression was fully inhibited by the protein synthesis inhibitor cycloheximide. CONCLUSION: Our experiments define an Nrf2-mediated pathway by which heme induces a homeostatic macrophage response following IPH.
Subject(s)
Heme Oxygenase-1/metabolism , Heme/metabolism , Hemorrhage/physiopathology , Homeostasis/physiology , Macrophages/physiology , NF-E2-Related Factor 2/metabolism , Plaque, Atherosclerotic/physiopathology , Antigens, CD/metabolism , Antigens, Differentiation, Myelomonocytic/metabolism , Cells, Cultured , Erythrocytes/pathology , Hemorrhage/pathology , Humans , Interleukin-10/metabolism , Macrophages/drug effects , Macrophages/pathology , Monocytes/pathology , Plaque, Atherosclerotic/pathology , RNA, Small Interfering/pharmacology , Receptors, Cell Surface/metabolism , Signal Transduction/physiologyABSTRACT
OBJECTIVE: To test whether ETS-related gene (Erg) inhibits tumor necrosis factor (TNF)-α-dependent endothelial activation and inflammation. METHODS AND RESULTS: Endothelial activation underlies many vascular diseases, including atherosclerosis. Endothelial activation by proinflammatory cytokines decreases expression of the ETS transcription factor Erg. By using human umbilical vein endothelial cells (HUVECs), we showed that Erg overexpression by adenovirus (AdErg) repressed basal and TNF-α-induced expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule (VCAM), and interleukin 8 (IL-8). Erg inhibited TNF-α-dependent activation of the ICAM-1 promoter, nuclear factor (NF)-κB activity, and NF-κB p65 phosphorylation. Basal NF-κB activity was also inhibited by Erg overexpression. Chromatin immunoprecipitation showed that Erg binds to the ICAM-1 proximal promoter region, which contains 7 putative ETS binding sites. To test the anti-inflammatory role of Erg in vivo, we used a murine model of TNF-α-dependent acute inflammation. The injection of AdErg into the paw decreased TNF-α-induced inflammation compared with control. Finally, staining of human coronary plaques showed loss of Erg expression from the endothelium overlaying active plaque shoulders. CONCLUSIONS: We have identified a novel physiological anti-inflammatory pathway under the control of the transcription factor Erg; this pathway inhibits NF-κB-dependent transcription and TNF-α-induced inflammation in vivo. These results suggest a novel approach to anti-inflammatory therapies.
Subject(s)
Endothelial Cells/immunology , Inflammation Mediators/metabolism , Inflammation/prevention & control , NF-kappa B/metabolism , Trans-Activators/metabolism , Animals , Base Sequence , Binding Sites , Cells, Cultured , Coronary Artery Disease/metabolism , Disease Models, Animal , Down-Regulation , Humans , Inflammation/genetics , Inflammation/immunology , Intercellular Adhesion Molecule-1/genetics , Intercellular Adhesion Molecule-1/metabolism , Interleukin-8/genetics , Interleukin-8/metabolism , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Phosphorylation , Promoter Regions, Genetic , RNA Interference , Time Factors , Trans-Activators/genetics , Transcription Factor RelA/metabolism , Transcriptional Regulator ERG , Transfection , Tumor Necrosis Factor-alpha/metabolism , Vascular Cell Adhesion Molecule-1/genetics , Vascular Cell Adhesion Molecule-1/metabolismABSTRACT
Identification of the genes underlying complex phenotypes and the definition of the evolutionary forces that have shaped eukaryotic genomes are among the current challenges in molecular genetics. Variation in gene copy number is increasingly recognized as a source of inter-individual differences in genome sequence and has been proposed as a driving force for genome evolution and phenotypic variation. Here we show that copy number variation of the orthologous rat and human Fcgr3 genes is a determinant of susceptibility to immunologically mediated glomerulonephritis. Positional cloning identified loss of the newly described, rat-specific Fcgr3 paralogue, Fcgr3-related sequence (Fcgr3-rs), as a determinant of macrophage overactivity and glomerulonephritis in Wistar Kyoto rats. In humans, low copy number of FCGR3B, an orthologue of rat Fcgr3, was associated with glomerulonephritis in the autoimmune disease systemic lupus erythematosus. The finding that gene copy number polymorphism predisposes to immunologically mediated renal disease in two mammalian species provides direct evidence for the importance of genome plasticity in the evolution of genetically complex phenotypes, including susceptibility to common human disease.
Subject(s)
Antigens, CD/genetics , Gene Dosage/genetics , Genetic Predisposition to Disease/genetics , Lupus Nephritis/genetics , Polymorphism, Genetic/genetics , Receptors, IgG/genetics , Animals , Base Sequence , Exons/genetics , GPI-Linked Proteins , Gene Duplication , Haplotypes , Humans , Lupus Nephritis/immunology , Lupus Nephritis/pathology , Molecular Sequence Data , Rats , Rats, Inbred WKY , Sequence Deletion/geneticsABSTRACT
C-reactive protein is involved in a plethora of pathophysiological conditions. Many genetic loci associated with C-reactive protein are annotated to lipid and glucose metabolism genes supporting common biological pathways between inflammation and metabolic traits. To identify novel pleiotropic loci, we perform multi-trait analysis of genome-wide association studies on C-reactive protein levels along with cardiometabolic traits, followed by a series of in silico analyses including colocalization, phenome-wide association studies and Mendelian randomization. We find 41 novel loci and 19 gene sets associated with C-reactive protein with various pleiotropic effects. Additionally, 41 variants colocalize between C-reactive protein and cardiometabolic risk factors and 12 of them display unexpected discordant effects between the shared traits which are translated into discordant associations with clinical outcomes in subsequent phenome-wide association studies. Our findings provide insights into shared mechanisms underlying inflammation and lipid metabolism, representing potential preventive and therapeutic targets.
Subject(s)
C-Reactive Protein , Genome-Wide Association Study , Humans , C-Reactive Protein/genetics , Polymorphism, Single Nucleotide , Genetic Pleiotropy , Genetic Loci , Inflammation/genetics , Genetic Predisposition to Disease , Mendelian Randomization AnalysisABSTRACT
BACKGROUND: Intermittent claudication (IC) is a common manifestation of peripheral arterial disease. Some patients with IC experience a rise in Urinary N-acetyl-ß-D-Glucosaminidase (NAG)/ Creatinine (Cr) ratio, a marker of renal injury, following exercise. In this study, we aim to investigate whether peripheral blood mononuclear cells (PBMC) from patients with IC who exhibit a rise in urinary NAG/ Cr ratio following exercise exhibit differential IL-10/ IL-12 ratio and gene expression compared to those who do not have a rise in NAG/ Cr ratio. METHODS: We conducted a single center observational cohort study of patients diagnosed with IC. Blood and urine samples were collected at rest and following a standardised treadmill exercise protocol. For comparative analysis patients were separated into those with any rise in NAG/Cr ratio (Group 1) and those with no rise in NAG/Cr ratio (Group 2) post exercise. Isolated PBMC from pre- and post-exercise blood samples were analysed using flow cytometry. PBMC were also cultured for 20 hours to perform further analysis of IL-10 and IL-12 cytokine levels. RNA-sequencing analysis was performed to identify differentially expressed genes between the groups. RESULTS: 20 patients were recruited (Group 1, n = 8; Group 2, n = 12). We observed a significantly higher IL-10/IL-12 ratio in cell supernatant from participants in Group 1, as compared to Group 2, on exercise at 20 hours incubation; 47.24 (IQR 9.70-65.83) vs 6.13 (4.88-12.24), p = 0.04. 328 genes were significantly differentially expressed between Group 1 and 2. The modulated genes had signatures encompassing hypoxia, metabolic adaptation to starvation, inflammatory activation, renal protection, and oxidative stress. DISCUSSION: Our results suggest that some patients with IC have an altered immune status making them 'vulnerable' to systemic inflammation and renal injury following exercise. We have identified a panel of genes which are differentially expressed in this group of patients.
Subject(s)
Acute Kidney Injury , Intermittent Claudication , Acetylglucosaminidase/urine , Acute Kidney Injury/metabolism , Biomarkers/urine , Creatinine/urine , Cytokines/genetics , Female , Gene Expression , Humans , Interleukin-10/genetics , Interleukin-12/genetics , Intermittent Claudication/genetics , Leukocytes, Mononuclear/metabolism , MaleABSTRACT
BACKGROUND: Antibody-based constructs for molecular imaging and therapeutic delivery provide promising opportunities for the diagnosis and treatment of atherosclerosis. OBJECTIVES: The authors aimed to generate and characterize immunoglobulin (Ig)G monoclonal autoantibodies in atherosclerosis for targeting of novel molecular determinants. METHODS: The authors created hybridomas from an unimmunized low-density lipoprotein (LDL) receptor-deficient (Ldlr-/-) mouse and selected an IgG2b isotype autoantibody, LO9, for further characterization. RESULTS: LO9 reacted well with native LDL bound to immobilized matrix components and less well to oxidized LDL. LO9 binding to immobilized native LDL was not neutralized by fluid-phase native LDL, indicating an adhesion-dependent epitope. The authors localized the epitope to a 20 amino-acid peptide sequence (P5) in the globular amino-terminus of apolipoprotein B. LO9 reacted with antigen in mouse atherosclerosis and in both human stable and ruptured coronary atherosclerosis. Furthermore, in vivo near-infrared fluorescence molecular tomographic imaging, and ex vivo confocal microscopy showed that intravenously injected LO9 localized beneath endothelium of the aortic arch in Ldlr-/- mice, in the vicinity of macrophages. CONCLUSIONS: The authors believe LO9 is the first example of an IgG autoantibody that reacts with a native LDL epitope revealed by adherence to tissue matrix. Antibodies against adherent native LDL have potential as molecular targeting agents for imaging of and therapeutic delivery to atherosclerosis.