Suppression of IL-1ß promotes beneficial accumulation of fibroblast-like cells in atherosclerotic plaques in clonal hematopoiesis.

Clonal hematopoiesis (CH) is an independent risk factor for atherosclerotic cardiovascular disease. Murine models of CH suggest a central role of inflammasomes and IL-1ß in accelerated atherosclerosis and plaque destabilization. Here we show using single-cell RNA sequencing in human carotid plaques that inflammasome components are enriched in macrophages, while the receptor for IL-1ß is enriched in fibroblasts and smooth muscle cells (SMCs). To address the role of inflammatory crosstalk in features of plaque destabilization, we conducted SMC fate mapping in Ldlr-/- mice modeling Jak2VF or Tet2 CH treated with IL-1ß antibodies. Unexpectedly, this treatment minimally affected SMC differentiation, leading instead to a prominent expansion of fibroblast-like cells. Depletion of fibroblasts from mice treated with IL-1ß antibody resulted in thinner fibrous caps. Conversely, genetic inactivation of Jak2VF during plaque regression promoted fibroblast accumulation and fibrous cap thickening. Our studies suggest that suppression of inflammasomes promotes plaque stabilization by recruiting fibroblast-like cells to the fibrous cap.

Genetic modification of inflammation- and clonal hematopoiesis-associated cardiovascular risk.

Clonal hematopoiesis of indeterminate potential (CHIP) is associated with an increased risk of cardiovascular diseases (CVDs), putatively via inflammasome activation. We pursued an inflammatory gene modifier scan for CHIP-associated CVD risk among 424,651 UK Biobank participants. We identified CHIP using whole-exome sequencing data of blood DNA and modeled as a composite, considering all driver genes together, as well as separately for common drivers (DNMT3A, TET2, ASXL1, and JAK2). We developed predicted gene expression scores for 26 inflammasome-related genes and assessed how they modify CHIP-associated CVD risk. We identified IL1RAP as a potential key molecule for CHIP-associated CVD risk across genes and increased AIM2 gene expression leading to heightened JAK2- and ASXL1-associated CVD risk. We show that CRISPR-induced Asxl1-mutated murine macrophages had a particularly heightened inflammatory response to AIM2 agonism, associated with an increased DNA damage response, as well as increased IL-10 secretion, mirroring a CVD-protective effect of IL10 expression in ASXL1 CHIP. Our study supports the role of inflammasomes in CHIP-associated CVD and provides evidence to support gene-specific strategies to address CHIP-associated CVD risk.

Hematopoietic NLRP3 and AIM2 Inflammasomes Promote Diabetes-Accelerated Atherosclerosis, but Increased Necrosis Is Independent of Pyroptosis.

Serum apolipoprotein C3 (APOC3) predicts incident cardiovascular events in people with type 1 diabetes, and silencing of APOC3 prevents both lesion initiation and advanced lesion necrotic core expansion in a mouse model of type 1 diabetes. APOC3 acts by slowing the clearance of triglyceride-rich lipoproteins, but lipid-free APOC3 has recently been reported to activate an inflammasome pathway in monocytes. We therefore investigated the contribution of hematopoietic inflammasome pathways to atherosclerosis in mouse models of type 1 diabetes. LDL receptor-deficient diabetes mouse models were transplanted with bone marrow from donors deficient in NOD, LRR and pyrin domain-containing protein 3 (NLRP3), absent in melanoma 2 (AIM2) or gasdermin D (GSDMD), an inflammasome-induced executor of pyroptotic cell death. Mice with diabetes exhibited inflammasome activation and consistently, increased plasma interleukin-1ß (IL-1ß) and IL-18. Hematopoietic deletions of NLRP3, AIM2, or GSDMD caused smaller atherosclerotic lesions in diabetic mice. The increased lesion necrotic core size in diabetic mice was independent of macrophage pyroptosis because hematopoietic GSDMD deficiency failed to prevent necrotic core expansion in advanced lesions. Our findings demonstrate that AIM2 and NLRP3 inflammasomes contribute to atherogenesis in diabetes and suggest that necrotic core expansion is independent of macrophage pyroptosis. ARTICLE HIGHLIGHTS: The contribution of hematopoietic cell inflammasome activation to atherosclerosis associated with type 1 diabetes is unknown. The goal of this study was to address whether hematopoietic NOD, LRR, and pyrin domain-containing protein 3 (NLRP3), absent in melanoma 2 (AIM2) inflammasomes, or the pyroptosis executioner gasdermin D (GSDMD) contributes to atherosclerosis in mouse models of type 1 diabetes. Diabetic mice exhibited increased inflammasome activation, with hematopoietic deletions of NLRP3, AIM2, or GSDMD causing smaller atherosclerotic lesions in diabetic mice, but the increased lesion necrotic core size in diabetic mice was independent of macrophage pyroptosis. Further studies on whether inflammasome activation contributes to cardiovascular complications in people with type 1 diabetes are warranted.

Endogenous SOD2 (Superoxide Dismutase) Regulates Platelet-Dependent Thrombin Generation and Thrombosis During Aging.

BACKGROUND: Reactive oxygen species (ROS) contribute to platelet hyperactivation during aging. Several oxidative pathways and antioxidant enzymes have been implicated; however, their mechanistic contributions during aging remain elusive. We hypothesized that mitochondria are an important source of platelet ROS and that mitochondrial SOD2 (superoxide dismutase) protects against mitochondrial ROS-driven platelet activation and thrombosis during aging. METHODS: We studied littermates of platelet-specific SOD2-knockout (SOD2fl/flPf4Cre, pSOD2-KO) and control (SOD2fl/fl) mice at young (4-5 months) or old (18-20 months) ages. We examined agonist-induced platelet activation, platelet-dependent thrombin generation potential, and susceptibility to in vivo thrombosis. RESULTS: Platelet αIIbß3 activation, aggregation, and adhesion were increased to similar extents in aged mice of both genotypes compared with young mice. In contrast, the age-dependent increases in mitochondrial and total cellular ROS, calcium elevation, and phosphatidylserine exposure were augmented in platelets from pSOD2-KO mice compared with control mice. Aged pSOD2-KO mice showed increased platelet-dependent thrombin generation compared with aged control mice. In vivo, aged pSOD2-KO mice exhibited enhanced susceptibility to carotid artery and pulmonary thrombosis compared to aged control mice. Adoptive transfer of platelets from aged pSOD2-KO but not aged control mice increased thrombotic susceptibility in aged host mice, suggesting a prothrombotic effect of platelet pSOD2 deficiency. Treatment with avasopasem manganese (GC4419), a SOD mimetic, decreased platelet mitochondrial pro-oxidants, cellular ROS levels, and inhibited procoagulant platelet formation and arterial thrombosis in aged mice. CONCLUSIONS: Platelet mitochondrial ROS contributes to age-related thrombosis and endogenous SOD2 protects from platelet-dependent thrombin generation and thrombosis during aging.

Erythroid lineage Jak2V617F expression promotes atherosclerosis through erythrophagocytosis and macrophage ferroptosis.

Elevated hematocrit is associated with cardiovascular risk; however, the causality and mechanisms are unclear. The JAK2V617F (Jak2VF) mutation increases cardiovascular risk in myeloproliferative disorders and in clonal hematopoiesis. Jak2VF mice with elevated WBCs, platelets, and RBCs display accelerated atherosclerosis and macrophage erythrophagocytosis. To investigate whether selective erythroid Jak2VF expression promotes atherosclerosis, we developed hyperlipidemic erythropoietin receptor Cre mice that express Jak2VF in the erythroid lineage (VFEpoR mice). VFEpoR mice without elevated blood cell counts showed increased atherosclerotic plaque necrosis, erythrophagocytosis, and ferroptosis. Selective induction of erythrocytosis with low-dose erythropoietin further exacerbated atherosclerosis with prominent ferroptosis, lipid peroxidation, and endothelial damage. VFEpoR RBCs had reduced antioxidant defenses and increased lipid hydroperoxides. Phagocytosis of human or murine WT or JAK2VF RBCs by WT macrophages induced ferroptosis, which was prevented by the ferroptosis inhibitor liproxstatin-1. Liproxstatin-1 reversed increased atherosclerosis, lipid peroxidation, ferroptosis, and endothelial damage in VFEpoR mice and in Jak2VF chimeric mice simulating clonal hematopoiesis, but had no impact in controls. Erythroid lineage Jak2VF expression led to qualitative and quantitative defects in RBCs that exacerbated atherosclerosis. Phagocytosis of RBCs by plaque macrophages promoted ferroptosis, suggesting a therapeutic target for reducing RBC-mediated cardiovascular risk.

Modulation of the NLRP3 inflammasome by Sars-CoV-2 Envelope protein.

Despite the initial success of some drugs and vaccines targeting COVID-19, understanding the mechanism underlying SARS-CoV-2 disease pathogenesis remains crucial for the development of further approaches to treatment. Some patients with severe Covid-19 experience a cytokine storm and display evidence of inflammasome activation leading to increased levels of IL-1ß and IL-18; however, other reports have suggested reduced inflammatory responses to Sars-Cov-2. In this study we have examined the effects of the Sars-Cov-2 envelope (E) protein, a virulence factor in coronaviruses, on inflammasome activation and pulmonary inflammation. In cultured macrophages the E protein suppressed inflammasome priming and NLRP3 inflammasome activation. Similarly, in mice transfected with E protein and treated with poly(I:C) to simulate the effects of viral RNA, the E protein, in an NLRP3-dependent fashion, reduced expression of pro-IL-1ß, levels of IL-1ß and IL-18 in broncho-alveolar lavage fluid, and macrophage infiltration in the lung. To simulate the effects of more advanced infection, macrophages were treated with both LPS and poly(I:C). In this setting the E protein increased NLRP3 inflammasome activation in both murine and human macrophages. Thus, the Sars-Cov-2 E protein may initially suppress the host NLRP3 inflammasome response to viral RNA while potentially increasing NLRP3 inflammasome responses in the later stages of infection. Targeting the Sars-Cov-2 E protein especially in the early stages of infection may represent a novel approach to Covid-19 therapy.

The AIM2 inflammasome exacerbates atherosclerosis in clonal haematopoiesis.

Clonal haematopoiesis, which is highly prevalent in older individuals, arises from somatic mutations that endow a proliferative advantage to haematopoietic cells. Clonal haematopoiesis increases the risk of myocardial infarction and stroke independently of traditional risk factors1. Among the common genetic variants that give rise to clonal haematopoiesis, the JAK2V617F (JAK2VF) mutation, which increases JAK-STAT signalling, occurs at a younger age and imparts the strongest risk of premature coronary heart disease1,2. Here we show increased proliferation of macrophages and prominent formation of necrotic cores in atherosclerotic lesions in mice that express Jak2VF selectively in macrophages, and in chimeric mice that model clonal haematopoiesis. Deletion of the essential inflammasome components caspase 1 and 11, or of the pyroptosis executioner gasdermin D, reversed these adverse changes. Jak2VF lesions showed increased expression of AIM2, oxidative DNA damage and DNA replication stress, and Aim2 deficiency reduced atherosclerosis. Single-cell RNA sequencing analysis of Jak2VF lesions revealed a landscape that was enriched for inflammatory myeloid cells, which were suppressed by deletion of Gsdmd. Inhibition of the inflammasome product interleukin-1ß reduced macrophage proliferation and necrotic formation while increasing the thickness of fibrous caps, indicating that it stabilized plaques. Our findings suggest that increased proliferation and glycolytic metabolism in Jak2VF macrophages lead to DNA replication stress and activation of the AIM2 inflammasome, thereby aggravating atherosclerosis. Precise application of therapies that target interleukin-1ß or specific inflammasomes according to clonal haematopoiesis status could substantially reduce cardiovascular risk.

Loss of MCU prevents mitochondrial fusion in G1-S phase and blocks cell cycle progression and proliferation.

The role of the mitochondrial Ca2+ uniporter (MCU) in physiologic cell proliferation remains to be defined. Here, we demonstrated that the MCU was required to match mitochondrial function to metabolic demands during the cell cycle. During the G1-S transition (the cycle phase with the highest mitochondrial ATP output), mitochondrial fusion, oxygen consumption, and Ca2+ uptake increased in wild-type cells but not in cells lacking MCU. In proliferating wild-type control cells, the addition of the growth factors promoted the activation of the Ca2+/calmodulin-dependent kinase II (CaMKII) and the phosphorylation of the mitochondrial fission factor Drp1 at Ser616 The lack of the MCU was associated with baseline activation of CaMKII, mitochondrial fragmentation due to increased Drp1 phosphorylation, and impaired mitochondrial respiration and glycolysis. The mitochondrial fission/fusion ratio and proliferation in MCU-deficient cells recovered after MCU restoration or inhibition of mitochondrial fragmentation or of CaMKII in the cytosol. Our data highlight a key function for the MCU in mitochondrial adaptation to the metabolic demands during cell cycle progression. Cytosolic CaMKII and the MCU participate in a regulatory circuit, whereby mitochondrial Ca2+ uptake affects cell proliferation through Drp1.

Glucose Metabolism Is Required for Platelet Hyperactivation in a Murine Model of Type 1 Diabetes.

Patients with type 1 diabetes mellitus (T1DM) have increased thrombosis and platelet activation. The mechanisms for platelet hyperactivation in diabetes are incompletely understood. T1DM is accompanied by hyperglycemia, dyslipidemia, and increased inflammation in addition to an altered hormonal milieu. In vitro analysis of platelets revealed that normal glucose reduces platelet activation whereas hyperglycemic conditions increase platelet activation. We therefore hypothesized that hyperglycemia increases platelet glucose utilization, which increases platelet activation to promote thrombosis. Glucose uptake and glycolysis were increased in platelets isolated from mice given streptozotocin (STZ) to induce T1DM in concert with induction of GLUT3. Platelets from STZ-induced diabetic mice exhibited increased activation after administration of protease-activated receptor 4 peptide and convulxin. In contrast, platelets isolated from GLUT1 and GLUT3 double-knockout (DKO) mice, which lack the ability to use glucose, failed to increase activation in hyperglycemic mice. Diabetic mice displayed decreased survival in a collagen/epinephrine-induced pulmonary embolism model of in vivo platelet activation relative to nondiabetic controls. Survival after pulmonary embolism was increased in diabetic DKO mice relative to nondiabetic controls. These data reveal that increased platelet glucose metabolism in vivo contributes to increased platelet activation and thrombosis in a model of T1DM.

Superoxide Dismutase 2 is dispensable for platelet function.

Increased intracellular reactive oxygen species (ROS) promote platelet activation. The sources of platelet-derived ROS are diverse and whether or not mitochondrial derived ROS, modulates platelet function is incompletely understood. Studies of platelets from patients with sickle cell disease, and diabetes suggest a correlation between mitochondrial ROS and platelet dysfunction. Therefore, we generated mice with a platelet specific knockout of superoxide dismutase 2 (SOD2-KO) to determine if increased mitochondrial ROS increases platelet activation. SOD2-KO platelets demonstrated decreased SOD2 activity and increased mitochondrial ROS, however total platelet ROS was unchanged. Mitochondrial function and content were maintained in non-stimulated platelets. However SOD2-KO platelets demonstrated decreased mitochondrial function following thrombin stimulation. In vitro platelet activation and spreading was normal and in vivo, deletion of SOD2 did not change tail-bleeding or arterial thrombosis indices. In pathophysiological models mediated by platelet-dependent immune mechanisms such as sepsis and autoimmune inflammatory arthritis, SOD2-KO mice were phenotypically identical to wildtype controls. These data demonstrate that increased mitochondrial ROS does not result in platelet dysfunction.

Deletion of GLUT1 and GLUT3 Reveals Multiple Roles for Glucose Metabolism in Platelet and Megakaryocyte Function.

Anucleate platelets circulate in the blood to facilitate thrombosis and diverse immune functions. Platelet activation leading to clot formation correlates with increased glycogenolysis, glucose uptake, glucose oxidation, and lactic acid production. Simultaneous deletion of glucose transporter (GLUT) 1 and GLUT3 (double knockout [DKO]) specifically in platelets completely abolished glucose uptake. In DKO platelets, mitochondrial oxidative metabolism of non-glycolytic substrates, such as glutamate, increased. Thrombosis and platelet activation were decreased through impairment at multiple activation nodes, including Ca2+ signaling, degranulation, and integrin activation. DKO mice developed thrombocytopenia, secondary to impaired pro-platelet formation from megakaryocytes, and increased platelet clearance resulting from cytosolic calcium overload and calpain activation. Systemic treatment with oligomycin, inhibiting mitochondrial metabolism, induced rapid clearance of platelets, with circulating counts dropping to zero in DKO mice, but not wild-type mice, demonstrating an essential role for energy metabolism in platelet viability. Thus, substrate metabolism is essential for platelet production, activation, and survival.

Glucose Transporter 3 Potentiates Degranulation and Is Required for Platelet Activation.

OBJECTIVE: On activation, platelets increase glucose uptake, glycolysis, and glucose oxidation and consume stored glycogen. This correlation between glucose metabolism and platelet function is not well understood and even less is known about the role of glucose metabolism on platelet function in vivo. For glucose to enter a cell, it must be transported through glucose transporters. Here we evaluate the contribution of GLUT3 (glucose transporter 3) to platelet function to better understand glucose metabolism in platelets. APPROACH AND RESULTS: Platelet-specific knockout of GLUT3 was generated by crossing mice harboring GLUT3 floxed allele to a PF4 (platelet factor 4)-driven Cre recombinase. In platelets, GLUT3 is localized primarily on α-granule membranes and under basal conditions facilitates glucose uptake into α-granules to be used for glycolysis. After activation, platelets degranulate and GLUT3 translocates to the plasma membrane, which is responsible for activation-mediated increased glucose uptake. In vivo, loss of GLUT3 in platelets increased survival in a collagen/epinephrine model of pulmonary embolism, and in a K/BxN model of autoimmune inflammatory disease, platelet-specific GLUT3 knockout mice display decreased disease progression. Mechanistically, loss of GLUT3 decreased platelet degranulation, spreading, and clot retraction. Decreased α-granule degranulation is due in part to an impaired ability of GLUT3 to potentiate exocytosis. CONCLUSIONS: GLUT3-mediated glucose utilization and glycogenolysis in platelets promotes α-granule release, platelet activation, and postactivation functions.

Metabolic pathways of inhaled glucocorticoids by the CYP3A enzymes.

Asthma is one of the most prevalent diseases in the world, for which the mainstay treatment has been inhaled glucocorticoids (GCs). Despite the widespread use of these drugs, approximately 30% of asthma sufferers exhibit some degree of steroid insensitivity or are refractory to inhaled GCs. One hypothesis to explain this phenomenon is interpatient variability in the clearance of these compounds. The objective of this research is to determine how metabolism of GCs by the CYP3A family of enzymes could affect their effectiveness in asthmatic patients. In this work, the metabolism of four frequently prescribed inhaled GCs, triamcinolone acetonide, flunisolide, budesonide, and fluticasone propionate, by the CYP3A family of enzymes was studied to identify differences in their rates of clearance and to identify their metabolites. Both interenzyme and interdrug variability in rates of metabolism and metabolic fate were observed. CYP3A4 was the most efficient metabolic catalyst for all the compounds, and CYP3A7 had the slowest rates. CYP3A5, which is particularly relevant to GC metabolism in the lungs, was also shown to efficiently metabolize triamcinolone acetonide, budesonide, and fluticasone propionate. In contrast, flunisolide was only metabolized via CYP3A4, with no significant turnover by CYP3A5 or CYP3A7. Common metabolites included 6ß-hydroxylation and Δ(6)-dehydrogenation for triamcinolone acetonide, budesonide, and flunisolide. The structure of Δ(6)-flunisolide was unambiguously established by NMR analysis. Metabolism also occurred on the D-ring substituents, including the 21-carboxy metabolites for triamcinolone acetonide and flunisolide. The novel metabolite 21-nortriamcinolone acetonide was also identified by liquid chromatography-mass spectrometry and NMR analysis.

Improved coiled-coil design enhances interaction with Bcr-Abl and induces apoptosis.

The oncoprotein Bcr-Abl drives aberrant downstream activity through trans-autophosphorylation of homo-oligomers in chronic myelogenous leukemia (CML).(1, 2) The formation of Bcr-Abl oligomers is achieved through the coiled-coil domain at the N-terminus of Bcr.(3, 4) We have previously reported a modified version of this coiled-coil domain, CCmut2, which exhibits disruption of Bcr-Abl oligomeric complexes and results in decreased proliferation of CML cells and induction of apoptosis.(5) A major contributing factor to these enhanced capabilities is the destabilization of the CCmut2 homodimers, increasing the availability to interact with and inhibit Bcr-Abl. Here, we included an additional mutation (K39E) that could in turn further destabilize the mutant homodimer. Incorporation of this modification into CCmut2 (C38A, S41R, L45D, E48R, Q60E) generated what we termed CCmut3, and resulted in further improvements in the binding properties with the wild-type coiled-coil domain representative of Bcr-Abl [corrected]. A separate construct containing one revert mutation, CCmut4, did not demonstrate improved oligomeric properties and indicated the importance of the L45D mutation. CCmut3 demonstrated improved oligomerization via a two-hybrid assay as well as through colocalization studies, in addition to showing similar biologic activity as CCmut2. The improved binding between CCmut3 and the Bcr-Abl coiled-coil may be used to redirect Bcr-Abl to alternative subcellular locations with interesting therapeutic implications.