Transfer of complex regional pain syndrome to mice via human autoantibodies is mediated by interleukin-1-induced mechanisms.

Neuroimmune interactions may contribute to severe pain and regional inflammatory and autonomic signs in complex regional pain syndrome (CRPS), a posttraumatic pain disorder. Here, we investigated peripheral and central immune mechanisms in a translational passive transfer trauma mouse model of CRPS. Small plantar skin-muscle incision was performed in female C57BL/6 mice treated daily with purified serum immunoglobulin G (IgG) from patients with longstanding CRPS or healthy volunteers followed by assessment of paw edema, hyperalgesia, inflammation, and central glial activation. CRPS IgG significantly increased and prolonged swelling and induced stable hyperalgesia of the incised paw compared with IgG from healthy controls. After a short-lasting paw inflammatory response in all groups, CRPS IgG-injected mice displayed sustained, profound microglia and astrocyte activation in the dorsal horn of the spinal cord and pain-related brain regions, indicating central sensitization. Genetic deletion of interleukin-1 (IL-1) using IL-1αß knockout (KO) mice and perioperative IL-1 receptor type 1 (IL-1R1) blockade with the drug anakinra, but not treatment with the glucocorticoid prednisolone, prevented these changes. Anakinra treatment also reversed the established sensitization phenotype when initiated 8 days after incision. Furthermore, with the generation of an IL-1ß floxed(fl/fl) mouse line, we demonstrated that CRPS IgG-induced changes are in part mediated by microglia-derived IL-1ß, suggesting that both peripheral and central inflammatory mechanisms contribute to the transferred disease phenotype. These results indicate that persistent CRPS is often contributed to by autoantibodies and highlight a potential therapeutic use for clinically licensed antagonists, such as anakinra, to prevent or treat CRPS via blocking IL-1 actions.

Carbon Monoxide Releasing Molecule A1 Reduces Myocardial Damage After Acute Myocardial Infarction in a Porcine Model.

ABSTRACT: Infarct size is a major determinant of outcomes after acute myocardial infarction (AMI). Carbon monoxide-releasing molecules (CORMs), which deliver nanomolar concentrations of carbon monoxide to tissues, have been shown to reduce infarct size in rodents. We evaluated efficacy and safety of CORM-A1 to reduce infarct size in a clinically relevant porcine model of AMI. We induced AMI in Yorkshire White pigs by inflating a coronary angioplasty balloon to completely occlude the left anterior descending artery for 60 minutes, followed by deflation of the balloon to mimic reperfusion. Fifteen minutes after balloon occlusion, animals were given an infusion of 4.27 mM CORM-A1 (n = 7) or sodium borate control (n = 6) over 60 minutes. Infarct size, cardiac biomarkers, ejection fraction, and hepatic and renal function were compared amongst the groups. Immunohistochemical analyses were performed to compare inflammation, cell proliferation, and apoptosis between the groups. CORM-A1-treated animals had significant reduction in absolute infarct area (158 ± 16 vs. 510 ± 91 mm2, P < 0.001) and infarct area corrected for area at risk (24.8% ± 2.6% vs. 45.2% ± 4.0%, P < 0.0001). Biochemical markers of myocardial injury also tended to be lower and left ventricular function tended to recover better in the CORM-A1 treated group. There was no evidence of hepatic or renal toxicity with the doses used. The cardioprotective effects of CORM-A1 were associated with a significant reduction in cell proliferation and inflammation. CORM-A1 reduces infarct size and improves left ventricular remodeling and function in a porcine model of reperfused MI by a reduction in inflammation. These potential cardioprotective effects of CORMs warrant further translational investigations.

X-ray Micro-Computed Tomography: An Emerging Technology to Analyze Vascular Calcification in Animal Models.

Vascular calcification describes the formation of mineralized tissue within the blood vessel wall, and it is highly associated with increased cardiovascular morbidity and mortality in patients with chronic kidney disease, diabetes, and atherosclerosis. In this article, we briefly review different rodent models used to study vascular calcification in vivo, and critically assess the strengths and weaknesses of the current techniques used to analyze and quantify calcification in these models, namely 2-D histology and the o-cresolphthalein assay. In light of this, we examine X-ray micro-computed tomography (µCT) as an emerging complementary tool for the analysis of vascular calcification in animal models. We demonstrate that this non-destructive technique allows us to simultaneously quantify and localize calcification in an intact vessel in 3-D, and we consider recent advances in µCT sample preparation techniques. This review also discusses the potential to combine 3-D µCT analyses with subsequent 2-D histological, immunohistochemical, and proteomic approaches in correlative microscopy workflows to obtain rich, multifaceted information on calcification volume, calcification load, and signaling mechanisms from within the same arterial segment. In conclusion we briefly discuss the potential use of µCT to visualize and measure vascular calcification in vivo in real-time.

Interleukin-1 mediates ischaemic brain injury via distinct actions on endothelial cells and cholinergic neurons.

The cytokine interleukin-1 (IL-1) is a key contributor to neuroinflammation and brain injury, yet mechanisms by which IL-1 triggers neuronal injury remain unknown. Here we induced conditional deletion of IL-1R1 in brain endothelial cells, neurons and blood cells to assess site-specific IL-1 actions in a model of cerebral ischaemia in mice. Tamoxifen treatment of IL-1R1 floxed (fl/fl) mice crossed with mice expressing tamoxifen-inducible Cre-recombinase under the Slco1c1 promoter resulted in brain endothelium-specific deletion of IL-1R1 and a significant decrease in infarct size (29%), blood-brain barrier (BBB) breakdown (53%) and neurological deficit (40%) compared to vehicle-treated or control (IL-1R1fl/fl) mice. Absence of brain endothelial IL-1 signalling improved cerebral blood flow, followed by reduced neutrophil infiltration and vascular activation 24 h after brain injury. Conditional IL-1R1 deletion in neurons using tamoxifen inducible nestin-Cre mice resulted in reduced neuronal injury (25%) and altered microglia-neuron interactions, without affecting cerebral perfusion or vascular activation. Deletion of IL-1R1 specifically in cholinergic neurons reduced infarct size, brain oedema and improved functional outcome. Ubiquitous deletion of IL-1R1 had no effect on brain injury, suggesting beneficial compensatory mechanisms on other cells against the detrimental effects of IL-1 on endothelial cells and neurons. We also show that IL-1R1 signalling deletion in platelets or myeloid cells does not contribute to brain injury after experimental stroke. Thus, brain endothelial and neuronal (cholinergic) IL-1R1 mediate detrimental actions of IL-1 in the brain in ischaemic stroke. Cell-specific targeting of IL-1R1 in the brain could therefore have therapeutic benefits in stroke and other cerebrovascular diseases.

Neurovascular dysfunction in vascular dementia, Alzheimer's and atherosclerosis.

Efficient blood supply to the brain is of paramount importance to its normal functioning and improper blood flow can result in potentially devastating neurological consequences. Cerebral blood flow in response to neural activity is intrinsically regulated by a complex interplay between various cell types within the brain in a relationship termed neurovascular coupling. The breakdown of neurovascular coupling is evident across a wide variety of both neurological and psychiatric disorders including Alzheimer's disease. Atherosclerosis is a chronic syndrome affecting the integrity and function of major blood vessels including those that supply the brain, and it is therefore hypothesised that atherosclerosis impairs cerebral blood flow and neurovascular coupling leading to cerebrovascular dysfunction. This review will discuss the mechanisms of neurovascular coupling in health and disease and how atherosclerosis can potentially cause cerebrovascular dysfunction that may lead to cognitive decline as well as stroke. Understanding the mechanisms of neurovascular coupling in health and disease may enable us to develop potential therapies to prevent the breakdown of neurovascular coupling in the treatment of vascular brain diseases including vascular dementia, Alzheimer's disease and stroke.

TWIST1 Integrates Endothelial Responses to Flow in Vascular Dysfunction and Atherosclerosis.

RATIONALE: Blood flow-induced shear stress controls endothelial cell (EC) physiology during atherosclerosis via transcriptional mechanisms that are incompletely understood. The mechanosensitive transcription factor TWIST is expressed during embryogenesis, but its role in EC responses to shear stress and focal atherosclerosis is unknown. OBJECTIVE: To investigate whether TWIST regulates endothelial responses to shear stress during vascular dysfunction and atherosclerosis and compare TWIST function in vascular development and disease. METHODS AND RESULTS: The expression and function of TWIST1 was studied in EC in both developing vasculature and during the initiation of atherosclerosis. In zebrafish, twist was expressed in early embryonic vasculature where it promoted angiogenesis by inducing EC proliferation and migration. In adult porcine and murine arteries, TWIST1 was expressed preferentially at low shear stress regions as evidenced by quantitative polymerase chain reaction and en face staining. Moreover, studies of experimental murine carotid arteries and cultured EC revealed that TWIST1 was induced by low shear stress via a GATA4-dependent transcriptional mechanism. Gene silencing in cultured EC and EC-specific genetic deletion in mice demonstrated that TWIST1 promoted atherosclerosis by inducing inflammation and enhancing EC proliferation associated with vascular leakiness. CONCLUSIONS: TWIST expression promotes developmental angiogenesis by inducing EC proliferation and migration. In addition to its role in development, TWIST is expressed preferentially at low shear stress regions of adult arteries where it promotes atherosclerosis by inducing EC proliferation and inflammation. Thus, pleiotropic functions of TWIST control vascular disease and development.

Neutrophil elastase promotes interleukin-1ß secretion from human coronary endothelium.

The endothelium is critically involved in the pathogenesis of atherosclerosis by producing pro-inflammatory mediators, including IL-1ß. Coronary arteries from patients with ischemic heart disease express large amounts of IL-1ß in the endothelium. However, the mechanism by which endothelial cells (ECs) release IL-1ß remains to be elucidated. We investigated neutrophil elastase (NE), a potent serine protease detected in vulnerable areas of human carotid plaques, as a potential "trigger" for IL-1ß processing and release. This study tested the hypothesis that NE potentiates the processing and release of IL-1ß from human coronary endothelium. We found that NE cleaves the pro-isoform of IL-1ß in ECs and causes significant secretion of bioactive IL-1ß via extracellular vesicles. This release was attenuated significantly by inhibition of neutrophil elastase but not caspase-1. Transient increases in intracellular Ca(2+) levels were observed prior to secretion. Inside ECs, and after NE treatment only, IL-1ß was detected within LAMP-1-positive multivesicular bodies. The released vesicles contained bioactive IL-1ß. In vivo, in experimental atherosclerosis, NE was detected in mature atherosclerotic plaques, predominantly in the endothelium, alongside IL-1ß. This study reveals a novel mechanistic link between NE expression in atherosclerotic plaques and concomitant pro-inflammatory bioactive IL-1ß secretion from ECs. This could reveal additional potential anti-IL-1ß therapeutic targets and provide further insights into the inflammatory process by which vascular disease develops.

Tribbles in inflammation.

Inflammation is part of the physiological innate immune response to invading pathogens and tissue injury. However, unresolved inflammation leads to human disease. The tribbles (TRIB) family of pseudokinase proteins has been shown to modulate key inflammatory signalling pathways, including the MAPK (mitogen-activated protein kinase) and PI3K (phosphoinositide 3-kinase) networks. This review summarizes our current knowledge on TRIBs in the context of inflammation, both at the level of molecular mechanisms and in disease development.

Streptococcus pneumoniae worsens cerebral ischemia via interleukin 1 and platelet glycoprotein Ibα.

OBJECTIVE: Bacterial infection contributes to diverse noninfectious diseases and worsens outcome after stroke. Streptococcus pneumoniae, the most common infection in patients at risk of stroke, is a major cause of prolonged hospitalization and death of stroke patients, but how infection impacts clinical outcome is not known. METHODS: We induced sustained pulmonary infection by a human S. pneumoniae isolate in naive and comorbid rodents to investigate the effect of infection on vascular and inflammatory responses prior to and after cerebral ischemia. RESULTS: S. pneumoniae infection triggered atherogenesis, led to systemic induction of interleukin (IL) 1, and profoundly exacerbated (50-90%) ischemic brain injury in rats and mice, a response that was more severe in combination with old age and atherosclerosis. Systemic blockade of IL-1 with IL-1 receptor antagonist (IL-1Ra) fully reversed infection-induced exacerbation of brain injury and functional impairment caused by cerebral ischemia. We show that infection-induced systemic inflammation mediates its effects via increasing platelet activation and microvascular coagulation in the brain after cerebral ischemia, as confirmed by reduced brain injury in response to blockade of platelet glycoprotein (GP) Ibα. IL-1 and platelet-mediated signals converge on microglia, as both IL-1Ra and GPIbα blockade reversed the production of IL-1α by microglia in response to cerebral ischemia in infected animals. INTERPRETATION: S. pneumoniae infection augments atherosclerosis and exacerbates ischemic brain injury via IL-1 and platelet-mediated systemic inflammation. These mechanisms may contribute to diverse cardio- and cerebrovascular pathologies in humans.

The pseudokinase tribbles homologue-3 plays a crucial role in cannabinoid anticancer action.

Δ(9)-Tetrahydrocannabinol (THC), the major active ingredient of marijuana, and other cannabinoids inhibit tumor growth in animal models of cancer. This effect relies, at least in part, on the up-regulation of several endoplasmic reticulum stress-related proteins including the pseudokinase tribbles homologue-3 (TRIB3), which leads in turn to the inhibition of the AKT/mTORC1 axis and the subsequent stimulation of autophagy-mediated apoptosis in tumor cells. Here, we took advantage of the use of cells derived from Trib3-deficient mice to investigate the precise mechanisms by which TRIB3 regulates the anti-cancer action of THC. Our data show that RasV(12)/E1A-transformed embryonic fibroblasts derived from Trib3-deficient mice are resistant to THC-induced cell death. We also show that genetic inactivation of this protein abolishes the ability of THC to inhibit the phosphorylation of AKT and several of its downstream targets, including those involved in the regulation of the AKT/mammalian target of rapamycin complex 1 (mTORC1) axis. Our data support the idea that THC-induced TRIB3 up-regulation inhibits AKT phosphorylation by regulating the accessibility of AKT to its upstream activatory kinase (the mammalian target of rapamycin complex 2; mTORC2). Finally, we found that tumors generated by inoculation of Trib3-deficient cells in nude mice are resistant to THC anticancer action. Altogether, the observations presented here strongly support that TRIB3 plays a crucial role on THC anti-neoplastic activity. This article is part of a Special Issue entitled Lipid Metabolism in Cancer.

miRNA-21 is dysregulated in response to vein grafting in multiple models and genetic ablation in mice attenuates neointima formation.

AIMS: The long-term failure of autologous saphenous vein bypass grafts due to neointimal thickening is a major clinical burden. Identifying novel strategies to prevent neointimal thickening is important. Thus, this study aimed to identify microRNAs (miRNAs) that are dysregulated during neointimal formation and determine their pathophysiological relevance following miRNA manipulation. METHODS AND RESULTS: We undertook a microarray approach to identify dysregulated miRNAs following engraftment in an interpositional porcine graft model. These profiling experiments identified a number of miRNAs which were dysregulated following engraftment. miR-21 levels were substantially elevated following engraftment and these results were confirmed by quantitative real-time PCR in mouse, pig, and human models of vein graft neointimal formation. Genetic ablation of miR-21 in mice or grafted veins dramatically reduced neointimal formation in a mouse model of vein grafting. Furthermore, pharmacological knockdown of miR-21 in human veins resulted in target gene de-repression and a significant reduction in neointimal formation. CONCLUSION: This is the first report demonstrating that miR-21 plays a pathological role in vein graft failure. Furthermore, we also provided evidence that knockdown of miR-21 has therapeutic potential for the prevention of pathological vein graft remodelling.

"How to Release or Not Release, That Is the Question." A Review of Interleukin-1 Cellular Release Mechanisms in Vascular Inflammation.

Cardiovascular disease remains the leading cause of death worldwide, characterized by atherosclerotic activity within large and medium-sized arteries. Inflammation has been shown to be a primary driver of atherosclerotic plaque formation, with interleukin-1 (IL-1) having a principal role. This review focuses on the current state of knowledge of molecular mechanisms of IL-1 release from cells in atherosclerotic plaques. A more in-depth understanding of the process of IL-1's release into the vascular environment is necessary for the treatment of inflammatory disease processes, as the current selection of medicines being used primarily target IL-1 after it has been released. IL-1 is secreted by several heterogenous mechanisms, some of which are cell type-specific and could provide further specialized targets for therapeutic intervention. A major unmet challenge is to understand the mechanism before and leading to IL-1 release, especially by cells in atherosclerotic plaques, including endothelial cells, vascular smooth muscle cells, and macrophages. Data so far indicate a heterogeneity of IL-1 release mechanisms that vary according to cell type and are stimulus-dependent. Unraveling this complexity may reveal new targets to block excess vascular inflammation.

A cardinal role for cathepsin d in co-ordinating the host-mediated apoptosis of macrophages and killing of pneumococci.

The bactericidal function of macrophages against pneumococci is enhanced by their apoptotic demise, which is controlled by the anti-apoptotic protein Mcl-1. Here, we show that lysosomal membrane permeabilization (LMP) and cytosolic translocation of activated cathepsin D occur prior to activation of a mitochondrial pathway of macrophage apoptosis. Pharmacological inhibition or knockout of cathepsin D during pneumococcal infection blocked macrophage apoptosis. As a result of cathepsin D activation, Mcl-1 interacted with its ubiquitin ligase Mule and expression declined. Inhibition of cathepsin D had no effect on early bacterial killing but inhibited the late phase of apoptosis-associated killing of pneumococci in vitro. Mice bearing a cathepsin D(-/-) hematopoietic system demonstrated reduced macrophage apoptosis in vivo, with decreased clearance of pneumococci and enhanced recruitment of neutrophils to control pulmonary infection. These findings establish an unexpected role for a cathepsin D-mediated lysosomal pathway of apoptosis in pulmonary host defense and underscore the importance of apoptosis-associated microbial killing to macrophage function.

Barriers to equality, diversity and inclusion in research and academia stubbornly persist. So, what are we doing about it?

Despite an appetite for change, equality, diversity and inclusivity (EDI)-related issues continue to ripple through the world of research and academia, from inequity at the point of entry into education, through to lack of diversity and equality in senior roles. Many academic institutes and governments are taking action to solve these issues, and we welcome the growing number of inclusive practices in the science communication arena. Building from this, we - at the University of Sheffield, UK - have assessed our own situation, responded to pressures applied by research councils, and listened to our staff and student voice. Our new 'One University' initiative puts EDI on a par with research, innovation and education as a core university priority, and our Gender, Disability and Race Action Plans allow us to make measurable and impactful changes. Tackling EDI issues needs a collaborative approach, action at an institutional- or sector-wide level and clear commitment from senior leaders.

Reverse diastolic flow of the fetal middle cerebral artery associated with placental chorangiomatosis and asymptomatic concealed placental abruption.

Reverse diastolic flow of the fetal middle cerebral artery is a rare, yet ominous finding which has been associated with adverse perinatal outcomes including: intracranial hemorrhage, growth restriction, fetal-maternal hemorrhage, severe anemia, hydrops, hepatic anomaly, subsequent stillbirth, and early neonatal death. We report a case in which following notation of a nonreassuring fetal heart rate at 32 weeks' gestation, sonographic documentation of persistent reverse diastolic flow of the fetal middle cerebral artery was noted in association with sonographic findings of vascular placental dysmorphology and an asymptomatic concealed placental abruption. Subsequent fetal heart rate tracing consistent with uteroplacental insufficiency led to immediate Cesarean birth of an anemic yet nonacidotic, nonhypoxic neonate, who did well following management of respiratory distress syndrome and partial exchange transfusion. Placental abruption was confirmed at delivery. Histopathology of the placenta confirmed the presence of localized chorangiomatosis ("wandering" chorangioma). The association of reverse diastolic flow of the fetal middle cerebral artery, placental chorangiomatosis and placental abruption has not been reported previously. We conclude that in the presence of prenatal sonographic findings of placental dysmorphology and or placental abruption, insonation of the fetal middle cerebral artery should be performed to assess the possibility of increased peak systolic velocity and possible reverse diastolic flow, both associated with fetal anemia and increased likelihood of an adverse perinatal outcome.

Transient increase in atherosclerotic plaque macrophage content following Streptococcus pneumoniae pneumonia in ApoE-deficient mice.

Introduction: Despite epidemiological associations between community acquired pneumonia (CAP) and myocardial infarction, mechanisms that modify cardiovascular disease during CAP are not well defined. In particular, largely due to a lack of relevant experimental models, the effect of pneumonia on atherosclerotic plaques is unclear. We describe the development of a murine model of the commonest cause of CAP, Streptococcus pneumoniae pneumonia, on a background of established atherosclerosis. We go on to use our model to investigate the effects of pneumococcal pneumonia on atherosclerosis. Methods: C57BL/6J and ApoE-/- mice were fed a high fat diet to promote atherosclerotic plaque formation. Mice were then infected with a range of S. pneumoniae serotypes (1, 4 or 14) with the aim of establishing a model to study atherosclerotic plaque evolution after pneumonia and bacteremia. Laser capture microdissection of plaque macrophages enabled transcriptomic analysis. Results: Intratracheal instillation of S. pneumoniae in mice fed a cholate containing diet resulted in low survival rates following infection, suggestive of increased susceptibility to severe infection. Optimization steps resulted in a final model of male ApoE-/- mice fed a Western diet then infected by intranasal instillation of serotype 4 (TIGR4) S. pneumoniae followed by antibiotic administration. This protocol resulted in high rates of bacteremia (88.9%) and survival (88.5%). Pneumonia resulted in increased aortic sinus plaque macrophage content 2 weeks post pneumonia but not at 8 weeks, and no difference in plaque burden or other plaque vulnerability markers were found at either time point. Microarray and qPCR analysis of plaque macrophages identified downregulation of two E3 ubiquitin ligases, Huwe1 and Itch, following pneumonia. Treatment with atorvastatin failed to alter plaque macrophage content or other plaque features. Discussion: Without antibiotics, ApoE-/- mice fed a high fat diet were highly susceptible to mortality following S. pneumoniae infection. The major infection associated change in plaque morphology was an early increase in plaque macrophages. Our results also hint at a role for the ubiquitin proteasome system in the response to pneumococcal infection in the plaque microenvironment.

Serpins in rice: protein sequence analysis, phylogeny and gene expression during development.

BACKGROUND: Most members of the serpin family of proteins are potent, irreversible inhibitors of specific serine or cysteine proteinases. Inhibitory serpins are distinguished from members of other families of proteinase inhibitors by their metastable structure and unique suicide-substrate mechanism. Animal serpins exert control over a remarkable diversity of physiological processes including blood coagulation, fibrinolysis, innate immunity and aspects of development. Relatively little is known about the complement of serpin genes in plant genomes and the biological functions of plant serpins. RESULTS: A structurally refined amino-acid sequence alignment of the 14 full-length serpins encoded in the genome of the japonica rice Oryza sativa cv. Nipponbare (a monocot) showed a diversity of reactive-centre sequences (which largely determine inhibitory specificity) and a low degree of identity with those of serpins in Arabidopsis (a eudicot). A new convenient and functionally informative nomenclature for plant serpins in which the reactive-centre sequence is incorporated into the serpin name was developed and applied to the rice serpins. A phylogenetic analysis of the rice serpins provided evidence for two main clades and a number of relatively recent gene duplications. Transcriptional analysis showed vastly different levels of basal expression among eight selected rice serpin genes in callus tissue, during seedling development, among vegetative tissues of mature plants and throughout seed development. The gene OsSRP-LRS (Os03g41419), encoding a putative orthologue of Arabidopsis AtSerpin1 (At1g47710), was expressed ubiquitously and at high levels. The second most highly expressed serpin gene was OsSRP-PLP (Os11g11500), encoding a non-inhibitory serpin with a surprisingly well-conserved reactive-centre loop (RCL) sequence among putative orthologues in other grass species. CONCLUSIONS: The diversity of reactive-centre sequences among the putatively inhibitory serpins of rice point to a range of target proteases with different proteolytic specificities. Large differences in basal expression levels of the eight selected rice serpin genes during development further suggest a range of functions in regulation and in plant defence for the corresponding proteins.

Paigen diet-fed apolipoprotein E knockout mice develop severe pulmonary hypertension in an interleukin-1-dependent manner.

Inflammatory mechanisms are proposed to play a significant role in the pathogenesis of pulmonary arterial hypertension (PAH). Previous studies have described PAH in fat-fed apolipoprotein E knockout (ApoE(-/-)) mice. We have reported that signaling in interleukin-1-receptor-knockout (IL-1R1(-/-)) mice leads to a reduction in diet-induced systemic atherosclerosis. We subsequently hypothesized that double-null (ApoE(-/-)/IL-1R1(-/-)) mice would show a reduced PAH phenotype compared with that of ApoE(-/-) mice. Male IL-1R1(-/-), ApoE(-/-), and ApoE(-/-)/IL-1R1(-/-) mice were fed regular chow or a high-fat diet (Paigen diet) for 8 weeks before phenotyping for PAH. No abnormal phenotype was observed in the IL-1R1(-/-) mice. Fat-fed ApoE(-/-) mice developed significantly increased right ventricular systolic pressure and substantial pulmonary vascular remodeling. Surprisingly, ApoE(-/-)/IL-1R1(-/-) mice showed an even more severe PAH phenotype. Further molecular investigation revealed the expression of a putative, alternatively primed IL-1R1 transcript expressed within the lungs but not aorta of ApoE(-/-)/IL-1R1(-/-) mice. Treatment of ApoE(-/-) and ApoE(-/-)/IL-1R1(-/-) mice with IL-1-receptor antagonist prevented progression of the PAH phenotype in both strains. Blocking IL-1 signaling may have beneficial effects in treating PAH, and alternative IL-1-receptor signaling in the lung may be important in driving PAH pathogenesis.

Dietary phosphate modulates atherogenesis and insulin resistance in apolipoprotein E knockout mice--brief report.

OBJECTIVE: Epidemiological studies link higher serum phosphate and the phosphatonin fibroblast growth factor 23 with cardiovascular events and atheroma, and they link lower serum phosphate with insulin resistance and the metabolic syndrome. We investigated whether manipulating dietary phosphate influences atherogenesis or insulin sensitivity in mice. METHODS AND RESULTS: Apolipoprotein E knockout mice were fed an atherogenic diet with low (0.2%), standard (0.6%), or high (1.6%) phosphate content. Serum phosphate and fibroblast growth factor 23 significantly increased with increasing dietary phosphate intake, but lipid profile and blood pressure were unaffected. After 20 weeks, mice on the higher phosphate diet had significantly more atheroma at the aortic sinus (42±1.9% versus 30±1.5% for high versus low phosphate, P<0.01). Compared with standard and high-phosphate diet groups, mice on a low-phosphate diet had more adipose tissue and a 4-fold increase in insulin resistance measured by homeostatic model assessment (43.7±9.3 versus 8.9±0.7 for low versus high phosphate, P<0.005). CONCLUSIONS: A high-phosphate diet accelerates atherogenesis in apolipoprotein E(-/-) mice, whereas low phosphate intake induces insulin resistance. These data indicate for the first time that controlling dietary phosphate intake may influence development of both atherosclerosis and the metabolic syndrome.

OxLDL induces the release of IL-1ß from primed human endothelial and smooth muscle cells via different caspase -1-dependent mechanisms.

Atherosclerosis is characterised by abnormal lipid and cell accumulation within arterial layers that leads to disturbed blood flow. Modified cholesterol forms such as oxidised low-density lipoprotein (oxLDL) enter cells altering their phenotype, triggering over-exuberant repair and arterial occlusion, myocardial infarction or stroke. We hypothesised that oxLDL enters vascular wall cells and induces interleukin-1ß (IL-1ß) secretion, potentially via a caspase-1/NLRP3 mechanism. Human coronary artery endothelial cells (HCAEC) and smooth muscle cells (HCASMC), isolated from different donors, were cultured and stimulated (primed) with pro-inflammatory cytokines TNFα and IL-1α (10 ng/mL each, for 48 h), followed by incubation with human oxLDL (10-50 ug/mL) for up to 6 h. Inhibitors of caspase-1 (YVAD), NLRP3 (MCC950) and gasdermin D (disulfiram) were added 1 h before oxLDL. Cell lysates and culture supernatants were collected and analysed for IL-1ß using ELISA. Microscopy imaging showed oxLDL entered stimulated cells and formed particles. OxLDL at 20 and 50 ug/mL induced the maximum release of IL-1ß from stimulated HCASMCs and HCAECs, respectively, compared to control. Inhibition of either NLRP3, caspase-1 or gasdermin D significantly reduced the release of IL-1ß (4-fold, P < 0.0001; 14-fold, P < 0.0001, 1.5-fold, P < 0.0003, respectively) in HCAEC. In contrast, in HCASMCs, only caspase-1 inhibition reduced the release of IL-1ß (2.1-fold, P < 0.0001). HCAECs and HCASMCs elicited the release of IL-1ß in response to the same stimulus via different mechanisms. In HCAECs, released IL-1ß potentially exits via a GSDMD-induced membrane pore. These data suggest that caspase-1 or gasdermin D inhibition is likely to be effective vessel wall cell-specific strategies for the reduction of atherosclerosis.