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Breast cancer bone metastasis is currently incurable, ~75% of patients with late-stage breast cancer develop disease recurrence in bone and available treatments are only palliative. We have previously shown that production of the pro-inflammatory cytokine interleukin-1B (IL-1B) by breast cancer cells drives bone metastasis in patients and in preclinical in vivo models. In the current study, we have investigated how IL-1B from tumour cells and the microenvironment interact to affect primary tumour growth and bone metastasis through regulation of the immune system, and whether targeting IL-1 driven changes to the immune response improves standard of care therapy for breast cancer bone metastasis. Using syngeneic IL-1B/IL1R1 knock out mouse models in combination with genetic manipulation of tumour cells to overexpress IL-1B/IL1R1, we found that IL-1B signalling elicited an opposite response in primary tumours compared with bone metastases. In primary tumours, IL-1B inhibited growth, by impairing the infiltration of innate immune cell subsets with potential anti-cancer functions but promoted enhanced tumour cell migration. In bone, IL-1B stimulated the development of osteolytic metastases. In syngeneic models of breast cancer, combining standard of care treatments (Doxorubicin and Zoledronic acid) with the IL-1 receptor antagonist Anakinra inhibited both primary tumour growth and metastasis. Anakinra had opposite effects on the immune response compared to standard of care treatment, and its anti-inflammatory signature was maintained in the combination therapy. These data suggest that targeting IL-1B signalling may provide a useful therapeutic approach to inhibit bone metastasis and improve efficacy of current treatments for breast cancer patients.

The pro-inflammatory cytokine interleukin-1 (IL-1) plays a key role in many physiological processes and during the inflammatory and immune response to most common diseases. IL-1 exists as two agonists, IL-1α and IL-1β that bind to the only signaling IL-1 type 1 receptor (IL-1R1), while a second decoy IL-1 type 2 receptor (IL-1R2) binds both forms of IL-1 without inducing cell signaling. The field of immunology and inflammation research has, over the past 35 years, unraveled many mechanisms of IL-1 actions, through in vitro manipulation of the IL-1 system or by using genetically engineered mouse models that lack either member of the IL-1 family in ubiquitous constitutive manner. However, the limitation of global mouse knockout technology has significantly hampered our understanding of the precise mechanisms of IL-1 actions in animal models of disease. Here we report and review the recent generation of new conditional mouse mutants in which exons of Il1a, Il1b, Il1r1, and Il1r2 genes flanked by loxP sites (fl/fl) can be deleted in cell-/tissue-specific constitutive or inducible manner by Cre recombinase expression. Hence, IL-1αfl/fl, IL-1βfl/fl, IL-1R1fl/fl, and IL-1R2fl/fl mice constitute a new toolbox that will provide a step change in our understanding of the cell-specific role of IL-1 and its receptor in health and disease and the potential development of targeted IL-1 therapies.

Neurovascular coupling is a critical brain mechanism whereby changes to blood flow accompany localised neural activity. The breakdown of neurovascular coupling is linked to the development and progression of several neurological conditions including dementia. In this study, we examined cortical haemodynamics in mouse preparations that modelled Alzheimer’s disease (J20-AD) and atherosclerosis (PCSK9-ATH) between 9 and 12 m of age. We report novel findings with atherosclerosis where neurovascular decline is characterised by significantly reduced blood volume, altered levels of oxyhaemoglobin and deoxyhaemoglobin, in addition to global neuroinflammation. In the comorbid mixed model (J20-PCSK9-MIX), we report a 3 x increase in hippocampal amyloid-beta plaques. A key finding was that cortical spreading depression (CSD) due to electrode insertion into the brain was worse in the diseased animals and led to a prolonged period of hypoxia. These findings suggest that systemic atherosclerosis can be detrimental to neurovascular health and that having cardiovascular comorbidities can exacerbate pre-existing Alzheimer’s-related amyloid-plaques.

Introduction Ischemic stroke is commonly caused by large artery atherosclerosis. Patients with a high atherosclerotic burden (stenosis) and inflamed or ulcerated plaque are at increased risk of early recurrent ischaemic events. Treatment with anti-inflammatory agents may therefore reduce stroke incidence and recurrence in patients with this condition, but in vitro human studies of the possible mechanisms are lacking. We hypothesised that an anti-inflammatory compound (denoted drug X*) alters the expression of macrophage specific genes, including scavenger receptors in in vitro models of human carotid plaque macrophages. Methods CD14+ monocytes were collected from whole blood donations (volunteers) and differentiated to monocyte derived macrophages (MDMs). Macrophage polarisation with M1 (100 ng/ml LPS + 20 ng/ml INF-γ) and M2a (20 ng/ml IL-4) markers was performed in complete media supplemented with 3 different clinically relevant concentrations of drug X. Gene expression (RT-qPCR) analysis (n=10) was carried out in the differentiated hMDMs to test for macrophage specific genes and scavenger receptors. Western Blot analysis (n=7) and immunofluorescence stain (IF) (n=5) was performed on drug X- treated MDMs to validate changes in the expression of Oxidized Low Density Lipoprotein Receptor 1 (OLR1). The physiological effect of drug X was also tested on oxLDL (25 μg/ml) uptake by macrophages (n=5). Results Our data suggest that drug X may re-program macrophages to a less inflammatory state (an M2 state) by significantly altering Mannose Receptor C-type 1 (MRC1) gene expression (p:0.0183). Drug X also alters the expression level of Macrophage Scavenger Receptor 1 (MSR1) gene, provoking the pacification of the M1 state and resulting in a beneficial effect on LDL uptake (p: 0.0845). Using OLR1 gene expression as a readout, in both M1 and M2a macrophages, the average OLR1 expression was reduced by treatment with drug X (p: 0.0003). This implies that M1 macrophages can bind less oxLDL which could reduce the formation of atherosclerotic plaque in the vessel wall. OLR1 protein expression was also reduced (p:0.0003) following 24-hour exposure of the macrophages to drug X. In functional tests, oxLDL uptake by drug X treated hMDMs was reduced by 44% (p:0.003). Conclusions The gene expression of macrophage specific genes and scavenger receptors in human M1 and M2a macrophages was significantly altered following 24-hour exposure to clinically relevant concentrations of drug X in vitro. Drug X reduced ‘active’ and pathogenic cell behaviours e.g., lipid uptake and promoted the polarisation to the M2 state. These data demonstrate that human macrophages can be reprogrammed to a less pathogenic state in vitro and they provide a potential mechanism for the effectiveness of drug X in the stabilisation of carotid atherosclerotic plaques in humans. * Drug X is used for ongoing IP/patent assessment Conflict of Interest No

The dynamic regulation of endothelial pathophenotypes in pulmonary hypertension (PH) remains undefined. Cellular senescence is linked to PH with intracardiac shunts; however, its regulation across PH subtypes is unknown. Since endothelial deficiency of iron-sulfur (Fe-S) clusters is pathogenic in PH, we hypothesized that a Fe-S biogenesis protein, frataxin (FXN), controls endothelial senescence. An endothelial subpopulation in rodent and patient lungs across PH subtypes exhibited reduced FXN and elevated senescence. In vitro, hypoxic and inflammatory FXN deficiency abrogated activity of endothelial Fe-S–containing polymerases, promoting replication stress, DNA damage response, and senescence. This was also observed in stem cell–derived endothelial cells from Friedreich’s ataxia (FRDA), a genetic disease of FXN deficiency, ataxia, and cardiomyopathy, often with PH. In vivo, FXN deficiency–dependent senescence drove vessel inflammation, remodeling, and PH, whereas pharmacologic removal of senescent cells in Fxn-deficient rodents ameliorated PH. These data offer a model of endothelial biology in PH, where FXN deficiency generates a senescent endothelial subpopulation, promoting vascular inflammatory and proliferative signals in other cells to drive disease. These findings also establish an endothelial etiology for PH in FRDA and left heart disease and support therapeutic development of senolytic drugs, reversing effects of Fe-S deficiency across PH subtypes.

The mechanisms linking shear stress, endothelial physiology and plaque biology are currently poorly understood, but their elucidation could identify new strategies to reduce plaque growth and rupture. To address this, we use eNOS as high shear stress marker coupled to immunofluorescent staining, optical clearing and light-sheet microscopy, to develop a system for analysing the spatial distribution of proteins in murine plaques and correlate them with local WSS. Results Confocal microscopy of concavity mounted slides revealed strong eNOS staining at the outer curvature of WT mice but significantly reduced staining at the inner curvature (N=5; P Light-sheet microscopy of cleared samples with preserved 3D structure confirmed elevated expression of eNOS at HSS regions of the outer curvature (figure 1A; N=5 WT; P Conflict of Interest Atherosclerosis

Optica 8(12), 1588 - 1595 (2021). doi:10.1364/OPTICA.441004, Two-dimensional (2D) Talbot array illuminators (TAIs) were designed, fabricated, and evaluated for high-resolution high-contrast x-ray phase imaging of soft tissue at 10���20 keV. The TAIs create intensity modulations with a high compression ratio on the micrometer scale at short propagation distances. Their performance was compared with various other wavefront markers in terms of period, visibility, flux efficiency, and flexibility to be adapted for limited beam coherence and detector resolution. Differential x-ray phase contrast and dark-field imaging were demonstrated with a one-dimensional, linear phase stepping approach yielding 2D phase sensitivity using unified modulated pattern analysis (UMPA) for phase retrieval. The method was employed for x-ray phase computed tomography reaching a resolution of 3 ��m on an unstained murine artery. It opens new possibilities for three-dimensional, non-destructive, and quantitative imaging of soft matter such as virtual histology. The phase modulators can also be used for various other x-ray applications such as dynamic phase imaging, super-resolution structured illumination microscopy, or wavefront sensing., Published by OSA, Washington, DC

The use of animal models is fundamental to furthering our understanding of human disease mechanisms, as well as identifying potential therapeutic targets. Diseases of ageing often involve multiple body systems; however, multi-systemic features are not fully recapitulated in the many of the animal models available. Therefore, combining pre-clinical models to better reflect the multimorbidities observed at the clinical level is critical. This review will highlight some of the key pre-clinical experimental models associated with cardiovascular (atherosclerosis, coronary heart disease), cerebrovascular (stroke, vascular dementia), metabolic (obesity, type-2 diabetes mellitus) and neurological (amyotrophic lateral sclerosis, frontotemporal dementia, Parkinson's, epilepsy) diseases, and whether these models encompass known multimorbidities. In addition to this, we discuss established pre-clinical models that combine two or more conditions, within the context of dementia.

Arterial medial calcification is an independent risk factor for mortality in chronic kidney disease. We previously reported that knock-down of PKCα expression increases high phosphate-induced mineral deposition by vascular smooth muscle cellsin vitro. This new study tests the hypothesis that PKCα regulates uremia-induced medial calcificationin vivo. Female wild-type and PKCα−/−mice underwent a two-stage subtotal nephrectomy and were fed a high phosphate diet for 8 weeks. X-ray micro computed tomography demonstrated that uremia-induced medial calcification was increased in the abdominal aorta and aortic arch of PKCα−/−mice compared to wild-types. Blood urea nitrogen was also increased in PKCα−/−mice compared to wild-types; there was no correlation between blood urea nitrogen and calcification in PKCα−/−mice. Phosphorylated SMAD2 immunostaining was detected in calcified aortic arches from uremic PKCα−/−mice; the osteogenic marker Runx2 was also detected in these areas. No phosphorylated SMAD2 staining were detected in calcified arches from uremic wild-types. PKCα knock-down increased TGF-β1-induced SMAD2 phosphorylation in vascular smooth muscle cellsin vitro, whereas the PKCα activator prostratin decreased SMAD2 phosphorylation. In conclusion, loss of PKCα increases uremia-induced medial calcification. The PKCα/TGF-β signaling axis could therefore represent a new therapeutic target for arterial medial calcification in chronic kidney disease.

Endothelial cell (EC) sensing of fluid shear stress regulates atherosclerosis, a disease of arteries that causes heart attack and stroke. Atherosclerosis preferentially develops at regions of arteries exposed to low oscillatory shear stress (LOSS), whereas high shear regions are protected. We show using inducible EC-specific genetic deletion in hyperlipidaemic mice that the Notch ligands JAG1 and DLL4 have opposing roles in atherosclerosis. While endothelial Jag1 promoted atherosclerosis at sites of LOSS, endothelial Dll4 was atheroprotective. Analysis of porcine and murine arteries and cultured human coronary artery EC exposed to experimental flow revealed that JAG1 and its receptor NOTCH4 are strongly upregulated by LOSS. Functional studies in cultured cells and in mice with EC-specific deletion of Jag1 show that JAG1-NOTCH4 signalling drives vascular dysfunction by repressing endothelial repair. These data demonstrate a fundamental role for JAG1-NOTCH4 in sensing LOSS during disease, and suggest therapeutic targeting of this pathway to treat atherosclerosis.

Early impairments to neurovascular coupling have been proposed to be a key pathogenic factor in the onset and progression of Alzheimer’s disease (AD). Studies have shown impaired neurovascular function in several mouse models of AD, including the J20-hAPP mouse. In this study, we aimed to investigate early neurovascular changes using wild-type (WT) controls and J20-hAPP mice at 6-9 months of age, by measuring cerebral haemodynamics and neural activity to physiological sensory stimulations. A thinned cranial window was prepared to allow access to cortical vasculature and imaged using 2D-optical imaging spectroscopy (2D-OIS). After chronic imaging sessions where the skull was intact, a terminal acute imaging session was performed where an electrode was inserted into the brain to record simultaneous neural activity. We found that cerebral haemodynamic changes were significantly enhanced in J20-hAPP mice compared with controls in response to physiological stimulations, potentially due to the significantly higher neural activity (hyperexcitability) seen in the J20-hAPP mice. Thus, neurovascular coupling remained preserved under a chronic imaging preparation. Further, under hyperoxia, the baseline blood volume and saturation of all vascular compartments in the brains of J20-hAPP mice were substantially enhanced compared to WT controls, but this effect disappeared under normoxic conditions. This study highlights novel findings not previously seen in the J20-hAPP mouse model, and may point towards a potential therapeutic strategy by driving an increased baseline blood flow to the brain, thereby potentially enhancing the clearance of beta-amyloid.

ObjectiveAtherosclerosis is a major risk factor for dementia. The aims of this study were to determine if experimental atherosclerosis leads to altered neurovascular function and causes neurovascular damage.Approach and ResultsWe analysed cerebral blood volume in male C57BL6/J mice injected with an adeno-associated virus (AAV) vector for mutated proprotein convertase subtilisin/kexin type 9 (PCSK9D377Y) fed a Western diet for 35 weeks to induce atherosclerosis (ATH) and 9-12m male wild-type (WT) C57BL/6J. We imaged blood volume responses to sensory stimulation and vascular reactivity gas challenges in the cortex of the brain through a thinned cranial window using 2D-optical imaging spectroscopy (2D-OIS). Neural activity was also recorded with multi-channel electrodes. Stimulation-evoked cortical haemodynamics, in terms of cerebral blood volume, were significantly reduced in ATH mice compared to WT and evoked neural activity was also significantly lower. However, vascular reactivity as assessed by 10% hypercapnia, remained intact in ATH mice. Immunohistochemistry in ATH mice revealed a reduced number of cortical neurons and pericytes in the cortex, but increased astrogliosis. qRT-PCR revealed significantly enhanced TNFα & IL1β in ATH mice compared to WT as well as significant upregulation of eNOS.ConclusionSystemic atherosclerosis causes significant neurovascular decline by 9m in atherosclerotic mice characterised by reduced neural activity, associated with loss of neurons and subsequent reduced cortical haemodynamics in response to physiological stimulations. The altered neurovascular function in ATH mice is chiefly mediated by TNFα.HighlightsSystemic atherosclerosis leads to significantly reduced stimulus-evoked hemodynamic responses in the cortex by 9m of age in the rAAV8-mPCSK9-D377Y mouse model of atherosclerosis compared to wild-type controls.Reduced cerebral haemodynamics are related to reduced neural activity in the cortex that could be due to a loss of cortical neurons potentially caused by significant TNFa-mediated neuroinflammation.

Neutrophils are implicated in the pathogenesis of atherosclerosis but are seldom detected in atherosclerotic plaques. We investigated whether neutrophil-derived microvesicles may influence arterial pathophysiology. Here we report that levels of circulating neutrophil microvesicles are enhanced by exposure to a high fat diet, a known risk factor for atherosclerosis. Neutrophil microvesicles accumulate at disease-prone regions of arteries exposed to disturbed flow patterns, and promote vascular inflammation and atherosclerosis in a murine model. Using cultured endothelial cells exposed to disturbed flow, we demonstrate that neutrophil microvesicles promote inflammatory gene expression by delivering miR-155, enhancing NF-κB activation. Similarly, neutrophil microvesicles increase miR-155 and enhance NF-κB at disease-prone sites of disturbed flow in vivo. Enhancement of atherosclerotic plaque formation and increase in macrophage content by neutrophil microvesicles is dependent on miR-155. We conclude that neutrophils contribute to vascular inflammation and atherogenesis through delivery of microvesicles carrying miR-155 to disease-prone regions., The role of neutrophils in the development of atherosclerosis has long been an enigma, with few neutrophils detected within the plaque. Here, the authors show that microvesicles released from neutrophils increase vascular inflammation and enhance atherosclerotic plaque formation through delivery of miR-155.

Context Hypoglycemia is emerging as a risk for cardiovascular events in diabetes. We hypothesized that hypoglycemia activates the innate immune system, which is known to increase cardiovascular risk. Objective To determine whether hypoglycemia modifies subsequent innate immune system responses. Design and Setting Single-blinded, prospective study of three independent parallel groups. Participants and Interventions Twenty-four healthy participants underwent either a hyperinsulinemic-hypoglycemic (2.5 mmol/L), euglycemic (6.0 mmol/L), or sham-saline clamp (n = 8 for each group). After 48 hours, all participants received low-dose (0.3 ng/kg) intravenous endotoxin. Main Outcome Measures We studied in-vivo monocyte mobilization and monocyte-platelet interactions. Results Hypoglycemia increased total leukocytes (9.98 ± 1.14 × 109/L vs euglycemia 4.38 ± 0.53 × 109/L, P < 0.001; vs sham-saline 4.76 ± 0.36 × 109/L, P < 0.001) (mean ± SEM), mobilized proinflammatory intermediate monocytes (42.20 ± 7.52/μL vs euglycemia 20.66 ± 3.43/μL, P < 0.01; vs sham-saline 26.20 ± 3.86/μL, P < 0.05), and nonclassic monocytes (36.16 ± 4.66/μL vs euglycemia 12.72 ± 2.42/μL, P < 0.001; vs sham-saline 19.05 ± 3.81/μL, P < 0.001). Following hypoglycemia vs euglycemia, platelet aggregation to agonist (area under the curve) increased (73.87 ± 7.30 vs 52.50 ± 4.04, P < 0.05) and formation of monocyte-platelet aggregates increased (96.05 ± 14.51/μL vs 49.32 ± 6.41/μL, P < 0.05). Within monocyte subsets, hypoglycemia increased aggregation of intermediate monocytes (10.51 ± 1.42/μL vs euglycemia 4.19 ± 1.08/μL, P < 0.05; vs sham-saline 3.81± 1.42/μL, P < 0.05) and nonclassic monocytes (9.53 ± 1.08/μL vs euglycemia 2.86 ± 0.72/μL, P < 0.01; vs sham-saline 3.08 ± 1.01/μL, P < 0.05), with platelets compared with controls. Hypoglycemia led to greater leukocyte mobilization in response to subsequent low-dose endotoxin challenge (10.96 ± 0.97 vs euglycemia 8.21 ± 0.85 × 109/L, P < 0.05). Conclusions Hypoglycemia mobilizes monocytes, increases platelet reactivity, promotes interaction between platelets and proinflammatory monocytes, and potentiates the subsequent immune response to endotoxin. These changes may contribute to increased cardiovascular risk observed in people with diabetes., Using an in vivo human experimental model, we show that hypoglycemia primes the innate immune system, leading to a more profound inflammatory response to a subsequent inflammatory stimulus.

Background: Bone morphogenetic protein receptor type 2 (BMPR2) mutations are present in patients with heritable and idiopathic pulmonary arterial hypertension (PAH). Circulating levels of Interleukin-1 (IL-1) are raised in patients and animal models. Whether interplay between BMP and IL- 1 signalling can explain the local manifestation of PAH in the lung remains unclear. Methods: Cell culture, siRNA and mRNA microarray analysis of RNA isolated from human Pulmonary artery (PASMC) and Aortic (AoSMC) smooth muscle cells were used. R899X+/- BMPR2 transgenic mice fed western diet for six weeks were given daily injections of IL-1s prior to assessment for PAH and tissue collection. Results: PASMC have reduced inflammatory activation in response to IL-1s compared with AoSMCs, however PASMC with reduced BMPR2 demonstrated an exaggerated response. Mice treated with IL-1s had higher white blood cell counts, and significantly raised serum protein levels of IL-6 and OPG plasma levels recapitulating in vitro data. Phenotypically, IL-1s treated mice demonstrated increased pulmonary vascular remodelling. Conclusions: IL-1s induces an exaggerated pulmonary artery specific transcriptomic inflammatory response when BMPR2 signalling is reduced.

RationaleTransforming growth factor-beta (TGFβ) is tightly regulated at multiple levels, with regulation at the receptor level now recognized as a key determinant of the cellular response to this pleiotropic cytokine. TGFβ promotes saphenous vein graft neointima formation after coronary artery bypass graft (CABG) surgery, inducing smooth muscle cell (SMC) hyperplasia and fibrosis by signaling via activin receptor-like kinase 5(ALK5). However, the role of the alternate TGFβ receptor ALK1 remains completely unknown.ObjectiveTo define the receptor pathways activated by TGFβ in SMCs and their mechanistic importance during CABG neointima formation.Methods and resultsRadioligand co-IP assays revealed direct interactions between TGFβ, ALK5 and ALK1 in primary saphenous vein graft SMC (HSVSMC) from patients undergoing CABG. Knockdown and pharmacological inhibition of ALK5 or ALK1 in HSVSMC significantly attenuated TGFβ-induced phosphorylation of receptor-regulated (R)-Smads 2/3 and 1/5, respectively. Microarray profiling followed by qRT-PCR validation showed that TGFβ induced distinct transcriptional networks downstream of ALK5 or ALK1, associated with HSVSMC contractility and migration, respectively and confirmed using migration assays as well as qRT-PCR and western blot assays of contractile SMC markers. scRNAseq analysis of TGFβ-treated HSVSMC identified distinct subgroups of cells showing ALK5 or ALK1 transcriptional responses, while RNA velocity analyses indicated divergence in differentiation towards ALK5 or ALK1-dominant lineages. ALK1, ALK5 and their downstream effectors pSmad1/5 and pSmad2/3 were localized to αSMA+ neointimal SMCs in remodelled mouse vein grafts. Pharmacological inhibition or genetic ablation of Smad1/5 substantially reducing neointima formation following acute vascular injury. Notably, expression and activation of ALK1, ALK5 and their respective downstream R-Smads was already evident in hyperplastic saphenous veins prior to grafting.ConclusionsWhilst canonical TGFβ signaling via ALK5 promotes a contractile HSVSMC phenotype, transactivation of ALK1 by TGFβ induces neointima formation by driving cell migration. Restoring the balance between ALK1 and ALK5 in HSVSMC may represent a novel therapeutic strategy for vein graft failure.

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-1αβ 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-1β floxed(fl/fl) mouse line, we demonstrated that CRPS IgG-induced changes are in part mediated by microglia-derived IL-1β, 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.

Trib1 controls atherosclerotic plaque macrophage function by up-regulating OLR1, promoting foam cell formation and atherosclerosis., Macrophages drive atherosclerotic plaque progression and rupture; hence, attenuating their atherosclerosis-inducing properties holds promise for reducing coronary heart disease (CHD). Recent studies in mouse models have demonstrated that Tribbles 1 (Trib1) regulates macrophage phenotype and shows that Trib1 deficiency increases plasma cholesterol and triglyceride levels, suggesting that reduced TRIB1 expression mediates the strong genetic association between the TRIB1 locus and increased CHD risk in man. However, we report here that myeloid-specific Trib1 (mTrib1) deficiency reduces early atheroma formation and that mTrib1 transgene expression increases atherogenesis. Mechanistically, mTrib1 increased macrophage lipid accumulation and the expression of a critical receptor (OLR1), promoting oxidized low-density lipoprotein uptake and the formation of lipid-laden foam cells. As TRIB1 and OLR1 RNA levels were also strongly correlated in human macrophages, we suggest that a conserved, TRIB1-mediated mechanism drives foam cell formation in atherosclerotic plaque and that inhibiting mTRIB1 could be used therapeutically to reduce CHD.

Intimal calcification is the formation of mineralised tissue within atherosclerotic lesions and can lead to an increased risk of plaque rupture and mortality in man. Even though experimental data are sparse, it had been suggested that inhibiting protein kinase Cα (PKCα) may be of therapeutic benefit in atherosclerosis. However, we recently discovered that loss of PKCα increases mineral deposition by vascular smooth muscle cells (VSMCs) in a transforming growth factor-β (TGFβ)-dependent manner in vitro, suggesting that vascular calcification and its devastating consequences could be increased if patients with cardiovascular disease are treated with PKCα inhibitors. This study tests the hypothesis that PKCα regulates atherosclerosis and intimal calcification in vivo. Nine to ten week-old male PKCα knock-out (PKCα-/-) mice crossed with ApoE-/- mice (PKCα-/-ApoE-/-) were fed a high-fat, high-cholesterol diet (Western diet) for 8, 18 and 28 weeks. Male ApoE-/- mice fed the same diet were used as controls. En face Oil Red O staining was significantly increased (∼1.5-fold, P Aortic sinus lesion size was not significantly different between PKCα-/-ApoE-/- and ApoE-/- mice at any time point. However, a significant increase in intimal calcification (∼2-fold, P In conclusion, our study has identified that PKCα may play a protective role in both atherosclerosis and intimal calcification. This suggests that inhibiting PKCα may not be of any therapeutic benefit in atherosclerosis. Activation of PKCα could instead represent a new therapeutic target for atherosclerosis-induced intimal calcification. Conflict of interest None

Medial calcification is the formation of mineralised tissue within the smooth muscle layer of the vessel wall, and frequently occurs in patients with chronic kidney disease. Calcification within the medial layer of the vessel wall can reduce aortic and arterial elasticity, which impairs cardiovascular haemodynamics and results in a significantly elevated risk of morbidity and mortality in the form of hypertension, cardiac hypertrophy and sudden cardiac death. Protein kinase Cα (PKCα) belongs to the PKC family of serine/threonine kinases and we recently discovered that knocking-down PKCα expression increases high phosphate-induced mineral deposition by vascular smooth muscle cells (VSMCs) in vitro. This study tests the hypothesis that PKCα regulates uraemia-induced medial calcification in vivo. PKCα-/- mice were generated on the calcification-susceptible DBA/2 background (PKCα-/-) using CRISPR/Cas9 technology. To induce uraemia, wild-type DBA/2 and PKCα-/- mice underwent a two-stage sub-total nephrectomy and were fed a high phosphate (1.5%) diet for 8 weeks. Renal function was measured by blood urea nitrogen (BUN). Calcification in the ascending aorta/aortic arch and abdominal aorta were analysed and quantified by micro CT and histology. On average, 68.6 ± 3% (SD, n=3) of renal mass was removed from wild-type and 65 ± 3.2% (n=5) was removed from PKCα-/- mice (P>0.05). Loss of PKCα significantly increased uraemia-induced medial calcification in the abdominal aorta (∼20-fold increase, P We have shown previously that inhibiting transforming growth factor-β (TGF-β) signalling with SB431542 prevents the increase in calcification observed in PKCα-siRNA treated VSMCs. Therefore, to determine the mechanism by which loss of PKCα exerts its effects we examined the relationship between PKCα and TGF-β signalling in vitro and in vivo. Our results show that knock-down of PKCα using siRNA increased TGF-β1-induced Smad2 phosphorylation in VSMCs in vitro (P In conclusion, our study suggests that PKCα may play a protective role in uraemia-induced medial calcification. The PKCα/TGFβ signalling axis could therefore represent a new therapeutic target for uraemia-induced medial calcification. Conflict of interest None

Introduction Atherosclerosis is a complex inflammatory disease in which major arteries narrow, and an atherosclerotic plaque develops, modulated by genetic and environmental factors. Macrophages participate in all stages of the plaque formation and progression. Different macrophage subtypes within carotid plaques have been shown to be altered in unstable vs. stable plaques. M1 proinflammatory macrophages are mostly abundant in symptomatic and unstable plaques. In contrast, M2 macrophages are associated with regulatory and wound-healing properties and generally found in stable lesions. In the future, the characterisation of these changes in situ in the carotid plaques, associated with in vitro macrophage studies, and blood composition may lead to the identification of diagnostic markers to direct the best treatment for stroke patients. Methods Carotid plaques were obtained from patients with a recent stroke or TIA and with a greater than 50% stenosis of the internal carotid artery. Plaques were removed during carotid endarterectomy and fixed in 10% (v/v) neutral buffered formalin for maximum 36h. Macrophage phenotypes within the shoulder regions were characterised with immunofluorescence microscopy. A novel immunofluorescence staining, image acquisition and analysis technique was developed to identify single M1 (CD68+CD86+MRC1-), single M2 (CD68+CD86-MRC1+), double positive (CD68+CD86+MRC1+) and double negative (CD68+CD86-MRC1-) macrophages in the human carotid atherosclerotic plaques. Results 20 carotid plaques were collected from patients with recent stroke or TIA (73% male, 70% smokers, 87% hypertensive). In 17 plaques the shoulder region (either side of a thinned fibrous cap) was sufficiently intact for histological analysis. Dense infiltration with macrophages was seen in all 17 specimens. The population comprised double positive (CD86+MRC1+) (71%), M1 single positive (CD86+MRC1-) (19%), and M2 single positive (CD86-MRC1+) (4%). Double negative (CD86-MRC1-) macrophages represented 6% of all macrophages. Amongst 10 unstable plaques (defined as AHA Grade 6 or above) the majority (80%) of shoulder macrophages were M1 single positive. In contrast, amongst 7 stable plaques, the majority of plaque shoulder macrophages (65%) were M2 single positive. Conclusions Macrophages within symptomatic carotid plaques can express both M1 (CD86) and M2 (MRC1) markers, or can express neither. Pro-inflammatory macrophage phenotypes cluster in areas of unstable carotid plaque. Larger studies are needed to confirm the role of dual staining macrophages and double negative macrophages. If such in situ macrophages can be manipulated towards one phenotype with pharmaceutical interventions, this could offer a potential new approach to plaque stabilising therapies. Conflict of interest No

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.

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Despite decades of research, our understanding of the processes controlling late-stage atherosclerotic plaque stability remains poor. A prevailing hypothesis is that reducing inflammation may improve advanced plaque stability, as recently tested in the Canakinumab Anti-inflammatory Thrombosis Outcome Study (CANTOS) trial, in which post-myocardial infarction subjects were treated with an IL-1β antibody. Here, we performed intervention studies in which smooth muscle cell (SMC) lineage-tracing Apoe-/- mice with advanced atherosclerosis were treated with anti-IL-1β or IgG control antibodies. Surprisingly, we found that IL-1β antibody treatment between 18 and 26 weeks of Western diet feeding induced a marked reduction in SMC and collagen content, but increased macrophage numbers in the fibrous cap. Moreover, although IL-1β antibody treatment had no effect on lesion size, it completely inhibited beneficial outward remodeling. We also found that SMC-specific knockout of Il1r1 (encoding IL-1 receptor type 1) resulted in smaller lesions nearly devoid of SMCs and lacking a fibrous cap, whereas macrophage-selective loss of IL-1R1 had no effect on lesion size or composition. Taken together, these results show that IL-1β has multiple beneficial effects in late-stage murine atherosclerosis, including promotion of outward remodeling and formation and maintenance of an SMC- and collagen-rich fibrous cap.

Background Hypertension is a complex condition and a common cardiovascular risk factor. Dietary docosahexaenoic acid ( DHA ) modulates atherosclerosis and hypertension, possibly via an inflammatory mechanism. IL‐1 (interleukin 1) has an established role in atherosclerosis and inflammation, although whether IL ‐1 inhibition modulates blood pressure is unclear. Methods and Results Male apoE −/− (apolipoprotein E–null) mice were fed either a high fat diet or a high fat diet plus DHA (300 mg/kg per day) for 12 weeks. Blood pressure and cardiac function were assessed, and effects of DHA on wall shear stress and atherosclerosis were determined. DHA supplementation improved left ventricular function, reduced wall shear stress and oscillatory shear at ostia in the descending aorta, and significantly lowered blood pressure compared with controls (119.5±7 versus 159.7±3 mm Hg, P DHA feeding in mice demonstrated a 4‐fold reduction in lesion burden in distal aortas and in brachiocephalic arteries ( P DHA treatment selectively decreased plaque endothelial IL ‐1β ( P Conclusions Our findings revealed that raised blood pressure can be reduced by inhibiting IL ‐1 indirectly by administration of DHA in the diet through a mechanism that involves a reduction in wall shear stress and local expression of the proinflammatory cytokine IL ‐1β.

Introduction Genome Wide Association Studies have identified Tribbles-1 (TRIB1) as a regulator of plasma lipid levels and a risk factor for MI. Studies using Trib1 full body- and liver specific knockout mice have shown that hepatic expression of Trib1 reduces circulating lipids1. Additionally, Trib1 has been shown to be a regulator of alternatively activated macrophage polarisation2. However, there has been no study to directly evaluate the role of myeloid Trib1 (mTrib1) in atherogenesis. Methods To determine the role of mTrib1 in atherosclerosis, we developed myeloid-Trib1 deficient (mTrib1KO) and overexpression (mTrib1Tg) mouse strains. To distinguish between metabolic and inflammatory drivers of atherosclerosis, bone marrow from these strains were transplanted into lethally irradiated ApoE-/- mice and fed on a western diet for 12 weeks. Additionally, we also induced atherosclerosis in mTrib1Tg and mTrib1WT strains by injecting rAAV8/mPCSK9 and fed western diet for 12 weeks. Results mTrib1KO→ApoE-/- mice were protected while mTrib1Tg→ApoE-/- mice presented with increased atherosclerotic burden in both the aorta (p Conclusion We conclude that Trib1 is a potent regulator of atherosclerosis, the over-expression of which promotes atherogenesis through elevated oxLDL uptake and subsequent foam cell formation in plaque macrophages. References . Burkhardt et al. JCI (2010);120:4410–4414. . Satoh et al. Nature (2013);7442:524–8.

Introduction Atherosclerosis is a pathological chronic inflammatory process where lipoprotein, cholesterol, calcium and apoptotic cells build up a lipid-rich plaque within an artery. Inflammatory cells such as macrophages are a major component of the atherosclerotic plaque and participate in all stages of plaque formation and progression. The plaque can be classified as histologically stable or unstable, depending on the extent of inflammation, composition and the thickness of fibrous cap. Macrophage subtypes within carotid plaques have been shown to be altered in unstable vs. stable plaques. Broadly, M1 macrophages are proinflammatory and promote atherogenesis as well as enhancing plaque instability. In contrast, M2 macrophages have regulatory and wound-healing properties including collagen synthesis and promote tissue repair. Detailed characterisation of these macrophage subtypes and correlative analysis with in vivo carotid plaque MRI and blood composition may lead to the identification of diagnostic markers to direct the best treatment for stroke patients. Methodology Carotid plaques were obtained from patients undergoing carotid endarterectomy for recently symptomatic carotid stenosis. A validated clinical histology grading was used to define degree of overall plaque instability. The plaque ’shoulder’ regions were identified on H and E stained sections and were deemed the regions of interest (ROI). Macrophage phenotypes within the shoulder regions were characterised with immunofluorescence (IF) microscopy. An IF protocol to simultaneously stain for M1 and M2 macrophages alongside an image acquisition and analysis strategy was then developed. We used single IF with a pan macrophage polarisation marker (CD68), and dual IF with M1 (CD86) and M2 marker (MR: Mannose Receptor). Plaques were imaged using multi-colour fluorescence microscopy (Leica AF6000). After image registration, the regions of positive staining for CD68 were overlapped with the dual staining (CD86/MR) section and the prevalence of CD86 +and MR +macrophages were measured. Results Of the 16 collected plaques, an intact shoulder was identified in 13 samples and in these regions, macrophages were abundant. A median of 31.1% (4.5%–70.6%) macrophages stained for both M1 (CD86) and M2 (MR) markers. There was a higher prevalence of CD86 +MR + macrophages in shoulder regions from unstable versus stable plaques but this did not reach significance. Single positive M2 (MR +CD86-) cells were of higher prevalence in stable plaques. Hemosiderin-laden macrophages were also identified via their strong auto-fluorescence. Conclusions M1 (CD86+) and dual staining (CD86 +MR+) macrophage populations were predominant in the unstable plaques whereas single positive M2 (MR +CD86-) cells were predominant in stable plaques. Future analysis on a larger number of carotid plaques is required to better understand the prevalence and potential influence of different macrophage populations on plaque instability.

Background Atherosclerosis is a chronic vascular inflammatory disease characterised by disturbed arterial blood flow due to the atheromatous plaque build-up within arterial layers. Lipid rich plaques contain various forms of cholesterol, such as acetylated low density lipoprotein (AcLDL), that undergo many modification processes and which enter vascular cells, initiating over exuberant repair processes leading to arterial occlusion and life threatening myocardial infarction or stroke. Endothelial cells (EC) strongly drive the pathogenesis of atherosclerosis by producing pro-inflammatory cytokines, such as interleukin-1 beta (IL-1β). We have recently shown that the release of IL-1β from EC occurs via extracellular lysosomal derived vesicles, but the stimuli and mechanisms by which IL-1β is released remain to be fully elucidated. We hypothesise that AcLDL enters the arterial endothelium and induces IL-1 secretion, potentially via a caspase-1/NLRP3 mechanism. Methods Human coronary artery endothelial cells (HCAEC) isolated from three different donors were cultured and stimulated with pro-inflammatory cytokines TNF-α and IL-1β (10 ng/ml each, for 48 hours), followed by incubation with native human AcLDL cholesterol at multiple concentrations (10–200 ug/ml) for 6 hours. Cell lysates and culture supernatants were collected and analysed for IL-1β using ELISA. Cell viability was also measured. Results AcLDL induced the release of IL-1β from stimulated HCAECs in a dose dependent manner with maximum release (155.4 pg/ml, n=3) at concentration of 50 ug/ml. This was 4 fold greater than that released by cytokine-stimulated (38.49 pg/ml, n=3) and 3 fold greater than that released by neutrophil elastase (49.45 pg/ml, n=3). This release was not caused by toxicity of the AcLDL: cell viability was confirmed by lactate dehydrogenase cell viability assay. Conclusion AcLDL is capable of eliciting IL-1β release in activated HCAECs, without causing toxicity.

Neutrophils have been implicated in the pathogenesis of atherosclerosis, a lipid-driven disease of arteries, but they are seldom found in atherosclerotic plaques. To resolve this longstanding paradox, we investigated whether neutrophil-derived microvesicles may influence arterial pathophysiology. Clinical and pre-clinical studies revealed that levels of circulating neutrophil microvesicles were enhanced by exposure to a high fat diet, a known risk factor for atherosclerosis. Neutrophil microvesicles accumulated at disease-prone regions of arteries that are exposed to complex flow patterns, and they promoted vascular inflammation and atherosclerosis in a murine model. Using cultured endothelial cells exposed to disturbed flow, it was demonstrated that neutrophil microvesicles promoted inflammatory gene expression by delivering a microRNA (miR-155) that enhanced NF-κB activation. Similary, neutrophil microvesicles increased miR-155 and enhanced NF-κB at disease-prone sites of disturbed flow in arteries of mice. We conclude that delivery of microvesicles carrying miR-155 to disease-prone regions of arteries provides a novel mechanism by which neutrophils contribute to vascular inflammation and atherogenesis.

The endothelium is critically involved in the pathogenesis of atherosclerosis by producing proinflammatory mediators, including interleukin-1 beta (IL-1β). Coronary arteries from patients with ischaemic heart disease express large amounts of IL-1β in endothelium. However, the mechanism by which endothelial cells (ECs) release IL-1β 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-1β processing and release. This study tested the hypothesis that NE potentiates the processing and release of IL-1β from human coronary endothelium. We found that NE cleaves the pro-isoform of IL-1β in ECs and causes significant secretion of bioactive IL-1β via extracellular vesicles. This release was significantly attenuated by inhibition of neutrophil elastase, but not caspase-1. Transient increases in intracellular Ca2+ levels were observed prior to secretion. Inside ECs, and after NE treatment only, IL-1β was detected within LAMP-1 positive multivesicular bodies (MVBs). The released vesicles contained bioactive IL-1β. In vivo, in experimental atherosclerosis, NE was detected in mature atherosclerotic plaques, predominantly in the endothelium, alongside IL-1β. This study reveals a novel mechanistic link between NE expression in atherosclerotic plaques and concomitant pro-inflammatory bioactive IL-1β secretion from ECs; this could reveal additional potential anti-IL-1β therapeutic targets and provide further insight into the inflammatory process by which vascular disease develops.

The success of percutaneous coronary intervention (PCI) has been limited by restenosis and stent thrombosis. Delayed or incomplete endothelial regeneration is believed to be a key factor responsible for these events. Developing a stent with an accelerated healing profile may be of benefit. We aimed to evaluate the feasibility and safety of seeding a bare metal stent (BMS) with human trophoblastic endovascular progenitor cells (hTEC) derived from human embryonic stem cells. A porcine coronary artery model was used to compare the rate and extent of endothelial regeneration and the degree of neointimal proliferation. Characterisation of hTEC confirmed a mixed progenitor and endothelial cell phenotype. The biodistribution and fate of hTEC were studied using radiolabelled 111Indium oxine and fluorescent in situ hybridisation. Scanning electron microscopy showed earlier endothelial coverage in hTEC-seeded stents as compared to similar BMS. hTEC-seeded BMS achieved complete stent coverage in three days. Quantitative coronary angiography, intravascular ultrasound assessment and histomorphometry showed no difference in neointimal hyperplasia between hTEC-seeded and control BMS. hTEC seeding of coronary stents is a novel and safe approach to accelerate endothelial regeneration without increasing neointimal proliferation.

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) Ib?. IL-1 and platelet-mediated signals converge on microglia, as both IL-1Ra and GPIb? blockade reversed the production of IL-1? 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.

Aims Platelets have a fundamental role in atherothrombosis, but their role in early atherogenesis is unclear. The P2Y12 receptor is responsible for amplifying and sustaining platelet activation and P2Y12 inhibition is crucial in modulating the vessel wall response to injury. We therefore examined the role of platelet vs. vessel wall P2Y12 in early atherogenesis and considered the use of P2Y12 antagonists ticagrelor and clopidogrel in modulating this process. Methods and results ApoE−/− and ApoE−/−P2Y12−/− male mice underwent bone marrow transplantation and were fed a western diet for 4 weeks before assessing atherosclerotic burden. Compared with ApoE−/− controls, platelet P2Y12 deficiency profoundly reduced platelet reactivity but had no effect on atheroma formation, whereas vessel wall P2Y12 deficiency significantly attenuated atheroma in the aortic sinus and brachiocephalic artery (both P < 0.001). ApoE−/− and ApoE−/−P2Y12−/− male mice fed western diet plus either twice-daily doses of ticagrelor (100 mg/kg) or daily clopidogrel (20 mg/kg) for 4 weeks exhibited no significant reduction in atheroma compared with control mice fed mannitol. Attenuated P-selectin expression confirmed platelet P2Y12 inhibition in drug-treated mice. Conclusions Despite its major contribution to platelet reactivity, platelet P2Y12 has no effect on early atheroma formation, whereas vessel wall P2Y12 is important in this process. Ticagrelor and clopidogrel effectively reduced platelet reactivity but were unable to inhibit early atherogenesis, demonstrating that these P2Y12 inhibitors may not be effective in preventing early disease.