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.

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-1β (IL-1β) 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-1β 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-1β from stimulated HCASMCs and HCAECs, respectively, compared to control. Inhibition of either NLRP3, caspase-1 or gasdermin D significantly reduced the release of IL-1β (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-1β (2.1-fold, P < 0.0001). HCAECs and HCASMCs elicited the release of IL-1β in response to the same stimulus via different mechanisms. In HCAECs, released IL-1β 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.

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.

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.

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 <0.001, n=4 per group). Analysis of atheroma following DHA feeding in mice demonstrated a 4‐fold reduction in lesion burden in distal aortas and in brachiocephalic arteries ( P <0.001, n=12 per group). In addition, DHA treatment selectively decreased plaque endothelial IL ‐1β ( P <0.01). 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.

Idiopathic pulmonary arterial hypertension (IPAH) is increasingly diagnosed in elderly patients who also have an increased risk of co-morbid atherosclerosis. Apolipoprotein E-deficient (ApoE−/−) mice develop atherosclerosis with severe PAH when fed a high-fat diet (HFD) and have increased levels of endothelin (ET)-1. ET-1 receptor antagonists (ERAs) are used for the treatment of PAH but less is known about whether ERAs are beneficial in atherosclerosis. We therefore examined whether treatment of HFD-ApoE−/− mice with macitentan, a dual ETA/ETB receptor antagonist, would have any effect on both atherosclerosis and PAH. ApoE−/− mice were fed chow or HFD for eight weeks. After four weeks of HFD, mice were randomized to a four-week treatment of macitentan by food (30 mg/kg/day dual ETA/ETB antagonist), or placebo groups. Echocardiography and closed-chest right heart catheterization were used to determine PAH phenotype and serum samples were collected for cytokine analysis. Thoracic aortas were harvested to assess vascular reactivity using wire myography, and histological analyses were performed on the brachiocephalic artery and aortic root to assess atherosclerotic burden. Macitentan treatment of HFD-fed ApoE−/− mice was associated with a beneficial effect on the PAH phenotype and led to an increase in endothelial-dependent relaxation in thoracic aortae. Macitentan treatment was also associated with a significant reduction in interleukin 6 (IL-6) concentration but there was no significant effect on atherosclerotic burden. Dual blockade of ETA/ETB receptors improves endothelial function and improves experimental PAH but had no significant effect on atherosclerosis.

Coronary angioplasty initially employed balloon dilatation only. This technique revolutionized the treatment of coronary artery disease, although outcomes were compromised by acute vessel closure, late constrictive remodeling, and restenosis due to neointimal proliferation. These processes were studied in animal models, which contributed to understanding the biology of endovascular arterial injury. Coronary stents overcome acute recoil, with improvements in the design and metallurgy since then, leading to the development of drug-eluting stents and bioresorbable scaffolds. These devices now undergo computer modeling and benchtop and animal testing before evaluation in clinical trials. Animal models, including rabbit, sheep, dog and pig are available, all with individual benefits and limitations. In smaller mammals, such as mouse and rabbit, the target for stenting is generally the aorta; whereas in larger animals, such as the pig, it is generally the coronary artery. The pig coronary stenting model is a gold-standard for evaluating safety; but insights into biomechanical properties, the biology of stenting, and efficacy in controlling neointimal proliferation can also be gained. Intra-coronary imaging modalities such as intravascular ultrasound and optical coherence tomography allow precise serial evaluation in vivo, and recent developments in genetically modified animal models of atherosclerosis provide realistic test beds for future stents and scaffolds.

Introduction Conventional pharmacological treatments for acute myocardial infarction (AMI), a life threatening complication of a sudden coronary occlusion, are limited by their efficiency and side effects. New therapeutic strategies are thus needed to improve outcomes. Carbon monoxide (CO) is cardio-protective at nanomolar concentrations. Carbon monoxide-releasing molecules (CORMs), capable of carrying and releasing controlled quantities of CO in cellular systems, are a promising therapeutic that overcome the limitations of CO gas. CORM-A1 is a water soluble and releases CO slowly through hydrolysis at physiological conditions. We investigated the efficacy and safety of CORM-A1 in reducing infarct size in a clinically relevant porcine model of re-perfused AMI. Methods Male Yorkshire White pigs (25–33 kg) underwent a balloon-induced coronary occlusion at the middle segment of left anterior descending artery beyond the first diagonal branch for 60min. From 15min post-occlusion, sodium borate (control) or CORM-A1 (4.27 mM, each) were infused over a period of 60min. Left ventricular (LV) function and blood pressure were assessed by cardiac catheterization. Cardiac biomarkers, hepatic and renal functions were compared between the groups. Seven days after AMI, animals were culled and in-situ double staining with Evans blue and 2,3,5-triphenyltetrazolium (TTC) performed to measure infarct size. Myocardial inflammation, proliferation and apoptosis were evaluated by immunohistochemistry, immunoblotting, and TUNEL assay. Results CORM-A1 treated pigs had a reduced infarcted area and improved LV function, but no significant change in blood pressure, compared to controls. Infarct size was 35 ± 7% of the area at risk (ischaemic area) in CORM-A1 pigs compared to 90 ± 5% in controls (p < 0.0001, n = 3–8/group). Myocardium from CORM-A1 treated animals had fewer TUNEL positive (30.5 ± 4.7 vs. 46.2 ± 6.6%, p < 0.05, n = 37), Ki67 positive (7.7 ± 2.3 vs. 29.0 ± 4.0%, p < 0.01, n = 3–7) and inflammation positive cells (4.3 ± 1.8 vs. 36.7 ± 7.1%, p < 0.01, n = 3–7) in the infarcted regions, compared to controls. CORM-A1 infused animals also had significantly reduced (2–3-fold) neovascular formation (vWF staining) in the infarcted areas compared to controls (22.0 ± 3.6 vs. 53.3 ± 8.9%, p < 0.01, n = 3–7). A similar pattern was seen in the ischaemic areas. These changes were associated with a down-regulation of HIF-1a expression in the myocardium of CORM-A1 treated animals. Conclusions Our data suggest CORM-A1 as a key modulator of myocardial repair following re-perfused AMI injury. Injury is reduced in CORM-A1 treated animals by reducing inflammation, proliferation and cell death whilst maintaining healthy repair via neovascularisation. This study suggests the development of CORM-A1 as a potential new therapeutic for treatment of patients with AMI and warrants further clinical studies.

Introduction Dietary omega-3 fatty acids have been associated with protection from atherosclerosis. However, the underlying mechanisms are incompletely understood. Blood flow generate a frictional force on endothelial cells called wall shear stress (WSS) that alters vascular wall function. The aim of this study was to determine whether docosahexaenoic acid (DHA), an omega-3 fatty acid, modulates vascular wall inflammation, blood flow velocity and WSS in experimental atherosclerosis. Methods ApoE–/– mice fed either high fat diet (control) or high fat diet plus DHA (300 mg/kg/day) for 12 weeks (n = 12/group). Blood flow velocity was assessed using pulsed wave doppler echocardiography and blood pressure was monitored using Visitech tail-cuff system. Atherosclerosis was measured in whole aorta using enface Oil red O stain, and in aortic roots and brachiocephalic sections stained with Alcian Blue & Elastic Van Gieson stain. Computational flow dynamics (CFD) was used to map WSS magnitude and oscillation in the aorta. Plasma cholesterol levels were quantified by gas chromatography. Results Plasma high density lipoprotein/total cholesterol ratio was significantly increased in DHA treated mice compared to controls (10.77 ± 1.86 vs. 6.63 ± 1.04, p < 0.05). DHA fed mice exhibited a 4–5 fold reduction in distal aortic and brachiocephalic atherosclerosis (p < 0.01) whereas lesion burden in the aortic arch was similar between groups. Dietary supplementation using DHA led to a reduction in blood pressure (119.5 ± 7.33 mmHg (+DHA) vs. 159.7 ± 2.482 mmHg (controls), p < 0.001 and a 12% decrease in aortic blood flow. CFD revealed that oscillatory shear in the descending aorta was reduced in DHA-fed mice compared to controls. Conclusions/Implications Our study suggests that dietary DHA can act systemically by enhancing levels of HDL. It can also act locally by reducing oscillatory shear stress in the descending aorta. Dietary DHA reduced lesion formation specifically in the descending aorta, an effect that can be explained by its dual effects on oscillatory shear and HDL. Therefore, the current study suggests novel and interacting protective mechanisms for DHA actions in atherogenesis with implications for the development of dietary interventions to prevent cardiovascular disease.

The endothelium is critically involved in the pathogenesis of atherosclerosis by producing pro-inflammatory mediators, including IL-1β. Coronary arteries from patients with ischemic heart disease express large amounts of IL-1β in the 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 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-1β was detected within LAMP-1-positive multivesicular bodies. 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 insights into the inflammatory process by which vascular disease develops.

ObjectiveBacterial 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.MethodsWe 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.ResultsS. 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.InterpretationS. 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. Ann Neurol 2014;75:670–683

Backgrounds Current evidence from epidemiological studies, clinical trials and animal based research has shown an inverse relationship between omega-3 fatty acids (n3FA) and cardiovascular events. Docosahexaenoic acid (DHA), a major n3FA in fish oil, has been studied in relation to inflammation but the exact molecular mechanisms involved remain unclear. Additionally, its role in atherosclerosis requires further elucidation. Plant-derived dietary a-Linolenic acid (ALA) has recently shown a favourable impact on hypertension in population-based studies. Objective The aim of this study was to determine whether there were significant differences in the effects of purified DHA and ALA on high blood pressure as a consequence of atheroma in mice fed high fat diet (HFD). Methods Apolipoprotein E knockout mice (ApoE-/-) were fed a HFD alone (control) or HFD containing either DHA or ALA (100 mg/kg/day) for 12 weeks, and their blood pressure and atherosclerotic plaque development were assessed. Results There was no significant changes in the body weights among the three groups. However, the main systolic blood pressure fell by 40 mm Hg (p < 0.0001, n = 4), and diastolic blood pressure by 15mm Hg (p < 0.01, n = 4) in the group received DHA than the control group, there was no significant changes in the group fed ALA. Furthermore, DHA fed mice had a significant increase in plasma HDL-C/T.Cholesterol ratio (10.77 ± 1.85 vs.5.54 ± 1.07 in control group, p < 0.05, n = 10), without significant changes in LDL-C and total Cholesterol levels. Interestingly, plasma cytokines MCP-1, IL-8 and RANTES were significantly decreased in the group received DHA compared to control (p < 0.01, n = 8–12). However, no significant impacts on plasma cytokines and lipid profiles were seen in the ALA fed group. ApoE-/- mice fed DHA showed significantly less atheroma in the distal vessels (Brachiocephalic arteries and descending part of aorta) than the apoE-/- mice on HFD alone or and ALA (p < 0.001, n = 12). Even though no significant changes were seen on plasma IL-1a and IL-1ß levels, there was a significant reduction in IL-1ß and IL-1a proteins in the aortic sinuses of DHA fed animals (20–30% decreased, p < 0.01, n = 8) compared to controls. Moreover, there was a significant decrease in Mac-3 (p < 0.01, n = 8) and TLR4 (p < 0.05, n = 6) positive areas of atherosclerotic plaque in the aortic roots of DHA than the control group. Conclusions This is the first demonstration that DHA but not ALA has profound impacts on high blood pressure induced by HFD. We have previously shown that blocking of IL-1 prevents the rise in blood pressure in response to HFD. Therefore, our study suggests that DHA modulates blood pressure by reducing inflammation in vessel walls through IL-1 linked and HDL-C dependent mechanism. These data raise the possibility of an entirely novel strategy to control atherosclerosis by diet modulation.

Endothelial cells (ECs) are critically involved in the pathogenesis of atherosclerosis by producing inflammatory mediators, including interleukin-1 beta (IL-1β). However, its mechanism of externalisation is yet to be elucidated. This study investigates the effect of neutrophil elastase (NE) on ECs in terms of IL-1β release and explores the underlying mechanism. Human coronary artery endothelial cells (HCAEC) were treated with a combination of tumour necrosis factor-alpha and interleukin-1 alpha then incubated with NE for 2 or 6 h. ELISA, western blotting, flow cytometry and live cell imaging were used. Paraffin-embedded sections of aortic sinus lesions of apoe-/- mice were immunostained for NE and VWF (Von Willebrand factor). NE is predominantly expressed in ECs in experimental atherosclerosis. In vitro, NE caused significant IL-1β release into supernatants after 6h (579 ± 90 vs. 80 ± 19 pg/ml in control; n = 4, p < 0.0001). The release was via microparticle shedding which is significantly attenuated by neutrophil elastase inhibitor III (62.64 ± 10 vs. 579.3 ± 90 pg/ml in NE-primed cells; n = 3, p<0.05); but, the levels of IL-1β in the supernatant or lysate did not change in the presence of caspase-1 inhibitor. NE enters ECs, cleaves proIL-1β (31kDa) inside cells resulting in release of active isoforms of IL-1β in microparticles. No remarkable effects were seen on NALP3 or caspase-1 expression in EC lysates. This is the first description of the secretion of IL-1β by microvesicle shedding from ECs which is caspase-1 independent. NE is detected in the endothelium of murine atherosclerotic plaques and, therefore, this is a plausible mechanism to promote local IL-1β release in the vasculature.

Background Pulmonary arterial hypertension is a fatal disease characterised by progressive narrowing of pulmonary arterioles, driven by aberrant cellular proliferation. Identification of key pathways in disease pathogenesis is required for the development of new-targeted therapies. We have previously reported tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) immunoreactivity within pulmonary vascular lesions from patients with idiopathic pulmonary arterial hypertension and animal models. Since TRAIL induces endothelial cell apoptosis and smooth muscle cell proliferation, we hypothesised that TRAIL is an important driver of disease in pulmonary arterial hypertension. Methods We characterised the expression of TRAIL in human and rodent pulmonary arterial hypertension and determined the effects of TRAIL on pulmonary artery smooth muscle cells (PASMCs) in vitro. Using genetic deletion, pharmacological overexpression, antibody blockade, and bone marrow transplant (BMT) chimera experiments we determined the direct pathogenic role of TRAIL in three independent rodent models of pulmonary arterial hypertension. We then tested the efficacy of inhibiting TRAIL in halting or regressing established disease in two preclinical models. Terminal phenotyping included cardiac catheterisation, echocardiography, and pulmonary vascular immunohistochemistry. Findings TRAIL mRNA and protein expression was upregulated in PASMCs from patients with pulmonary arterial hypertension. In vitro, TRAIL was a mitogen for PASMCs. TRAIL-deficient mice were protected from both hypoxia-induced and diet-induced pulmonary arterial hypertension. Antibody blockade prevented rats from developing toxin-induced disease. In BMT chimeras, only mice with expression of TRAIL restricted to tissue developed pulmonary arterial hypertension. In rodents with established pulmonary arterial hypertension, an anti-TRAIL antibody reversed pulmonary vascular remodelling, through reducing proliferation and inducing apoptosis, improved pulmonary haemodynamics, and significantly improved survival. Interpretation Our studies are the first to determine the importance of TRAIL in the pathogenesis of pulmonary arterial hypertension and demonstrate its potential for translation into a novel therapeutic target Funding British Heart Foundation.

Introduction Atherosclerosis is characterized by endothelial dysfunction, massive inflammatory cell recruitment and activation of leukocytes, accompanied by extracellular release of interleukin-1 beta (IL-1β), a potent pro-inflammatory mediator with proatherogenic effects. Even though IL-1β has been clearly implicated in atherogenesis, potentially via its secretion from activated endothelial cells (ECs), its mechanism of release remains unknown. The proximity of immune cells, monocytes and neutrophils to endothelium during plaque formation and rupture makes it likely that cross talk between cell types and release of soluble factors could cause IL-1 release from endothelial cells. Therefore, we hypothesised that a serine protease called neutrophil elastase (NE), produced by circulating inflammatory cells, might modulate IL-1 release from endothelial cells. Methods Human coronary artery endothelial cells (HCAEC) were treated with a combination of tumour necrosis factor-alpha (TNF-α; 10ng/ml) and interleukin-1 alpha (IL-1-α; 10ng/ml) for 48 hours to up-regulate intracellular IL-1β. The cells were then incubated with NE (1 µg/ml) for 2 or 6 hours in serum-free media. ELISA, western blotting, flow cytometry and live cell imaging were used to determine IL-1β expression, cellular changes and a potential mechanism. Results NE caused significant IL-1β release into supernatants after 6h (579±90 versus 80±19pg/ml in control; n=4, p<0.0001). Pre-treatment of cells for 30min with neutrophil elastase inhibitor type III (NEIII, 500 µM) significantly decreased IL-1β release (62.64±10 versus 579.3±90pg/ml in primed cells; n=3, p<0.05); but, the levels of IL-1β in the supernatant or lysate did not change in the presence of caspase-1 inhibitor (Ac-YVAD-CHO, 5 µM). Cell lysates contained the inactive, usually referred to as proform of IL-1β and supernatants contained pro and active IL-1β isoforms. Release and cleavage of IL-1β was independent of caspase-1 activity. Live cell imaging revealed that HCAEC shed microvesicles from their plasma membranes after NE stimulation and that these released microvesicles contained IL-1β. Conclusions This is the first description of the release of IL-1β from HCAEC. This is associated with EC microvesicle formation, and the release occurs via a caspase-1 independent mechanism. Neutrophil elastase has been detected in diseased human coronary arteries and, therefore, this is a plausible mechanism to promote local IL-1β release in the vessel wall.

Background and aims Pulmonary arterial hypertension (PAH) is a fatal disease characterised by progressive narrowing of pulmonary arterioles, driven by aberrant cellular proliferation. Identifying key pathways in disease pathogenesis is required for the development of new-targeted therapies. We have previously reported Tumour Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (TRAIL) immunoreactivity within pulmonary vascular lesions from patients with idiopathic PAH and animal models. Since TRAIL induces endothelial cell apoptosis and smooth muscle cell proliferation, we hypothesised that TRAIL is an important driver of disease in PAH. Methods Using the Paigen diet-fed ApoE-/- murine model, we first tested whether genetic deletion (ApoE-/-/TRAIL-/-) and/or anti-TRAIL antibody treatment could modulate disease progression. Bone marrow transplantation (BMT) from ApoE-/- into sub lethally irradiated ApoE-/-/TRAIL-/- mice, and vice versa was performed. Phenotyping included cardiac catheterisation (Right Ventricular Systolic Pressure) and immunohistological analyses of excised lung tissue. Results ApoE-/-/TRAIL-/- mice were protected from developing PAH (RVSP 28 mmHg v. 50 mmHg, P<0.001, n=6). Anti-TRAIL antibody treatment of ApoE-/- mice with established disease reversed PAH (RVSP 27 mmHg v. 88mmHg, P<0.05, n=4). Blocking TRAIL significantly decreased cellular proliferation and increased apoptosis within pulmonary arterioles. In chimaeras, only mice with expression of TRAIL restricted to tissue developed significant PAH (Mean RVSP 47 mmHg v. 26 mmHg p<0.01, n=4–6). Mice with TRAIL only expressed by bone marrow derived cells showed no significant signs of PAH. Conclusions Our studies are the first to determine the importance of TRAIL in the pathogenesis of PAH and demonstrate its potential for translation into a novel therapeutic target.

Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by the progressive narrowing and occlusion of small pulmonary arteries. Current therapies fail to fully reverse this vascular remodeling. Identifying key pathways in disease pathogenesis is therefore required for the development of new-targeted therapeutics. We have previously reported tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) immunoreactivity within pulmonary vascular lesions from patients with idiopathic PAH and animal models. Because TRAIL can induce both endothelial cell apoptosis and smooth muscle cell proliferation in the systemic circulation, we hypothesized that TRAIL is an important mediator in the pathogenesis of PAH. We demonstrate for the first time that TRAIL is a potent stimulus for pulmonary vascular remodeling in human cells and rodent models. Furthermore, antibody blockade or genetic deletion of TRAIL prevents the development of PAH in three independent rodent models. Finally, anti-TRAIL antibody treatment of rodents with established PAH reverses pulmonary vascular remodeling by reducing proliferation and inducing apoptosis, improves hemodynamic indices, and significantly increases survival. These preclinical investigations are the first to demonstrate the importance of TRAIL in PAH pathogenesis and highlight its potential as a novel therapeutic target to direct future translational therapies.