Ag Hameed

Inhibition of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) reverses experimental pulmonary hypertension.

Genetic deletion of TRAIL or antibody blockade prevents the development of pulmonary arterial hypertension and can reverse vascular remodeling in established disease.

Serum osteoprotegerin is increased and predicts survival in idiopathic pulmonary arterial hypertension

We previously reported that osteoprotegerin (OPG) is regulated by pathways associated with pulmonary arterial hypertension (PAH), and is present at elevated levels within pulmonary vascular lesions and sera from patients with idiopathic PAH (IPAH). Since OPG is a naturally secreted protein, we investigated the relationship between serum OPG and disease severity and outcome in patients with IPAH and animal models. OPG mRNA expression was measured in pulmonary artery smooth muscle cells (PASMC) from pulmonary arteries of patients with and without IPAH. Serum concentrations of OPG were measured in a retrospective and prospective group of patients. OPG levels were compared with phenotypic data and other putative PAH biomarkers. Prognostic significance was assessed and levels compared with healthy controls. Correlation of OPG and pulmonary vascular remodeling was also performed in rodent models of PAH. OPG mRNA was significantly increased 2-fold in PASMC isolated from explanted PAH lungs compared with control. Serum OPG concentrations were markedly elevated in IPAH compared with controls. In Cohort 1 OPG levels significantly correlated with mean right atrial pressure and cardiac index, while in Cohort 2 significant correlations existed between age-adjusted OPG levels and gas transfer. In both cohorts an OPG concentration above a ROC-derived threshold of 4728 pg/ml predicted poorer survival. In two rodent models, OPG correlated with the degree of pulmonary vascular remodeling. OPG levels are significantly elevated in patients with idiopathic PAH and are of prognostic significance. The role of OPG as a potential biomarker and therapeutic target merits further investigation.

Vitamin D Deficiency and Exogenous Vitamin D Excess Similarly Increase Diffuse Atherosclerotic Calcification in Apolipoprotein E Knockout Mice

Background Observational data associate lower levels of serum vitamin D with coronary artery calcification, cardiovascular events and mortality. However, there is little interventional evidence demonstrating that moderate vitamin D deficiency plays a causative role in cardiovascular disease. This study examined the cardiovascular effects of dietary vitamin D deficiency and of vitamin D receptor agonist (paricalcitol) administration in apolipoprotein E knockout mice. Methods Mice were fed atherogenic diets with normal vitamin D content (1.5IU/kg) or without vitamin D. Paricalcitol, or matched vehicle, was administered 3× weekly by intraperitoneal injection. Following 20 weeks of these interventions cardiovascular phenotype was characterized by histological assessment of aortic sinus atheroma, soluble markers, blood pressure and echocardiography. To place the cardiovascular assessments in the context of intervention effects on bone, structural changes at the tibia were assessed by microtomography. Results Vitamin D deficient diet induced significant reductions in plasma vitamin D (p<0.001), trabecular bone volume (p<0.01) and bone mineral density (p<0.005). These changes were accompanied by an increase in calcification density (number of calcifications per mm2) of von Kossa-stained aortic sinus atheroma (461 versus 200, p<0.01). Paricalcitol administration suppressed parathyroid hormone (p<0.001), elevated plasma calcium phosphate product (p<0.005) and induced an increase in calcification density (472 versus 200, p<0.005) similar to that seen with vitamin D deficiency. Atheroma burden, blood pressure, metabolic profile and measures of left ventricular hypertrophy were unaffected by the interventions. Conclusion Vitamin D deficiency, as well as excess, increases atherosclerotic calcification. This phenotype is induced before other measures of cardiovascular pathology associated clinically with vitamin D deficiency. Thus, maintenance of an optimal range of vitamin D signalling may be important for prevention of atherosclerotic calcification.

Hypoxia determines survival outcomes of bacterial infection through HIF-1alpha dependent re-programming of leukocyte metabolism

Infection outcomes are regulated by neutrophil responses to oxygen and nutrient availability. Hypoxic immune cell conditioning Oxygen deficiency, or hypoxia, has been shown to alter immune cell function. However, how these hypoxia-induced immune cell changes affect the host response to bacterial infection has remained unclear. Now, Thompson et al. report that although acute hypoxia accentuated morbidity and mortality as a result of bacterial infection in mice, chronic hypoxia before infection could actually prevent these pathological responses. This hypoxic preconditioning reduced neutrophil glucose utilization, decreasing the related pathology. If these findings hold true in humans, they suggest that immune targeting could aid patients with systemic hypoxia and chronic infections such as adult respiratory distress syndrome or chronic obstructive pulmonary disease. Hypoxia and bacterial infection frequently coexist, in both acute and chronic clinical settings, and typically result in adverse clinical outcomes. To ameliorate this morbidity, we investigated the interaction between hypoxia and the host response. In the context of acute hypoxia, both Staphylococcus aureus and Streptococcus pneumoniae infections rapidly induced progressive neutrophil-mediated morbidity and mortality, with associated hypothermia and cardiovascular compromise. Preconditioning animals through longer exposures to hypoxia, before infection, prevented these pathophysiological responses and profoundly dampened the transcriptome of circulating leukocytes. Specifically, perturbation of hypoxia-inducible factor (HIF) pathway and glycolysis genes by hypoxic preconditioning was associated with reduced leukocyte glucose utilization, resulting in systemic rescue from a global negative energy state and myocardial protection. Thus, we demonstrate that hypoxia preconditions the innate immune response and determines survival outcomes after bacterial infection through suppression of HIF-1α and neutrophil metabolism. In the context of systemic or tissue hypoxia, therapies that target the host response could improve infection-associated morbidity and mortality.

No Evidence for Cardiac Dysfunction in Kif6 Mutant Mice

A KIF6 variant in man has been reported to be associated with adverse cardiovascular outcomes after myocardial infarction. No clear biological or physiological data exist for Kif6. We sought to investigate the impact of a deleterious KIF6 mutation on cardiac function in mice. Kif6 mutant mice were generated and verified. Cardiac function was assessed by serial echocardiography at baseline, after ageing and after exercise. Lipid levels were also measured. No discernable adverse lipid or cardiac phenotype was detected in Kif6 mutant mice. These data suggest that dysfunction of Kif6 is linked to other more complex biological/biochemical parameters or is unlikely to be of material consequence in cardiac function.

T6 TRAIL is a potential novel therapeutic target in pulmonary arterial hypertension

Introduction and Objectives Pulmonary Arterial Hypertension (PAH) is a life threatening disease characterised by the progressive narrowing and occlusion of small pulmonary arteries, driven by the dysregulated growth of vascular cells. Current therapies are unable to reverse PAH and so identifying key pathways in disease pathogenesis should permit the development of more targeted therapeutics. The cytokine, Tumour Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (TRAIL) induces EC apoptosis and SMC proliferation in the systemic circulation, but hitherto has not been studied in PAH. We recently determined that TRAIL is a mitogen for human PA-SMCs in-vitro and was associated with pulmonary vascular lesions in humans and rodent models. We thus hypothesised that TRAIL is an important mediator in the pathogenesis of PAH and now describe the potential therapeutic benefits of targeting TRAIL in-vivo using two animal models. Methods To test whether blocking TRAIL could prevent the development of PAH, an anti-TRAIL antibody was delivered via osmotic mini-pump coincident with the induction of PAH in the MCT rat model. We also tested whether genetic deletion of TRAIL (ApoE−/−/TRAIL−/− mice) would confer protection to diet-induced PAH. To test whether inhibiting TRAIL could reverse established disease we again treated both models with an anti-TRAIL antibody starting from day 21 in the rat and 8 weeks in the ApoE−/− mouse. Phenotyping included echocardiography, closed chest cardiac catheterisation followed by immuno-histological and biochemical analyses of the lung. Results Antibody blockade (MCT) and genetic deletion (ApoE−/−) of TRAIL prevented the development of PAH in both models. Interestingly a PAH disease phenotype was restored in ApoE−/−/TRAIL−/− mice by the administration of recombinant TRAIL. In rodents with established PAH, an anti-TRAIL antibody, significantly increased survival and reduced pulmonary vascular remodelling in the fatal rat MCT model (p<0.05 cf control). In the murine model, an anti-TRAIL antibody treatment reversed both haemodynamics (RVSP 27 mm Hg vs 88 mm Hg, p<0.001) and pulmonary vascular remodelling. Conclusions Our preclinical investigations are the first to determine the importance of TRAIL to disease pathogenesis and highlight its potential as a novel and rational target to direct future translational therapies for PAH.

S104 Hypoxia preconditions the innate immune response to acute bacterial pulmonary infections

Introduction Systemic hypoxaemia and recurrent bacterial infections frequently co-exist in patients with acute and chronic lung disease and correlate with poor clinical outcomes. Inappropriate neutrophilic inflammation is regularly seen in these circumstances and the HIF/PHD pathway is implicated in the response of the innate immune system to both hypoxia and bacteria. Here we aimed to dissect and modify the interactions between hypoxia and innate host-pathogen response in the lung. Methods C57BL/6 mice were either housed in room air or ‘preconditioned’ by being housed in 10% oxygen for 7 days. They then received intratracheal 1x107 type 2 S. pneumoniae under recovery anaesthesia with subsequent exposure to hypoxia (10% O2) or room air (21% O2). Mice were assessed clinically, rectal temperatures recorded and culled for broncho-alveolar lavage (BAL) and tissue sampling (blood and lung) at various time points. Peripheral blood glucose was measured from tail vein venepuncture using a handheld blood glucose monitor. RNA from peripheral blood leucocytes was isolated and analysed using RNAseq. 18FDG-PET was performed on animals 14 h following infection to observe glucose utilisation. Histology was performed on formalin fixed sections for glycogen storage. Results Exposure to acute hypoxia resulted in significant morbidity (sickness (5.7 vs 2.1, p < 0.02) and hypothermia (31.8 vs 36.00C, p < 0.05)) and rapid 100% mortality by 48 h post infection. This response was independent of bacterial burden, and leukocyte recruitment. In keeping with a negative energy state, hypoxic mice displayed loss of liver glycogen, with increased serum ketone production and lower circulating glucose levels. Preconditioned mice showed marked protection from both the acute hypoxia-associated systemic phenotype and the negative energy state. Transfer of preconditioned bone marrow to naïve mice also rescued the pathophysiological response. RNAseq analysis of the circulating leukocyte population identified signal-induced suppression of HIF-1a pathway genes, which were linked to reduced leukocyte glucose utilisation in vivo by 18FDG-PET. Conclusions Hypoxic preconditioning reverses the morbidity and mortality associated with acute hypoxia following intrapulmonary bacterial challenge. This response is dependent on the preconditioning of the innate immune system by suppressing HIF1 alpha and altering circulating leukocyte metabolism. Support RSD is funded by the MRC. SRW is funded by the Wellcome Trust.

P143 Hypoxia induces hypothermia and sickness behaviour in mice following subcutaneous injection of live Staphylococcus aureus

Infections frequently cause or complicate illnesses associated with hypoxaemia and local tissue hypoxia. The influence of hypoxia on the interaction between host and pathogen is therefore of considerable interest. S. aureus is a major pathogen in critical care where patients may have profound hypoxaemia and at a tissue level, S. aureus frequently infects ischaemic wounds. We investigated the effect of systemic hypoxia on host-pathogen interactions using a subcutaneous S. aureus infection model in mice. C57BL/6 mice were shaved, injected with a low dose of S. aureus (5x107 SH1000) and placed in a hypoxic chamber (10% O2) or left in room air. At 6 or 12 hours mice were assessed clinically and rectal temperature recorded. Clinical assessment of mouse sickness behaviour was made by two independent observers blinded as to which oxygen tension the mice had been exposed. Mice were anaesthetised and tissue samples (blood, skin, lung, spleen, kidney, liver and brain) obtained for analysis. Mice injected with live bacteria and placed in hypoxia developed a phenotype of sickness behaviour and hypothermia. Infected hypoxic mice had significantly higher sickness scores and lower body temperature than infected normoxic mice or hypoxic mice injected with PBS (rectal temperature at 12 hours: hypoxic 33.4˚C ± 0.74, normoxic 37.7˚C ± 0.24, hypoxic PBS-injected 38.9˚C ± 0.26, p < 0.0001). Surprisingly, we found no evidence of bacteraemia, enhanced cytokine production, vascular leak or lung injury in the hypoxic infected mice. However, these animals had significant circulatory dysfunction, with hypotension, bradycardia and echocardiographic evidence of impaired left ventricular function. Interestingly, myeloid-cell deficiency of either HIF-1α or HIF-2α protected mice from the adverse systemic phenotype in this model, implicating the host innate immune response in the pathogenesis of the phenotype. These findings imply that hypoxia may adversely alter the host response to a minor bacterial challenge, leading to profound systemic illness and that in such a setting modulation of the HIF pathway may be a possible therapeutic option.

Tumour necrosis factor-related apoptosis-inducing ligand is a novel therapeutic target in pulmonary arterial hypertension

Abstract 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.

03 Tissue Trail Drives Pulmonary Vascular Remodeling and its Inhibition Reverses Experimental Pulmonary Arterial Hypertension

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.