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Cellular interactions in the pathogenesis of pulmonary hypertension

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S6 REGULATION OF ENDOTHELIN-1 PRODUCTION BY THE TRANSFORMING GROWTH FACTOR/BONE MORPHOGENETIC PROTEIN PATHWAY IN HUMAN PULMONARY ARTERY SMOOTH MUSCLE CELLS

1P. M. de Souza, 2N. W. Morrell, 2P. D. Upton, 1J. E. S. Park, 1S. J. Wort. 1Unit of Critical Care, NHLI, Imperial College, London, UK, 2University of Cambridge School of Clinical Medicine, Addenbrooke’s/CUHNHSFT and Papworth Hospitals, Cambridge, UK

Background Pulmonary artery hypertension is a fatal condition associated with remodelling of pulmonary resistance vessels. There is convincing evidence for the involvement of both the transforming growth factor β (TGFβ)/bone morphogenetic protein (BMP) and endothelin (ET-1) pathways in this remodelling process. However, it is unknown how these two pathways interact.

Aim To investigate the effect of TGFβ1, BMP2 and BMP4 on ET-1 release from normal human pulmonary artery smooth muscle cells (HPASMCs).

Methods HPASMCs were grown from resected and morphologically normal pulmonary arteries taken from patients with lung cancer at the Royal Brompton Hospital. Cells were treated with TGFβ1 and/or BMP2 and BMP4 (0, 1 and 10 ng/ml). Following 24 h incubation supernatants were collected and ET-1 concentrations determined by ELISA (R&D, Abingdon, UK). Data were analysed using Student t test.

Results TGFβ1 dose dependently increased ET-1 release from HPASMCs. TGFβ1 (1 ng/ml) significantly increased ET-1 generation by 553% compared with cells treated with medium alone (fig 1; n = 6)). BMP2 (10 ng/ml) and BMP4 (1 and 10 ng/ml) also significantly promoted ET-1 release by up to 20% compared with controls (fig 1; n = 6). When HPASMCs were co-treated with TGFβ1 and BMP2 or BMP4 there was a trend for BMP2 (10 ng/ml) and BMP4 (1 and 10 ng/ml) to attenuate TGFβ1-induced ET-1 release, with only BMP2 at 1 ng/ml significantly inhibiting this release by 24% (fig 1; n = 3).

Conclusion These findings suggest that there may be significant cross-talk between TGFβ/BMP and ET-1 in HPASMCs. Further work is needed to investigate the effect of bone morphogenetic protein type II receptor (BMPR-II) mutations on ET-1 release.

Abstract S6 Figure 1

The effect of transforming growth factor β (TGFβ), bone morphogenetic protein 2 (BMP2) and BMP4 alone or in combination on endothelin (ET-1) release. Data are from 3–6 donors where ***p<0.001, **p<0.01. *p<0.05 compared with cells treated with medium. # indicates p<0.05 compared with cells treated with TGFβ1 alone.

S7 TUMOUR NECROSIS FACTOR α, BONE MORPHOGENETIC PROTEIN 9 AND CICLOSPORIN AFFECT EXPRESSION OF BONE MORPHOGENETIC PROTEIN TYPE II RECEPTOR IN HUMAN PULMONARY ARTERY ENDOTHELIAL CELLS: POTENTIAL ROLES IN THE PATHOGENESIS OF PULMONARY ARTERIAL HYPERTENSION

M. Nikolic, L. Long, A. Crosby, N. W. Morrell. University of Cambridge, Department of Medicine, Cambridge, UK

Introduction and Objectives Mutations in the bone morphogenetic protein type II receptor gene (BMPR-II) are central to the pathogenesis of familial pulmonary arterial hypertension (PAH). They are found in 70% of familial and up to 40% of sporadic cases. In both familial and sporadic cases, BMPR-II expression is reduced irrespective of whether the BMPR-II gene is mutated or not. These findings suggest that the BMPR-II gene promoter and its regulation may play a key role. This study aimed to determine key pathways that affect BMPR-II gene expression in human pulmonary artery endothelial cells (HPAECs). Previous experiments in HeLa cells assessed a range of mediators and identified the inflammatory cytokine tumour necrosis factor α (TNFα) as a repressor of BMPR-II promoter activity. Inflammation has been widely implicated in the process of pulmonary vascular remodelling and we hypothesised that TNFα and other inflammatory cytokines decrease BMPR-II expression in disease-relevant cells, namely HPAECs.

Methods HPAECs were cultured to confluence, serum starved, and exposed to a panel of cytokines and growth factors implicated in cell signalling or vascular remodelling. Reagents which altered BMPR-II mRNA levels 2-fold were identified in reverse transcription-PCR (RT-PCR) screens, then tested more extensively by quantitative PCR (qPCR) and western blotting.

Results Angiotensin II, ciclosporin (CSA), β-oestradiol, bone morphogenetic protein 9 (BMP9), TNFα and thrombin significantly altered BMPR-II mRNA levels in screening experiments. However, only BMP9, CSA and TNFα significantly altered BMPR-II expression when the experiment was repeated in triplicate. BMP9 increased expression 8.74-fold, whereas CSA and TNFα decreased expression >2-fold. This was reproduced in western blotting.

Conclusions TNFα, BMP9 and CSA may be important regulators of BMPR-II expression. Further experiments are needed to establish whether this occurs through BMPR-II promoter binding and to identify important regulatory regions. The effect of TNFα on BMPR-II expression is in keeping with our previous findings in HeLa cells and with the inflammatory model of PAH. These results highlight the importance of controlling inflammation in primary and secondary prevention of PAH and may also provide future pharmacological targets.

S8 TRAIL EXPRESSION IS INCREASED IN THE RAT MONOCROTALINE MODEL OF PULMONARY ARTERIAL HYPERTENSION

A. G. Hameed, T. Vinogradova, S. E. Francis, C. M. H. Newman, D. C. Crossman, A. Lawrie. University of Sheffield, Sheffield, UK

Introduction and Objectives Despite improvements in the overall management of pulmonary arterial hypertension (PAH) the disorder still causes significant morbidity and mortality. Better insight into the molecular pathogenesis of pulmonary vascular remodelling could lead to the development of more targeted therapeutics. There is emerging evidence to support that tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) plays an important role in vascular biology. We were recently the first to demonstrate that TRAIL expression is upregulated within pulmonary vascular lesions of patients with idiopathic PAH. Moreover we have demonstrated that recombinant human TRAIL induces the proliferation and migration of human pulmonary artery smooth muscle cells (HPASMCs). The aim of this current study was to determine the expression profile of TRAIL through disease progression utilising the rat monocrotaline (MCT) model of PAH.

Methods Male Sprague–Dawley rats (200–260 g, n = 7 per group) were injected with either MCT (60 mg/kg) or saline as control. They subsequently underwent haemodynamic studies, with harvesting of serum, heart and lungs at day 2, 7, 14, 21 and 28 after injection. Segments of lung were immediately snap-frozen in liquid nitrogen for subsequent determination of TRAIL protein by western immunoblotting and subsequent densitometry with normalisation to β-actin.

Results A significant increase in right ventricular maximum dP/dT was observed 14 days after treatment with MCT. Right ventricular hypertrophy (RVH) and right ventricular systolic pressure (RVSP) developed, and remained significantly elevated from 21 days in the MCT-treated rats. The levels of TRAIL protein in whole lung protein lysates showed a trend for an increase at day 21 and were significantly higher at 28 days in the MCT-treated rats.

Conclusion TRAIL protein is upregulated in the rat MCT model of PAH but only in the latter stages of disease. Work is ongoing to determine the profile of TRAIL RNA expression in the lung, serum levels of TRAIL, and the localisation of TRAIL within the pulmonary vascular lesions of this model.

S9 OSTEOPROTEGERIN EXPRESSION IS INCREASED PRIOR TO HAEMODYNAMIC ALTERATIONS IN THE RAT MONOCROTALINE MODEL OF PULMONARY ARTERIAL HYPERTENSION

T. Vinogradova, A. G. Hameed, S. E. Francis, C. M. H. Newman, D. C. Crossman, A. Lawrie. University of Sheffield, Sheffield, UK

Introduction and Objectives Pulmonary arterial hypertension (PAH) is a life-threatening condition with high morbidity and poor life expectancy. Pathologically, PAH is characterised by pulmonary artery medial thickening, fibrosis and, in some cases, plexiform lesions. Better insight into the molecular pathogenesis of this process could lead to the development of novel targeted therapeutics. We have recently demonstrated that the secreted glycoprotein osteoprotegerin (OPG, TNFRSF11B) is increased both within lesions and in serum from patients with idiopathic PAH. We have also shown that OPG promotes the proliferation and migration of pulmonary artery smooth muscle cells and hypothesise that OPG plays an important role in the pathogenesis of PAH. The aim of this study was to determine the temporal expression profile of OPG during disease progression utilising the rat monocrotaline (MCT) model of PAH.

Methods Male Sprague–Dawley rats (200–260 g, n = 7 per group)were injected with either MCT (60 mg/kg) or saline as control. Cardiac catheterisation was performed to measure haemodynamic parameters prior to collection of serum and heart and lung tissue at day 2, 7, 14, 21 and 28 after treatment. OPG was measured in serum by sandwich ELISA and in whole lung protein lysates by western immunoblotting with normalisation to β-actin.

Results A significant increase in right ventricular maximum dP/dT was observed from 14 days after treatment with MCT, right ventricular hypertrophy (RVH) and right ventricular systolic pressure (RVSP) developed, and remained significantly elevated from 21 days in the MCT-treated rats. There was a trend for increased levels of serum OPG from day 7 and a significant increase from 14 days in MCT-treated rats. OPG protein expression within whole lung showed a trend towards an increase from 7 days and reached significance at 21 days in the MCT-treated rats.

Conclusion These data provide further evidence that OPG may be important in the pathogenesis of PAH. Serum OPG increased prior to haemodynamic changes in this model, suggesting that OPG may also be a useful biomarker for the early stages of PAH. Further studies are underway to determine whether OPG itself may be a target for therapy in PAH.

S10 REGULATION OF EXPRESSION AND FUNCTION OF OSTEOPROTEGERIN IN PULMONARY ARTERY SMOOTH MUSCLE CELLS IN VITRO

C. Paiva, S. E. Francis, C. M. H. Newman, D. C. Crossman, A. Lawrie. University of Sheffield, Sheffield, UK

Introduction and Objectives Pulmonary arterial hypertension (PAH) is characterised pathologically by the obliteration of small arteries in the lung. Abnormal proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) is key to this process. There are now well-established links between bone morphogenetic protein receptor type II (BMPR-II) mutations, the serotonin (5-hydroxytryptamine (5-HT)) pathway and inflammatory mechanisms in this process. We have recently reported that osteoprotegerin (OPG), also known as TNFRSF11B, is upregulated in serum and lesions of patients with idiopathic PAH. We have also shown that OPG induces both proliferation and migration of PASMCs in vitro. We hypothesise that OPG plays a central role in, and contributes to the pathogenesis of PAH. We now aim to determine the regulation of expression of OPG in PASMCs.

Methods Primary human PASMCs (Cascade Biologics) were used for all studies. Recombinant OPG and interleukin-1 (IL-1) were purchased from R&D Systems and 5-HT from Sigma. OPG mRNA levels were quantified using TaqMan PCR. Intracellular protein was assessed by western immunoblot, and conditioned medium was assayed by sandwich ELISA. To analyse the downstream signalling, protein lysates were isolated after 5, 10 and 60 min incubation with 50 ng/ml OPG.

Results OPG mRNA expression was raised at 4 h poststimulation with IL-1β and 5-HT. Intracellular OPG levels were increased following IL-1β and 5-HT stimulation at 24 and 48 h. OPG secretion into the culture medium significantly increased from 24 h poststimulation, suggesting that OPG is rapidly released from cells following translation. Stimulation with TGFβ increased OPG within conditioned media; however, no increase in either mRNA or intracellular protein was detected. Stimulation of PASMCs with OPG resulted in a significant increase in extracellular signal-regulated kinase (ERK) 1/2 phosphorylation at 5 min.

Conclusions We have demonstrated that signals from multiple pathways associated with PAH converge upon, and stimulate the expression and release of OPG from PASMCs. OPG induces the phosphorylation of ERK 1/2, and PASMC proliferation and migration. Further work is ongoing to establish whether ERK 1/2 phosphorylation is required for OPG-induced proliferation and migration.

S11 FAT-FED APOE/IL-1R1 DOUBLE-DEFICIENT MICE ARE PROTECTED FROM ATHEROSCLEROSIS BUT DEVELOP SEVERE PULMONARY HYPERTENSION

A. G. Hameed, S. E. Francis, M. Neilan, S. Parmar, D. C. Crossman, A. Lawrie. University of Sheffield, Sheffield, UK

Introduction and Objectives Pulmonary arterial hypertension (PAH) is a life-threatening condition with high morbidity and poor life expectancy. Inflammatory mechanisms are proposed to play a significant role in disease progression, particularly PAH associated with other diseases—for example, systemic sclerosis. Previous studies have demonstrated that members of the interleukin family of inflammatory cytokines are upregulated in PAH; treatment of rats with the interleukin-1 (IL-1) receptor antagonist has been shown to protect against development of monocrotaline-induced PAH, and IL-6 overexpression has been show to induce PAH in mice. Our group has recently demonstrated that ApoE−/−/IL-1R1−/− mice have reduced diet-induced atherosclerosis and lower systemic blood pressure compared with ApoE−/− mice on the same diet. Since fat-fed ApoE−/− mice have been shown to develop pulmonary hypertension, we hypothesised that ApoE−/−/IL-1R1−/− mice would exhibit a reduced PAH phenotype.

Methods ApoE−/− and ApoE−/−/IL-1R1−/− mice were fed either regular chow or Paigen diet for 8 weeks prior to echocardiography, right and left heart cardiac catheterisation and serum and tissue harvest to assess pulmonary hypertension phenotype.

Results The Paigen-fed ApoE mice developed increases in right ventricular systolic pressure (RVSP) in line with published data. Surprisingly, the ApoE−/−/IL-1R1−/−mice exhibited higher pressures (mean 75 mm Hg, p<0.05 n = 6). This finding was matched by significant increases in right ventricular hypertrophy compared with chow-fed controls. Analysis of lung sections by α-smooth muscle actin immunohistochemistry also revealed muscularisation of the distal pulmonary arteries. We have previously reported that high serum levels of osteoprotegerin (OPG) are associated with PAH in humans; this increase also correlates with development of PAH in our rat models. The ApoE−/− fat-fed mice exhibited a 2-fold increase (p<0.001, n = 9), and the ApoE−/−/IL-1R1−/− animals a 4-fold increase (p<0.0001, n = 5) in serum OPG compared with chow-fed controls.

Conclusions These studies further implicate IL-1 signalling in PAH; however, the mechanisms remain unclear. The data also support a role for OPG in PAH. Additional studies are underway to examine other key inflammatory pathways to determine whether they compensate for the lack of IL-1 signalling in this model, and so drive disease pathogenesis.

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