Dongmin Shao

The role of endothelin-1 in the pathogenesis of pulmonary arterial hypertension.

The term pulmonary arterial hypertension (PAH) describes a rare group of diseases characterized by raised pulmonary vascular resistance, resulting from vascular remodelling in the pre-capillary resistance arterioles (< 100 mm). Left untreated, patients die from right heart failure, with a mortality approaching most serious cancers. Endothelin-1(ET-1) is not only a potent vasoconstrictor, but causes proliferation of many of the vascular cells involved in vascular remodelling. Although produced mainly by the vascular endothelium, other cells such as smooth muscle, fibroblasts and macrophages are known sources of ET-1 when these cells are challenged by relevant stimuli. Plasma ET-1 levels are raised in patients with PAH and correlate with important clinical outcomes. Furthermore, ET-1 receptor antagonism has been demonstrated to improve both morbidity and mortality in conditions associated with PAH. We review the literature supporting the role for ET-1 in the pathogenesis of PAH.

Dexamethasone reverses monocrotaline-induced pulmonary arterial hypertension in rats

Pulmonary arterial hypertension (PAH) is associated with dysregulated bone morphogenetic protein receptor (BMPR)-II signaling and pulmonary vascular inflammation. We evaluated the effects of dexamethasone on monocrotaline (MCT)-induced PAH in rats for potential reversal of PAH at late time-points. Saline-treated control, MCT-exposed, MCT-exposed and dexamethasone-treated rats (5 mg·kg−1·day−1, 1.25 mg·kg−1 and 2.5 mg·kg−1·48 h−1) were evaluated at day 28 and day 35 following MCT for haemodynamic parameters, right ventricular hypertrophy, morphometry, immunohistochemistry, and IL6 and BMPR2 expression. Dexamethasone improved haemodynamics and pulmonary vascular remodelling, preventing PAH development at early (day 1–14 and 1–28) and reversing PAH at late (day 14–28 and 21–35) time-points following MCT, as well as improving survival in MCT-exposed rats compared with controls. Both MCT-induced pulmonary IL6 overexpression and interleukin (IL)-6-expressing adventitial inflammatory cell infiltration were reduced with dexamethasone. This was associated with pulmonary BMPR2 downregulation following MCT, which was increased with dexamethasone, in whole lung and control pulmonary artery smooth muscle cells. Dexamethasone also reduced proliferation of rat pulmonary artery smooth muscle cells in vitro. Experimental PAH can be prevented and reversed by dexamethasone, and survival is improved. In this model, mechanisms may involve reduction of IL-6-expressing inflammatory cells, restoration of pulmonary BMPR2 expression and reduced proliferation of vascular smooth muscle cells.

BMP-9 Induced Endothelial Cell Tubule Formation and Inhibition of Migration Involves Smad1 Driven Endothelin-1 Production

Background Bone morphogenetic proteins (BMPs) and their receptors, such as bone morphogenetic protein receptor (BMPR) II, have been implicated in a wide variety of disorders including pulmonary arterial hypertension (PAH). Similarly, endothelin-1 (ET-1), a mitogen and vasoconstrictor, is upregulated in PAH and endothelin receptor antagonists are used in its treatment. We sought to determine whether there is crosstalk between BMP signalling and the ET-1 axis in human pulmonary artery endothelial cells (HPAECs), possible mechanisms involved in such crosstalk and functional consequences thereof. Methodology/Principal Finding Using western blot, real time RT-PCR, ELISA and small RNA interference methods we provide evidence that in HPAECs BMP-9, but not BMP-2, -4 and -6 significantly stimulated ET-1 release under physiological concentrations. This release is mediated by both Smad1 and p38 MAPK and is independent of the canonical Smad4 pathway. Moreover, knocking down the ALK1 receptor or BMPR II attenuates BMP-9 stimulated ET-1 release, whilst causing a significant increase in prepro ET-1 mRNA transcription and mature peptide release. Finally, BMP-9 induced ET-1 release is involved in both inhibition of endothelial cell migration and promotion of tubule formation. Conclusions/Significance Although our data does not support an important role for BMP-9 as a source of increased endothelial ET-1 production seen in human PAH, BMP-9 stimulated ET-1 production is likely to be important in angiogenesis and vascular stability. However, increased ET-1 production by endothelial cells as a consequence of BMPR II dysfunction may be clinically relevant in the pathogenesis of PAH.

Nuclear Factor κ-B Is Activated in the Pulmonary Vessels of Patients with End-Stage Idiopathic Pulmonary Arterial Hypertension

Objectives To assess activation of the inflammatory transcription factor NF-kappa B (NF-κB) in human idiopathic pulmonary arterial hypertension (PAH). Background Idiopathic PAH is a severe progressive disease characterized by pulmonary vascular remodeling and excessive proliferation of vascular cells. Increasing evidence indicates that inflammation is important in disease pathophysiology. Methods NF-κB-p65 and CD68, CD20 and CD45 were measured by immunohistochemistry and confocal microscopy on lung specimens from patients with idiopathic PAH (n = 12) and controls undergoing lung surgery (n = 14). Clinical data were recorded for all patients including invasive pulmonary hemodynamics for the PAH patients. Immunohistochemical images were analyzed by blinded observers to include standard pulmonary vascular morphometry; absolute macrophage counts/mm2 and p65-positivity (p65+) using composite images and image-analysis software; and cytoplasmic:nuclear p65+ of individual pulmonary arterial endothelial and smooth muscle cells (PASMC) in 10–20 pulmonary arteries or arterioles per subject. The expression of ET-1 and CCL5 (RANTES) in whole lung was determined by RT-qPCR. Results Macrophage numbers were increased in idiopathic PAH versus controls (49.0±4.5 vs. 7.95±1.9 macrophages/100 mm2, p<0.0001): these macrophages demonstrated more nuclear p65+ than in macrophages from controls (16.9±2.49 vs. 3.5±1.25%, p<0.001). An increase in p65+ was also seen in perivascular lymphocytes in patients with PAH. Furthermore, NF-κB activation was increased in pulmonary arterial endothelial cells (62.3±2.9 vs. 14.4±3.8, p<0.0001) and PASMC (22.6±2.3 vs. 11.2±2.0, p<0.001) in patients with PAH versus controls, with similar findings in arterioles. Gene expression of both ET-1 mRNA ((0.213±0.069 vs. 1.06±0.23, p<0.01) and CCL5 (RANTES) (0.16±0.045 vs. 0.26±0.039, p<0.05) was increased in whole lung homogenates from patients with PAH. Conclusions NF-κB is activated in pulmonary macrophages, lymphocytes, endothelial and PASMC in patients with end-stage idiopathic PAH. Future research should determine whether NF-κB activation is a driver or bystander of pulmonary vascular inflammation and if the former, its potential role as a therapeutic target.

Viral Toll Like Receptor activation of pulmonary vascular smooth muscle cells results in endothelin-1 generation; relevance to pathogenesis of pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a rare but fatal condition in which raised pulmonary vascular resistance leads to right heart failure and death. Endothelin-1 is a potent endogenous vasoconstrictor, which is considered to be central to many of the events that lead to PAH, and is an important therapeutic target in the treatment of the condition. In many cases of PAH, the aetiology is unknown but inflammation is increasingly thought to play an important role and viruses have been implicated in the development of disease. The Toll Like Receptors (TLRs) play a key role in innate immune responses by initiating specific anti-bacterial and anti-viral defences in recognition of signature molecular motifs on the surface of invading pathogens. In this study, we set out to examine the expression of bacterial and viral TLRs in human pulmonary artery smooth muscle cells and to establish whether their activation could be relevant to PAH. We found that the viral TLR3 and bacterial TLRs 4 and 6 were most abundantly expressed in human pulmonary artery smooth muscle cells. Using specific TLR ligands, we found that activation of TLRs 3 and 4 resulted in IL-8 release by human pulmonary artery smooth muscle cells but that only TLR3 stimulation resulted in IP10 and endothelin-1 release. These data suggest that human pulmonary artery smooth muscle cells express significant levels of viral TLR3 and respond to its activation by releasing endothelin-1. This may have importance in understanding the association between viruses and the development of PAH.

S154 Is there a role for IL-33 in the pathogenesis of pulmonary arterial hypertension?

Introduction and objectives IL-33 is a 31KDa cytokine which is a member of the IL-1 family. It resides in the nucleus of endothelial and epithelial cells as a chromatin-associated factor in vivo. IL-33 is thought to be released from stressed or necrotic, but not apoptotic, endothelial or epithelial cells in response to cell injury or infection, acting as an endogenous ‘alarmin’, to alert the immune system of cell and tissue damage. IL-33 is then able to bind to its receptor (ST2) on immune cells, thereby stimulating immunoregulatory activity through induction of NF-κB and mitogen-activated protein kinases, as well as enhancing the production of the Th2 cytokines IL-5 and IL-13. There is increasing evidence that IL-33 may have a protective role in terms of endothelial integrity and function. As the pathogenesis of pulmonary arterial hypertension (PAH) is thought to involve endothelial cell dysfunction, we were interested to see whether there may be a role for IL-33 in this condition. Methods RT-PCR for IL-33 was performed on human pulmonary arterial cells (HPAECs) derived from normal healthy controls and patients with idiopathic PAH. siRNA IL-33 knockdown was performed using smartpool duplexes on normal HPAECs. RT-PCR was then performed using QuantiTec primer assays. Results IL-33 mRNA expression was decreased 2.1-fold in PAH patient samples (0.369±0.02, n=10) compared to controls (0.761±0.06, n=14) p<0.005. In normal human lung tissue, intense IL-33 staining was shown in the ECs nuclear in blood vessels. It is also worth to note that there was little or no IL-33 staining in non-ECs. siRNA knockdown IL-33 in HPAECs resulted in a 1.8±0.22, 1.3±0.13 and 1.4±0.20 (n=3) fold increase of mRNA IL-6, BMP-9 and ST2 mRNA, respectively; whereas RANTES, fractalkine and cathepsin-L mRNA was decreased by 1.5±0.03, 1.7±0.01 and 2.1±0.17 fold respectively (n=3) (Abstract S154 Figure 1).Abstract S154 Figure 1 Effect of IL-33 on gene expression. Conclusion IL-33mayplay an important role in the pathogenesis of PAH through regulating the expression ofcytokines and chemokines known to be involved in vascular remodelling. In particular, it is of interest that Il-33 may regulate IL-6 production and ST2 which acts as an endogenous IL-33 inhibitor.

Molecular Biological Aspects, Therapeutic Targets and New Treatment Strategies

The term pulmonary arterial hypertension (PAH) describes a rare group of diseases characterized by raised pulmonary vascular resistance, resulting from vascular remodelling in the pre-capillary resistance arterioles (<100 μm in cross-sectional diameter) [1]. Left untreated, patients die from right heart failure, with a mortality approaching most serious cancers. To date, most treatment has been focused on the endothelial cell vascular dysfunction seen in these disorders. As such, pulmonary vasodilators, such as endothelin (ET-1) receptor antagonists, prostacyclin (PGI2) analogues and phosphodiesterase type V inhibitors (enhancing endogenous nitric oxide (NO)) have improved both morbidity and mortality. Indeed, there is continuing development into new and improved drugs that target these established pathways. Recent examples are the dual ET-1 receptor antagonist, macitentan [2], and the soluble guanylate cyclase activator, riociguat [3]. However, none are a cure and the treated mortality rate is still unacceptable [4]. Further research into the molecular mechanisms underpinning the pathogenesis of PAH has led to the discovery of new putative pathways that may allow the targeting of vascular remodelling itself; such “reverse remodelling” may provide a cure for this devastating disease in the future and remains the “holy grail”.

P159 Peripheral Blood Mononuclear Cells from patients with Idiopathic Pulmonary Arterial Hypertension are Hyporesponsive to Inflammatory stimuli

Background Inflammation is a key feature of pulmonary arterial hypertension (PAH); circulating levels of plasma cytokines and chemokines are raised, some of which (including interleukin (IL)-6 and CXCL8) are associated with increased mortality (1). We wished to determine whether it was possible to detect an altered inflammatory phenotype in circulating inflammatory cells from PAH patients compared to controls. Methods Following ethical approval, patients with idiopathic PAH and age-matched non-smoking control subjects were selected. Peripheral blood mononuclear cells (PBMC) (105 cells) or citrated whole blood (WB) were plated into 96-well plates and treated with E. Coli LPS (0–1µg/ml) (Sigma) or recombinant TNF-a (0–10ng/ml) (R&D systems) for 24h at 37°C. Following centrifugation, plasma was aspirated and samples frozen at -20°c, and IL-6 and CXCL8 ELISAs later performed. Data are mean ± SEM. Results All patients had idiopathic PAH (n = 12) (7 in NYHA class III-IV) with 6MWD 370 ± 18m, mPAP 72 ± 5mmHg). All patients were taking advanced PAH therapies but not conventional anti-inflammatory therapies. There was no age difference between patients and controls (n = 9) (36.5 ± 3.7 vs. 36.2 ± 2.7 years, NS). At baseline, release of IL-6 and CXCL8 was higher from PBMC from PAH patients than controls (918.1 ± 280.5 vs. 195.2 ± 64.3 for IL-6 (p < 0.05) and 4203 ± 1354 vs. 1048 ± 288.4 p < 0.05 for CXCL8), although no difference was seen between groups from the whole blood assays. Following stimulation with LPS, however, release of both IL-6 and CXCL8 was lower from both WB and PBMC assays for PAH patients compared to controls. For example for LPS-induced IL-6 release from PBMC, both the maximal effect (Emax) was lower in PAH vs. controls (5148 ± 1320 vs. 9154 ± 2510 pg/ml, p < 0.05) and the log EC50 (half maximal effective concentration) was higher in PAH vs. controls (-2.70 ± 0.27 vs. -5.90 ± 1.16 µg/ml, p < 0.01) (Figure 1). Similar results were seen following stimulation with TNF-a. Conclusion The main findings of this study are that: (1) baseline CXCL8 and IL6 release was higher from PBMCs of patients with PAH compared to control donors; (2) PBMCs and WB from patients were hyporesponsive to LPS (and TNF-a), suggesting an altered inflammatory phenotype, at least in these models, to these stimuli. References Soon E. et al, Circulation. 2010;122(9):920–7 Abstract P159 Figure 1.

P29 Endothelial cell NF-kB activation is increased in human idiopathic PAH

Background Pulmonary arterial hypertension (PAH) is associated with pulmonary vascular inflammation, and several of the inflammatory genes involved are regulated by nuclear factor-kB (NF-kB). NF-kB is a heterodimer of p65 and p50 subunits which, upon activation, translocate into the nucleus and binds to target gene promoters. NF-kB activation in PAH has not been examined in detail to date. We assessed NF-kB activation by immunohistochemical analysis of nuclear p65. Methods Samples were obtained from South Paris University from patients with severe idiopathic PAH (IPAH) following lung transplantation (n=10) and from control subjects undergoing lobectomy or pneumonectomy (n=10). Tissue blocks were fixed and paraffin-embedded, 4-mm thick sections underwent immunoperoxidase double staining for macrophage (CD68+)/p65, using mouse anti-human CD68 (Dako; 1:100 dilution) and rabbit anti human p65 (Santa Cruz Biotechnology; 1:50 dilution), before detection with chromogen fast red and counterstaining with haematoxylin. Vessels of interest were defined and quantitative scoring of nuclear p65 immunostaining of ten randomly selected pulmonary arteries per slide performed by a researcher blinded to the groups. Data are expressed as median and IQR, and the groups were compared by the Kruskal–Wallis or Mann–Whitney U test. Results (1) PAH specimens showed co-localisation of p65 within CD68+ macrophages in 75.4 (64.8–84.6)% of samples. Airway epithelium, neutrophils and lymphocytes were also positive for p65. (2) Pulmonary arterial medial thickness was increased in PAH compared to controls, at 33.7 (18.8–67.9)% in vessels 100–250 mm external diameter (E.D.) and 27.2 (14.8–44.2)% in vessels 250–500 mm ED, vs 17.7 (11.2–30.3)% and 14.9 (11.8–17.8)% in controls (p<0.0001 between groups). (3) Nuclear p65 was present in pulmonary artery endothelial cells (EC) but not other vascular cells including pulmonary artery smooth muscle cells in PAH: 53.9 (0–100)% of vessels 100–250 mm E.D. and 53.1 (0–100)% of those 250–500 mm E.D. scored EC p65 positivity in PAH compared to 7.5 (0–25.0)% in 100–250 mm ED and 4.7 (0–21.1)% in 250–500 mm ED in controls (p<0.0001 between groups) (Abstract P29 Figure 1).Abstract P29 Figure 1 Percentage of positive endothelial cell (EC) nuclear p65 immunostaining subdivided according to pulmonary artery external diameter (E.D.). Conclusion NF-kB activation is present in macrophages and pulmonary arterial endothelial cells in pulmonary arteries of 100–500 mm ED in patients with PAH.

S152 Dexamethasone reverses established monocrotaline-induced pulmonary hypertension in rats and increases pulmonary BMPR2 expression

Background Pulmonary arterial hypertension (PAH) is associated with pulmonary vascular inflammation and dysregulated bone morphogenetic protein receptor type 2 (BMPR2) signalling in both human and experimental PAH. We evaluated the effects of dexamethasone on established monocrotaline-induced PAH in rats for potential reversal of PAH, at time points when pulmonary vascular remodelling has already developed (from day 14 after a single injection of monocrotaline at day 0), and for the effects on pulmonary IL6 and BMPR2 expression. Methods Saline-treated controls, monocrotaline-exposed, monocrotaline-exposed and dexamethasone-treated rats (5 mg/kg/day, 1.25 mg/kg and 2.5 mg/kg/48 h given from day 14–28 and day 21–35) were evaluated at day 28 and day 35 following monocrotaline for pulmonary vascular haemodynamic parameters, right ventricular hypertrophy, morphometry, immunohistochemistry, whole lung IL-6 and BMPR2 expression by quantitative real-time PCR (qRT-PCR). Results Dexamethasone significantly improved pulmonary haemodynamics and morphometric indices of pulmonary vascular remodelling, reversing PAH when given at day 14–28, day 21–35 following monocrotaline, as well as improving survival in monocrotaline-exposed rats compared to controls (log rank p<0.0001). Dexamethasone reduced both monocrotaline-induced whole lung IL-6 overexpression (p<0.05), as well as reducing IL-6-expressing adventitial inflammatory cell infiltration as assessed by immunohistochemistry. This was associated with pulmonary BMPR2 down-regulation (p<0.01) following monocrotaline, which was significantly increased following day 14–28 dexamethasone treatment in whole lung (p<0.05) (Abstract S152 Figure 1). Cellular BMPR2 was also increased following in vitro treatment of control pulmonary artery smooth muscle cells (PASMC) with ×10−8 molar dexamethasone (p<0.05), but not in PASMC isolated from pulmonary hypertensive rats. Dexamethasone (×10−8 and 10−7 molar) also reduced proliferation of PASMC isolated from both control and pulmonary hypertensive rats (p<0.05 for both doses).Abstract S152 Figure 1 Effects of monocrotaline and day 14–28 dexamethasone on whole-lung BMPR2 expression by qRT-PCR. Conclusion PAH in this well-characterised experimental model can be reversed by dexamethasone, and survival is improved. In this model, mechanisms may involve reduction of IL-6-expressing inflammatory cells, reduced proliferation of vascular smooth muscle cells, and restoration of pulmonary BMPR2 expression may be important.