David Welsh

The effects of hypoxia on the cells of the pulmonary vasculature

Pulmonary hypertension is associated with remodelling of pulmonary vessels. Chronic hypoxia is a common cause of pulmonary hypertension and pulmonary vascular remodelling. Vascular remodelling is characterised largely by fibroblast, smooth muscle and endothelial cell proliferation, which results in lumen obliteration. Chronic hypoxia elicits expression of mitogens, growth factors and cytokines by fibroblasts and endothelial cells, and also the suppression of endothelial nitric oxide synthase. Although hypoxic pulmonary vascular remodelling is associated with medial hypertrophy, many in vitro studies have found that hypoxia does not lead to a direct increase in smooth muscle cell proliferation. This paradox is not well understood and this review aims to examine the various reasons why this might be so. The present authors reviewed data from in vitro studies and also considered whether hypoxia could act on adjacent cells such as fibroblasts and endothelial cells to trigger smooth muscle cell proliferation. It is possible that hypoxia is sensed by fibroblasts, endothelial cells, or both, and relayed to adjacent pulmonary artery smooth muscle cells by intercellular signalling, causing proliferation. The present article reviews the data from in vitro studies of hypoxia on the three cellular components of the pulmonary vascular wall, namely endothelial cells, smooth muscle cells and fibroblasts.

Converging evidence in support of the serotonin hypothesis of dexfenfluramine-induced pulmonary hypertension with novel transgenic mice.

Background— The incidence of pulmonary arterial hypertension secondary to the use of indirect serotinergic agonists such as aminorex and dexfenfluramine led to the “serotonin hypothesis” of pulmonary arterial hypertension; however, the role of serotonin in dexfenfluramine-induced pulmonary arterial hypertension remains controversial. Here, we used novel transgenic mice lacking peripheral serotonin (deficient in tryptophan hydroxylase-1; Tph1−/− mice) or overexpressing the gene for the human serotonin transporter (SERT; SERT+ mice) to investigate this further. Methods and Results— Dexfenfluramine administration (5 mg · kg−1 · d−1 PO for 28 days) increased systolic right ventricular pressure and pulmonary vascular remodeling in wild-type mice but not in Tph1−/− mice, which suggests that dexfenfluramine-induced pulmonary arterial hypertension is dependent on serotonin synthesis. Dexfenfluramine was also administered to normoxic SERT+ mice and SERT+ mice exposed to chronic hypoxia. Dexfenfluramine and SERT overexpression had additive effects in increasing pulmonary vascular remodeling; however, in hypoxic SERT+ mice, dexfenfluramine reduced both systolic right ventricular pressure and pulmonary vascular remodeling. Pulmonary arterial fibroblasts from SERT+ mice, but not wild-type mice, proliferated in response to hypoxia. Dexfenfluramine inhibited hypoxia-induced proliferation of pulmonary arterial fibroblasts derived from SERT+ mice in a manner dependent on SERT activity. Dexfenfluramine also inhibited the hypoxia-mediated increase in phosphorylation of p38 mitogen-activated protein kinase in SERT+ pulmonary arterial fibroblasts. Conclusions— The results suggest that peripheral serotonin is critical for the development of dexfenfluramine-induced pulmonary arterial hypertension and that dexfenfluramine and SERT overexpression have additive effects on pulmonary vascular remodeling. We propose that dexfenfluramine can also inhibit hypoxia-induced pulmonary vascular remodeling via SERT activity and inhibition of hypoxia-induced p38 mitogen-activated protein kinase.

Ambulatory pulmonary artery pressure monitoring during sleep and exercise in normal individuals and patients with COPD

Background: Pulmonary hypertension is a common complication of chronic obstructive airways disease (COPD) and its presence implies a poor prognosis. However, it is difficult to measure and its specific contribution to symptoms is difficult to quantify. A micromanometer tipped pulmonary artery catheter was used to measure pulmonary artery pressure (PAP) during sleep and on exercise. Methods: Ten patients (five with COPD receiving long term oxygen therapy and five normal individuals) were studied. Pulmonary artery pressure was recorded continuously during two periods of sleep (breathing oxygen followed by air for the COPD group) and during exercise. Results: In the COPD group PAP during sleep on oxygen was significantly lower than PAP during sleep breathing air (mean (SD) difference 9.6 (5.3) mm Hg, 95% CI 4.9 to 14.3, p= 0.016). PAP during exercise was not significantly different from PAP during sleep breathing air (mean (SD) difference 0.8 (8.9) mm Hg, 95% CI –7.0 to 8.6, p= 0.851). In normal individuals the group mean (SD) PAP was 15 (5.9) mm Hg for the first nocturnal period and 15 (5.7) mm Hg for the second nocturnal period. PAP during exercise was not significantly different from PAP during sleep breathing air (mean (SD) difference 3.3 (2.2) mm Hg, 95% CI 1.1 to 5.5, p= 0.061). Conclusion: In patients with COPD, PAP rose significantly during sleep to levels similar to those measured during exercise, but this could be reversed with oxygen.

Inhibition of p38 MAPK reverses hypoxia-induced pulmonary artery endothelial dysfunction

Hypoxia-induced endothelial dysfunction plays a crucial role in the pathogenesis of hypoxic pulmonary hypertension. p38 MAPK expression is increased in the pulmonary artery following hypoxic exposure. Recent evidence suggests that increased p38 MAPK activity is associated with endothelial dysfunction. However, the role of p38 MAPK activation in pulmonary artery endothelial dysfunction is not known. Sprague-Dawley rats were exposed to 2 wk hypobaric hypoxia, which resulted in the development of pulmonary hypertension and vascular remodeling. Endothelium-dependent relaxation of intrapulmonary vessels from hypoxic animals was impaired due to a reduced nitric oxide (NO) generation. This was despite increased endothelial NO synthase immunostaining and protein expression. Hypoxia exposure increased superoxide generation and p38 MAPK expression. The inhibition of p38 MAPK restored endothelium-dependent relaxation, increased bioavailable NO, and reduced superoxide production. In conclusion, the pharmacological inhibition of p38 MAPK was effective in increasing NO generation, reducing superoxide burden, and restoring hypoxia-induced endothelial dysfunction in rats with hypoxia-induced pulmonary hypertension. p38 MAPK may be a novel target for the treatment of pulmonary hypertension.

The reversal of pulmonary vascular remodeling through inhibition of p38 MAPK-alpha: a potential novel anti-inflammatory strategy in pulmonary hypertension

The p38 mitogen-activated protein kinase (MAPK) system is increasingly recognized as an important inflammatory pathway in systemic vascular disease but its role in pulmonary vascular disease is unclear. Previous in vitro studies suggest p38 MAPKα is critical in the proliferation of pulmonary artery fibroblasts, an important step in the pathogenesis of pulmonary vascular remodeling (PVremod). In this study the role of the p38 MAPK pathway was investigated in both in vitro and in vivo models of pulmonary hypertension and human disease. Pharmacological inhibition of p38 MAPKα in both chronic hypoxic and monocrotaline rodent models of pulmonary hypertension prevented and reversed the pulmonary hypertensive phenotype. Furthermore, with the use of a novel and clinically available p38 MAPKα antagonist, reversal of pulmonary hypertension was obtained in both experimental models. Increased expression of phosphorylated p38 MAPK and p38 MAPKα was observed in the pulmonary vasculature from patients with idiopathic pulmonary arterial hypertension, suggesting a role for activation of this pathway in the PVremod A reduction of IL-6 levels in serum and lung tissue was found in the drug-treated animals, suggesting a potential mechanism for this reversal in PVremod. This study suggests that the p38 MAPK and the α-isoform plays a pathogenic role in both human disease and rodent models of pulmonary hypertension potentially mediated through IL-6. Selective inhibition of this pathway may provide a novel therapeutic approach that targets both remodeling and inflammatory pathways in pulmonary vascular disease.

Cellular responses to hypoxia in the pulmonary circulation.

Hypoxia can be defined as a reduction in available oxygen, whether in a whole organism or in a tissue or cell. It is a real life cause of pulmonary hypertension in humans both in terms of patients with chronic hypoxic lung disease and people living at high altitude. The effect of hypoxia on the pulmonary vasculature can be described in two ways; Hypoxic pulmonary vasoconstriction (HPV) (resulting from smooth muscle cell contraction) and pulmonary vascular remodelling (PVR) (resulting from pulmonary vascular cell proliferation). The pulmonary artery is made up of three resident cell types, the endothelial (intima), smooth muscle (media) and fibroblast (adventitia) cells. This review will examine the effects of hypoxia on the cells of the pulmonary vasculature and give an insight into the possible underlying mechanisms.

The role of microRNA-155/liver X receptor pathway in experimental and idiopathic pulmonary fibrosis

Background Idiopathic pulmonary fibrosis (IPF) is progressive and rapidly fatal. Improved understanding of pathogenesis is required to prosper novel therapeutics. Epigenetic changes contribute to IPF; therefore, microRNAs may reveal novel pathogenic pathways. Objectives We sought to determine the regulatory role of microRNA (miR)‐155 in the profibrotic function of murine lung macrophages and fibroblasts, IPF lung fibroblasts, and its contribution to experimental pulmonary fibrosis. Methods Bleomycin‐induced lung fibrosis in wild‐type and miR‐155−/− mice was analyzed by histology, collagen, and profibrotic gene expression. Mechanisms were identified by in silico and molecular approaches and validated in mouse lung fibroblasts and macrophages, and in IPF lung fibroblasts, using loss‐and‐gain of function assays, and in vivo using specific inhibitors. Results miR‐155−/− mice developed exacerbated lung fibrosis, increased collagen deposition, collagen 1 and 3 mRNA expression, TGF‐&bgr; production, and activation of alternatively activated macrophages, contributed by deregulation of the miR‐155 target gene the liver X receptor (LXR)&agr; in lung fibroblasts and macrophages. Inhibition of LXR&agr; in experimental lung fibrosis and in IPF lung fibroblasts reduced the exacerbated fibrotic response. Similarly, enforced expression of miR‐155 reduced the profibrotic phenotype of IPF and miR‐155−/− fibroblasts. Conclusions We describe herein a molecular pathway comprising miR‐155 and its epigenetic LXR&agr; target that when deregulated enables pathogenic pulmonary fibrosis. Manipulation of the miR‐155/LXR pathway may have therapeutic potential for IPF. Graphical abstract Figure. No caption available.

Low-Dose Fluvastatin Reverses the Hypoxic Pulmonary Adventitial Fibroblast Phenotype in Experimental Pulmonary Hypertension

Hypoxic pulmonary hypertension is a worldwide public health problem. Statins attenuate hypoxic pulmonary hypertension in animal models, but the mechanism of action and applicability of these results to human treatment are not established. In hypoxic models, pulmonary artery fibroblast proliferation contributes substantially to pulmonary vascular remodeling. We previously showed that acute hypoxic pulmonary adventitial fibroblast proliferation can be selectively inhibited by statins and p38 mitogen-activated protein (MAP) kinase inhibitors. Here we used complementary chronic hypoxic and acute hypoxic coculture models to obtain necessary preclinical information regarding the utility of fluvastatin in the treatment of chronic hypoxic pulmonary hypertension. The effects of fluvastatin, cholesterol pathway intermediates, and related inhibitors on hypoxic adventitial fibroblast proliferation, p38 MAP kinase phosphorylation, and pulmonary artery smooth muscle cell proliferation were determined, using complementary chronic hypoxic rat and acute hypoxic bovine cell models. Fluvastatin reversed the proliferative phenotypic switch in adventitial fibroblasts from chronic hypoxic animals. This effect was circulation-specific, and implicated a Rac1-p38 MAP kinase signaling pathway. Coculture and conditioned media experiments also implicated this statin-sensitive signaling pathway in the release of pulmonary artery smooth muscle cell mitogens by hypoxic pulmonary adventitial fibroblasts. Treprostinil, sildenafil, and bosentan exerted no effect on the hypoxic fibroblast phenotype. Phenotypic changes (increased proliferation and mitogen release) in pulmonary artery fibroblasts during chronic hypoxia are dependent on a Rac1-p38 MAP kinase signaling pathway. The inhibition of these phenotypic changes with fluvastatin may be therapeutically relevant in high-altitude residents and in patients with hypoxic lung disease.

S155 The role of ST2 in a model of pulmonary hypertension

Background Pulmonary arterial hypertension (PAH) is a fatal condition involving remodelling of the pulmonary vessel wall leading to elevated pulmonary arterial pressure and eventually right heart failure. ST2 is a transmembrane receptor with ligand interleukin-33 (IL-33). p38 MAP kinase is an intracellular signalling molecule shown to be involved in the vascular remodelling associated with PAH; and is also involved in ST2/IL-33 signalling. ST2/IL-33 signalling has been shown to reduce fibrosis in the heart following pressure overload in animal models. Aims To determine whether ST2/IL-33 signalling was involved in the proliferation of mouse pulmonary artery fibroblasts and if p38 MAP kinase was involved. Methods Two cell types were used—wild type (WT) and ST2 knockout (ST2−/−) mouse pulmonary artery fibroblasts. Proliferation was assessed by [3H] Thymidine incorporation. Expression of p38 MAP kinase was detected by Western blotting. Cells were cultured at various serum concentrations in normoxia and hypoxia. A p38 MAP kinase inhibitor (SB203580) was used to assess its role in cell proliferation. The effect of IL-33 on cell proliferation and expression of p38 MAP kinase was studied. Results WT and ST2−/− cells proliferated in response to increasing serum concentration. WT cells hyperproliferated in response to hypoxia. ST2−/− cells hyperproliferated in normoxia and hypoxia compared to WT cells. The hyperproliferation of ST2−/− cells in normoxia and hypoxia could be reduced by p38 MAP kinase inhibition (p38) (see Abstract S155 Figure 1). Phosphorylated p38 MAP kinase was detected in all ST2−/− cells and in WT cells in hypoxia with 10% serum, demonstrating p38 MAP kinase is involved in the hyperproliferation observed. Hyperproliferation in response to hypoxia in WT cells could be blocked by addition of IL-33. IL-33 stimulation also decreased the phosphorylation of p38 MAP kinase. There was no effect of IL-33 on ST2−/− cells.Abstract S155 Figure 1 Proliferation in Response to pp38 MAP Kinase Inhibition in Normoxia ST2-/- cells were cultured in normoxia at serum concentrations 0, 0.3, 1, 3, 5 and 10%, with and without the p38 MAP kinase inhibitor SB203580. Proliferation was reduced in the presence of the inhibitor with statistical significance at serum concentrations ?1% serum suggesting that p38 MAP kinase is involved in the hyperproliferation seen in ST2-/- cells. Conclusions Mouse pulmonary artery fibroblasts hyperproliferate in response to hypoxia and in the absence of the ST2 receptor. This hyperproliferation involves phosphorylation of p38 MAP kinase. This phosphorylation and excessive cell proliferation can be blocked by ST2/IL-33 signalling. ST2 may be a potentially novel therapeutic target in the PAH.

S36 P38 MAPK: An Important Pathway in the Pathobiology of Pulmonary Hypertension and Pulmonary Vascular Remodelling

The p38 MAPK pathway is increasingly recognised as important in inflammation leading to systemic vascular disease but its role in pulmonary vascular disease is unclear. Our group has previously identified the p38MAPKα isoform to be critical in hypoxic-induced proliferation of pulmonary artery fibroblasts, a key step in the pathogenesis of pulmonary vascular remodelling. This study sought to investigate the role of p38MAPK in animal models of pulmonary hypertension and in human disease. Methods Sprague Dawley rats were exposed to chronic hypoxia for 14 days and received a selective p38 MAPKα inhibitor from day 1 in a prevention strategy, or after 14 days in a treatment strategy. Both prevention and treatment methods were further employed in a second monocrotaline animal model. Haemodynamic measurements (right ventricular systolic pressure RVSP, right ventricular hypertrophy RVH) were performed, and lungs were removed for immunohistochemistry (IHC) and biochemical analysis. Multiplex ELISA was used to analyse cytokine profile in rat serum. Primary PAF were isolated and siRNA techniques employed to knockdown p38MAPKα. IHC for p38 MAPKα was performed on human tissue from patients with idiopathic pulmonary arterial hypertension. Results siRNA to p38MAPKα inhibited the hypoxic induced proliferation of PAFs. Increased levels of total p38 MAPK activity and increased expression of the alpha isoform was found in the lungs of both chronic hypoxic and MCT animals compared to normal. Using the p38 MAPK inhibitor in the chronic hypoxic and monocrotaline in vivo prevention study resulted in lower RVSP and RVH in the drug treated animals (p<0.005). In the reversal study of both animal models the inhibitor reversed established pulmonary hypertension as determined by RVSP and RVH (p<0.001). Both serum and whole lung levels of IL-6 were lower in the drug treated animals compared to normal. Increased expression of p38 MAPK was observed in lungs from IPAH patients compared to control. Conclusions Our study suggests p38 MAPK alpha is important in pulmonary hypertension. Inhibition of this pathway can prevent the development of PH and perhaps more clinically relevant, can reverse established disease in vivo. Reduction in IL-6 may be a mechanism underlying this process. Abstract S36 Figure 1