Andrew Pearce

An inhibitor of NADPH oxidase-4 attenuates established pulmonary fibrosis in a rodent disease model.

Idiopathic pulmonary fibrosis is a chronic progressive disease of increasing prevalence for which there is no effective therapy. Increased oxidative stress associated with an oxidant-antioxidant imbalance is thought to contribute to disease progression. NADPH oxidases (Nox) are a primary source of reactive oxygen species within the lung and cardiovascular system. We demonstrate that the Nox4 isoform is up-regulated in the lungs of patients with IPF and in a rodent model of bleomycin-induced pulmonary fibrosis and vascular remodeling. Nox4 is constitutively active, and therefore increased expression levels are likely to contribute to disease pathology. Using a small molecule Nox4/Nox1 inhibitor, we demonstrate that targeting Nox4 results in attenuation of an established fibrotic response, with reductions in gene transcripts for the extracellular matrix components collagen 1α1, collagen 3α1, and fibronectin and in principle pathway components associated with pulmonary fibrosis and hypoxia-mediated vascular remodeling: transforming growth factor (TGF)-β1, plasminogen activator inhibitor-1, hypoxia-inducible factor, and Nox4. TGF-β1 is a principle fibrotic mediator responsible for inducing up-regulation of profibrotic pathways associated with disease pathology. Using normal human lung-derived primary fibroblasts, we demonstrate that inhibition of Nox4 activity using a small molecule antagonist attenuates TGF-β1-mediated up-regulation in expression of profibrotic genes and inhibits the differentiation of fibroblast to myofibroblasts, that is associated with up-regulation in smooth muscle actin and acquisition of a contractile phenotype. These studies support the view that targeting Nox4 may provide a therapeutic approach for attenuating pulmonary fibrosis.

Imatinib Attenuates Hypoxia-induced Pulmonary Arterial Hypertension Pathology via Reduction in 5-Hydroxytryptamine through Inhibition of Tryptophan Hydroxylase 1 Expression

RATIONALE Whether idiopathic, familial, or secondary to another disease, pulmonary arterial hypertension (PAH) is characterized by increased vascular tone, neointimal hyperplasia, medial hypertrophy, and adventitial fibrosis. Imatinib, a potent receptor tyrosine kinase inhibitor, reverses pulmonary remodeling in animal models of PAH and improves hemodynamics and exercise capacity in selected patients with PAH. OBJECTIVES Here we use both imatinib and knockout animals to determine the relationship between platelet-derived growth factor receptor (PDGFR) and serotonin signaling and investigate the PAH pathologies each mediates. METHODS We investigated the effects of imatinib (100 mg/kg) on hemodynamics, vascular remodeling, and downstream molecular signatures in the chronic hypoxia/SU5416 murine model of PAH. MEASUREMENTS AND MAIN RESULTS Treatment with imatinib reduced all measures of PAH pathology observed in hypoxia/SU5416 mice. In addition, 5-hydroxytryptamine (5-HT) and tryptophan hydroxylase 1 (Tph1) expression were reduced compared with the normoxia/SU5416 control group. Imatinib attenuated hypoxia-induced increases in Tph1 expression in pulmonary endothelial cells in vitro via inhibition of the PDGFR-β pathway. To better understand the consequences of this novel mode of action for imatinib, we examined the development of PAH after hypoxic/SU5416 exposure in Tph1-deficient mice (Tph1(-/-)). The extensive changes in pulmonary vascular remodeling and hemodynamics in response to hypoxia/SU5416 were attenuated in Tph1(-/-) mice and further decreased after imatinib treatment. However, imatinib did not significantly further impact collagen deposition and collagen 3a1 expression in hypoxic Tph1(-/-) mice. Post hoc subgroup analysis suggests that patients with PAH with greater hemodynamic impairment showed significantly reduced 5-HT plasma levels after imatinib treatment compared with placebo. CONCLUSIONS We report a novel mode of action for imatinib, demonstrating TPH1 down-regulation via inhibition of PDGFR-β signaling. Our data reveal interplay between PDGF and 5-HT pathways within PAH, demonstrating TPH1-dependent imatinib efficacy in collagen-mediated mechanisms of fibrosis.

Attenuation of leukocyte recruitment via CXCR1/2 inhibition stops the progression of PAH in mice with genetic ablation of endothelial BMPR-II

Previous studies from our group have demonstrated that bone morphogenetic protein receptor-II (BMPR-II), expressed on pulmonary artery endothelial cells, imparts profound anti-inflammatory effects by regulating the release of proinflammatory cytokines and promoting barrier function by suppressing the transmigration of leukocytes into the pulmonary vessel wall. Here we demonstrate that, in mice with endothelial-specific loss of BMPR-II expression (L1Cre(+);Bmpr2(f/f)), reduction in barrier function and the resultant pulmonary hypertension observed in vivo are the result of increased leukocyte recruitment through increased CXCR1/2 signaling. Loss of endothelial expressed BMPR-II leads to elevated plasma levels of a wide range of soluble mediators important in regulating leukocyte migration and extravasation, including the CXCR1/2 ligand, KC. Treatment of L1Cre(+);Bmpr2(f/f) mice with the CXCR1/2 antagonist SCH527123 inhibits leukocyte transmigration into lung and subsequently reverses the pulmonary hypertension. Our data have uncovered a previously unrecognized regulatory function of BMPR-II, which acts to regulate the expression of CXCR2 on endothelial cells, suggesting that increased CXCR2 signaling may also be a feature of the human pathology and that CXCR1/2 pathway antagonists may represent a novel therapeutic approach for treating pulmonary hypertension because of defects in BMPR-II expression.

Impaired Bone Morphogenetic Protein Receptor II Signaling in a Transforming Growth Factor-β–Dependent Mouse Model of Pulmonary Hypertension and in Systemic Sclerosis

RATIONALE Up to 10% of patients with systemic sclerosis (SSc) develop pulmonary arterial hypertension (PAH). This risk persists throughout the disease and is time dependent, suggesting that SSc is a susceptibility factor. Outcome for SSc-PAH is poor compared with heritable or idiopathic forms, despite clinical and pathological similarities. Although susceptibility in heritable PAH and idiopathic PAH is strongly associated with gene mutations leading to reduced expression of bone morphogenetic protein receptor (BMPR) II, these mutations have not been observed in SSc-PAH. OBJECTIVES To explore BMPRII expression and function in a mouse model of SSc (TβRIIΔk-fib) that is susceptible to developing pulmonary hypertension and in SSc lung. METHODS BMPRII and downstream signaling pathways were profiled in lung tissue and fibroblasts from the TβRIIΔk-fib model, which develops pulmonary vasculopathy with pulmonary hypertension that is exacerbated by SU5416. Complementary studies examined SSc or control lung tissue and fibroblasts. MEASUREMENTS AND MAIN RESULTS Our study shows reduced BMPRII, impaired signaling, and altered receptor turnover activity in a transforming growth factor (TGF)-β-dependent mouse model of SSc-PAH. Similarly, a significant reduction in BMPRII expression is observed in SSc lung tissue and fibroblasts. Increased proteasomal degradation of BMPRII appears to underlie this and may result from heightened TGF-β activity. CONCLUSIONS We found reduced BMPRII protein in patients with SSc-PAH and a relevant mouse model associated with increased proteasomal degradation of BMPRII. Collectively, these results suggest that impaired BMP signaling, resulting from TGF-β-dependent increased receptor degradation, may promote PAH susceptibility in SSc and provide a unifying mechanism across different forms of PAH.

A functional analysis of Wnt inducible signalling pathway protein −1 (WISP-1/CCN4)

Wnt-1 inducible signalling pathway protein 1 (WISP-1/CCN4) is an extracellular matrix protein that belongs to the Cyr61 (cysteine-rich protein 61), CTGF (connective tissue growth factor) and NOV (CCN) family and plays a role in multiple cellular processes. No specific WISP-1 receptors have been identified but emerging evidence suggests WISP-1 mediates its downstream effects by binding to integrins. Here we describe a functional analysis of integrin receptor usage by WISP-1. Truncated WISP-1 proteins were produced using a baculovirus expression system. Full length WISP-1 and truncated proteins were evaluated for their ability to induce adhesion in A549 epithelial cells and β-catenin activation and CXCL3 secretion in fibroblasts (NRK49-F cells). Subsequent inhibition of these responses by neutralising integrin antibodies was evaluated. A549 cells demonstrated adhesion to full-length WISP-1 whilst truncated proteins containing VWC, TSP or CT domains also induced adhesion, with highest activity observed with proteins containing the C-terminal TSP and CT domains. Likewise the ability to induce β-catenin activation and CXCL3 secretion was retained in truncations containing C-terminal domains. Pre-treatment of A549s with either integrin αVβ5, αVβ3 or β1 neutralising antibodies partially inhibited full length WISP-1 induced adhesion whilst combining integrin αVβ5 and β1 antibodies increased the potency of this effect. Incubation of NRK49-F cells with integrin neutralising antibodies failed to effect β-catenin translocation or CXCL3 secretion. Analysis of natural WISP-1 derived from human lung tissue showed the native protein is a high order oligomer. Our data suggest that WISP-1 mediated adhesion of A549 cells is an integrin-driven event regulated by the C-terminal domains of the protein. Activation of β-catenin signalling and CXCL3 secretion also resides within the C-terminal domains of WISP-1 but are not regulated by integrins. The oligomeric nature of native WISP-1 may drive a high avidity interaction with these receptors in vivo.

Optimization of Platelet-Derived Growth Factor Receptor (PDGFR) Inhibitors for Duration of Action, as an Inhaled Therapy for Lung Remodeling in Pulmonary Arterial Hypertension.

A series of potent PDGFR inhibitors has been identified. The series was optimized for duration of action in the lung. A novel kinase occupancy assay was used to directly measure target occupancy after i.t. dosing. Compound 25 shows 24 h occupancy of the PDGFR kinase domain, after a single i.t. dose and has efficacy at 0.03 mg/kg, in the rat moncrotaline model of pulmonary arterial hypertension. Examination of PK/PD data from the optimization effort has revealed in vitro:in vivo correlations which link duration of action in vivo with low permeability and high basicity and demonstrate that nonspecific binding to lung tissue increases with lipophilicity.

Chronic Obstructive Pulmonary Disease (COPD)

Chronic obstructive pulmonary disease (COPD) is a major health burden predicted to become the third most common cause of death and the fifth most common cause of disability worldwide by 2020 [1]. An international population-based COPD study in 2007 identified 10 % prevalence of mild or severe COPD in 12 different countries [2].