Tonio Pera

Arginase and pulmonary diseases

Recent studies have indicated that arginase, which converts l-arginine into l-ornithine and urea, may play an important role in the pathogenesis of various pulmonary disorders. In asthma, chronic obstructive pulmonary disease (COPD) and cystic fibrosis, increased arginase activity in the airways may contribute to obstruction and hyperresponsiveness of the airways by inducing a reduction in the production of bronchodilatory nitric oxide (NO) that results from its competition with constitutive (cNOS) and inducible (iNOS) NO synthases for their common substrate. In addition, reduced l-arginine availability to iNOS induced by arginase may result in the synthesis of both NO and the superoxide anion by this enzyme, thereby enhancing the production of peroxynitrite, which has procontractile and pro-inflammatory actions. Increased synthesis of l-ornithine by arginase may also contribute to airway remodelling in these diseases. l-Ornithine is a precursor of polyamines and l-proline, and these metabolic products may promote cell proliferation and collagen production, respectively. Increased arginase activity may also be involved in other fibrotic disorders of the lung, including idiopathic pulmonary fibrosis. Finally, through its action of inducing reduced levels of vasodilating NO, increased arginase activity has been associated with primary and secondary forms of pulmonary hypertension. Drugs targeting the arginase pathway could have therapeutic potential in these diseases.

Tiotropium inhibits pulmonary inflammation and remodelling in a guinea pig model of COPD

Airway remodelling and emphysema are major structural abnormalities in chronic obstructive pulmonary disease (COPD). In addition, pulmonary vascular remodelling may occur and contribute to pulmonary hypertension, a comorbidity of COPD. Increased cholinergic activity in COPD contributes to airflow limitation and, possibly, to inflammation and airway remodelling. This study aimed to investigate the role of acetylcholine in pulmonary inflammation and remodelling using an animal model of COPD. To this aim, guinea pigs were instilled intranasally with lipopolysaccharide (LPS) twice weekly for 12 weeks and were treated, by inhalation, with the long-acting muscarinic receptor antagonist tiotropium. Repeated LPS exposure induced airway and parenchymal neutrophilia, and increased goblet cell numbers, lung hydroxyproline content, airway wall collagen and airspace size. Furthermore, LPS increased the number of muscularised microvessels in the adventitia of cartilaginous airways. Tiotropium abrogated the LPS-induced increase in neutrophils, goblet cells, collagen deposition and muscularised microvessels, but had no effect on emphysema. In conclusion, tiotropium inhibits remodelling of the airways as well as pulmonary inflammation in a guinea pig model of COPD, suggesting that endogenous acetylcholine plays a major role in the pathogenesis of this disease.

Cigarette smoke and lipopolysaccharide induce a proliferative airway smooth muscle phenotype

BackgroundA major feature of chronic obstructive pulmonary disease (COPD) is airway remodelling, which includes an increased airway smooth muscle (ASM) mass. The mechanisms underlying ASM remodelling in COPD are currently unknown. We hypothesized that cigarette smoke (CS) and/or lipopolysaccharide (LPS), a major constituent of CS, organic dust and gram-negative bacteria, that may be involved in recurrent airway infections and exacerbations in COPD patients, would induce phenotype changes of ASM.MethodsTo this aim, using cultured bovine tracheal smooth muscle (BTSM) cells and tissue, we investigated the direct effects of CS extract (CSE) and LPS on ASM proliferation and contractility.ResultsBoth CSE and LPS induced a profound and concentration-dependent increase in DNA synthesis in BTSM cells. CSE and LPS also induced a significant increase in BTSM cell number, which was associated with increased cyclin D1 expression and dependent on activation of ERK 1/2 and p38 MAP kinase. Consistent with a shift to a more proliferative phenotype, prolonged treatment of BTSM strips with CSE or LPS significantly decreased maximal methacholine- and KCl-induced contraction.ConclusionsDirect exposure of ASM to CSE or LPS causes the induction of a proliferative, hypocontractile ASM phenotype, which may be involved in airway remodelling in COPD.

Pharmacology of airway smooth muscle proliferation

Airway smooth muscle thickening is a pathological feature that contributes significantly to airflow limitation and airway hyperresponsiveness in asthma. Ongoing research efforts aimed at identifying the mechanisms responsible for the increased airway smooth muscle mass have indicated that hyperplasia of airway smooth muscle, due in part to airway myocyte proliferation, is likely a major factor. Airway smooth muscle proliferation has been studied extensively in culture and in animal models of asthma, and these studies have revealed that a variety of receptors and mediators contributes to this response. This review aims to provide an overview of the receptors and mediators that control airway smooth muscle cell proliferation, with emphasis on the intracellular signalling mechanisms involved.

Quaking, an RNA-Binding Protein, Is a Critical Regulator of Vascular Smooth Muscle Cell Phenotype

Rationale: RNA-binding proteins are critical post-transcriptional regulators of RNA and can influence pre-mRNA splicing, RNA localization, and stability. The RNA-binding protein Quaking (QKI) is essential for embryonic blood vessel development. However, the role of QKI in the adult vasculature, and in particular in vascular smooth muscle cells (VSMCs), is currently unknown. Objective: We sought to determine the role of QKI in regulating adult VSMC function and plasticity. Methods and Results: We identified that QKI is highly expressed by neointimal VSMCs of human coronary restenotic lesions, but not in healthy vessels. In a mouse model of vascular injury, we observed reduced neointima hyperplasia in Quaking viable mice, which have decreased QKI expression. Concordantly, abrogation of QKI attenuated fibroproliferative properties of VSMCs, while potently inducing contractile apparatus protein expression, rendering noncontractile VSMCs with the capacity to contract. We identified that QKI localizes to the spliceosome, where it interacts with the myocardin pre-mRNA and regulates the splicing of alternative exon 2a. This post-transcriptional event impacts the Myocd_v3/Myocd_v1 mRNA balance and can be modulated by mutating the quaking response element in exon 2a of myocardin. Furthermore, we identified that arterial damage triggers myocardin alternative splicing and is tightly coupled with changes in the expression levels of distinct QKI isoforms. Conclusions: We propose that QKI is a central regulator of VSMC phenotypic plasticity and that intervention in QKI activity can ameliorate pathogenic, fibroproliferative responses to vascular injury.

Airway smooth muscle in asthma: Linking contraction and mechanotransduction to disease pathogenesis and remodelling

Asthma is an obstructive airway disease, with a heterogeneous and multifactorial pathogenesis. Although generally considered to be a disease principally driven by chronic inflammation, it is becoming increasingly recognised that the immune component of the pathology poorly correlates with the clinical symptoms of asthma, thus highlighting a potentially central role for non-immune cells. In this context airway smooth muscle (ASM) may be a key player, as it comprises a significant proportion of the airway wall and is the ultimate effector of acute airway narrowing. Historically, the contribution of ASM to asthma pathogenesis has been contentious, yet emerging evidence suggests that ASM contractile activation imparts chronic effects that extend well beyond the temporary effects of bronchoconstriction. In this review article we describe the effects that ASM contraction, in combination with cellular mechanotransduction and novel contraction-inflammation synergies, contribute to asthma pathogenesis. Specific emphasis will be placed on the effects that ASM contraction exerts on the mechanical properties of the airway wall, as well as novel mechanisms by which ASM contraction may contribute to more established features of asthma such as airway wall remodelling.

Arginase Inhibition Prevents Inflammation and Remodeling in a Guinea Pig Model of Chronic Obstructive Pulmonary Disease

Airway inflammation and remodeling are major features of chronic obstructive pulmonary disease (COPD), whereas pulmonary hypertension is a common comorbidity associated with a poor disease prognosis. Recent studies in animal models have indicated that increased arginase activity contributes to features of asthma, including allergen-induced airway eosinophilia and mucus hypersecretion. Although cigarette smoke and lipopolysaccharide (LPS), major risk factors for COPD, may increase arginase expression, the role of arginase in COPD is unknown. This study aimed to investigate the role of arginase in pulmonary inflammation and remodeling using an animal model of COPD. Guinea pigs were instilled intranasally with LPS or saline twice weekly for 12 weeks and pretreated by inhalation of the arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH) or vehicle. Repeated LPS exposure increased lung arginase activity, resulting in increased l-ornithine/l-arginine and l-ornithine/l-citrulline ratios. Both ratios were reversed by ABH. ABH inhibited the LPS-induced increases in pulmonary IL-8, neutrophils, and goblet cells as well as airway fibrosis. Remarkably, LPS-induced right ventricular hypertrophy, indicative of pulmonary hypertension, was prevented by ABH. Strong correlations were found between arginase activity and inflammation, airway remodeling, and right ventricular hypertrophy. Increased arginase activity contributes to pulmonary inflammation, airway remodeling, and right ventricular hypertrophy in a guinea pig model of COPD, indicating therapeutic potential for arginase inhibitors in this disease.

The RNA-Binding Protein Quaking is a Critical Regulator of Vascular Smooth Muscle Cell Phenotype

Rationale: RNA-binding proteins are critical post-transcriptional regulators of RNA and can influence pre-mRNA splicing, RNA localization, and stability. The RNA-binding protein Quaking (QKI) is essential for embryonic blood vessel development. However, the role of QKI in the adult vasculature, and in particular in vascular smooth muscle cells (VSMCs), is currently unknown. Objective: We sought to determine the role of QKI in regulating adult VSMC function and plasticity. Methods and Results: We identified that QKI is highly expressed by neointimal VSMCs of human coronary restenotic lesions, but not in healthy vessels. In a mouse model of vascular injury, we observed reduced neointima hyperplasia in Quaking viable mice, which have decreased QKI expression. Concordantly, abrogation of QKI attenuated fibroproliferative properties of VSMCs, while potently inducing contractile apparatus protein expression, rendering noncontractile VSMCs with the capacity to contract. We identified that QKI localizes to the spliceosome, where it interacts with the myocardin pre-mRNA and regulates the splicing of alternative exon 2a. This post-transcriptional event impacts the Myocd_v3/Myocd_v1 mRNA balance and can be modulated by mutating the quaking response element in exon 2a of myocardin. Furthermore, we identified that arterial damage triggers myocardin alternative splicing and is tightly coupled with changes in the expression levels of distinct QKI isoforms. Conclusions: We propose that QKI is a central regulator of VSMC phenotypic plasticity and that intervention in QKI activity can ameliorate pathogenic, fibroproliferative responses to vascular injury.

Crosstalk between beta-2-adrenoceptor and muscarinic acetylcholine receptors in the airway

The M3 and M2 muscarinic acetylcholine receptors (mAChRs) and beta-2-adrenoceptors (β2ARs) are important regulators of airway cell function, and drugs targeting these receptors are among the first line drugs in the treatment of the obstructive lung diseases asthma and chronic obstructive lung disease (COPD). Cross-regulation or crosstalk between mAChRs and β2ARs in airway smooth muscle (ASM) helps determine the contractile state of the muscle, thus airway diameter and resistance to airflow. In this review we will detail mAChR and β2AR-signaling and crosstalk, focusing on events in the ASM cell but also addressing the function of these receptors in other cell types that impact airway physiology. We conclude by discussing how recent advances in GPCR pharmacology offer a unique opportunity to fine tune mAChR and β2AR signaling and their crosstalk, and thereby produce superior therapeutics for obstructive lung and other diseases.

Role for TAK1 in cigarette smoke-induced proinflammatory signaling and IL-8 release by human airway smooth muscle cells

Chronic obstructive pulmonary disease (COPD) is an inflammatory disease, characterized by a progressive decline in lung function. Airway smooth muscle (ASM) mass may be increased in COPD, contributing to airflow limitation and proinflammatory cytokine production. Cigarette smoke (CS), the major risk factor of COPD, causes ASM cell proliferation, as well as interleukin-8 (IL-8)-induced neutrophilia. In various cell types, transforming growth factor-β-activated kinase 1 (TAK1) plays a crucial role in MAP kinase and NF-κB activation, as well as IL-8 release induced by IL-1β, TNF-α, and lipopolysaccharide. The role of TAK1 in CS-induced IL-8 release is not known. The aim of this study was to investigate the role of TAK1 in CS-induced NF-κB and MAP kinase signaling and IL-8 release by human ASM cells. Stimulation of these cells with CS extract (CSE) increased IL-8 release and ERK-1/2 phosphorylation, as well as Iκ-Bα degradation and p65 NF-κB subunit phosphorylation. CSE-induced ERK-1/2 phosphorylation and Iκ-Bα degradation were both inhibited by pretreatment with the specific TAK1 inhibitor LL-Z-1640-2 (5Z-7-oxozeaenol; 100 nM). Similarly, expression of dominant-negative TAK1 inhibited CSE-induced ERK-1/2 phosphorylation. In addition, inhibitors of TAK1 and the NF-κB (SC-514; 50 μM) and ERK-1/2 (U-0126; 3 μM) signaling inhibited the CSE-induced IL-8 release by ASM cells. These data indicate that TAK1 plays a major role in CSE-induced ERK-1/2 and NF-κB signaling and in IL-8 release by human ASM cells. Furthermore, they identify TAK1 as a novel target for the inhibition of CS-induced inflammatory responses involved in the development and progression of COPD.