Associate editor: D. Spina
Attacking the multi-tiered proteolytic pathology of COPD: New insights from basic and translational studies

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Abstract

Protease activity in inflammation is complex. Proteases released by cells in response to infection, cytokines, or environmental triggers like cigarette smoking cause breakdown of the extracellular matrix (ECM). In chronic inflammatory diseases like chronic obstructive pulmonary disease (COPD), current findings indicate that pathology and morbidity are driven by dysregulation of protease activity, either through hyperactivity of proteases or deficiency or dysfunction their antiprotease regulators. Animal studies demonstrate the accuracy of this hypothesis through genetic and pharmacologic tools. New work shows that ECM destruction generates peptide fragments active on leukocytes via neutrophil or macrophage chemotaxis towards collagen and elastin derived peptides respectively. Such fragments now have been isolated and characterized in vivo in each case. Collectively, this describes a biochemical circuit in which protease activity leads to activation of local immunocytes, which in turn release cytokines and more proteases, leading to further leukocyte infiltration and cyclical disease progression that is chronic. This circuit concept is well known, and is intrinsic to the protease–antiprotease hypothesis; recently analytic techniques have become sensitive enough to establish fundamental mechanisms of this hypothesis, and basic and clinical data now implicate protease activity and peptide signaling as pathologically significant pharmacologic targets. This review discusses targeting protease activity for chronic inflammatory disease with special attention to COPD, covering important basic and clinical findings in the field; novel therapeutic strategies in animal or human studies; and a perspective on the successes and failures of agents with a focus on clinical potential in human disease.

Introduction

In the lungs, chronic inflammatory diseases including COPD, chronic bronchitis, and asthma are increasingly prevalent as humans become more regularly exposed to particulate material in the environment and increased prevalence of cigarette smoking, all of which leads to activation of the immune system (Crystal, 1997, Kobzik, 1999). In the disease asthma, this immune activation manifests as a hypersensitivity response to a particular antigen, which causes airway obstruction by bronchoconstriction in the regions of the tracheobronchial tree possessing smooth muscle. Fortunately, the condition is often self-limited and symptoms are reduced without a ‘trigger’ antigen. For COPD, there is a more sustained and nonspecific response to repeated chemical and particulate exposure (especially to cigarette smoke), with permanent airway remodeling and alveolar space destruction eventually leading to decreased lung elasticity with airflow obstruction at the level of the bronchiole with air retention distal to this collapse.

Cystic fibrosis (CF) is another disease which manifests with chronic pulmonary inflammation as a hallmark, due to genetically determined abnormalities in ion and water transport in the alveolus causing desiccation of the airspace. CF patients are thus more prone to inflammation and infection after environmental exposure to normally innocuous pathogens, leading to permanent tissue remodeling with recurrent infections being common. In each disease, inflammation is the primary culprit for structural changes which result in pulmonary compromise, the progression of symptoms, lifelong disease burden, and in the severely affected, respiratory failure with the possibility of death.

For the purposes of this article, we will deal primarily with chronic obstructive pulmonary disease (COPD), as this disease has a large public health impact and has been the focus of much research in both the clinical and laboratory setting over the last decade (Barnes et al., 2003). In the introduction, we will discuss the disease burden of COPD and current treatments for the disease; then in the next section we will shift focus to the scientific developments in our understanding the pathobiology of this complex disease, specifically discussing protease activity and tissue breakdown and the recent developments in this area of investigation. We will then present recent preclinical and clinical data in the use of new pharmacologic agents whose mechanisms of action target protease activity at the enzymatic level with a summary of the various animal and human studies in pulmonary inflammation and COPD. We will then finish with an analysis of the accumulated biochemical and in vivo data and a discussion of the most promising possible therapeutic agents and their spectra of clinical applications that may be explored with such agents.

COPD is now the most common cause of death among pulmonary disorders (“From the global strategy for the diagnosis, management and prevention of COPD, global initiative for chronic obstructive lung disease (gold)”, 2007); the global burden of disease is increasing with prevalence in the US more than doubling over the past 3 decades and US deaths from the disease also more than doubling between 1980 and 2000 (“From the global strategy for the diagnosis, management and prevention of COPD, global initiative for chronic obstructive lung disease gold), (2007, Global surveillance, prevention and control of chronic respiratory diseases: a comprehensive approach, 2007). The disease is largely considered attributable to cigarette smoking or environmental exposure to smoke or particulate matter which leads to the inflammatory phenotype we will discuss.

Diagnosis of COPD is made by lung spirometry, which measures the dynamics of air movement in the lungs and airway thereby giving data about a patient's respiratory system mechanics. Obstruction is diagnosed by a reduced forced expiratory volume over the first second of exhalation (FEV1), and can be interpreted as a raw value in liters, as a percentage of predicted based on age and sex matched peers, or in terms of a ratio to total lung volume or forced vital capacity (FVC; giving the FEV1/FVC ratio). Essentially, the lower the FEV1, the greater is the patient's loss of expiratory force, and the greater is the degree of obstruction.

As a disease, COPD represents a global health problem whose incidence and prevalence is increasing over time. The disease prevalence estimates range from 11 to 17 million people in the US being affected with the disease, with still more undiagnosed cases. The incidence of COPD in the US has therefore been estimated to be on the order of 5% of the population, with most patients diagnosed after age 40. Globally, COPD is projected to affect approximately 340 million people. The costs of disease management are likewise increasing with a recent estimate of ∼ $1800 per patient year (Miravitlles et al., 2003), which can be extrapolated to a staggering ∼ $30 billion in the USA annually with its costs expected to rise further as the US population's elderly proportion increases over the next few decades.

As advances in management strategies have been made for these conditions over the last 20 years, our pharmacologic arsenal is still limited to a few classes of agents: bronchodilators or β-adrenergic receptor (β-AR) agonists, anticholinergics, phosphodiesterase inhibitors, glucocorticosteroids, and supplemental oxygen, which are used in various combinations for long term disease management. High potency glucocorticosteroids and antimicrobial therapies are used in combination with these agents during acute COPD exacerbations and respiratory infections. More intensive mechanical interventions such as positive pressure ventilation are used during respiratory failure or severe exacerbations requiring hospitalization.

In general, β-AR agonists such as albuterol (short acting) and salmeterol (long acting) have their effect via bronchiolar smooth muscle, inducing the relaxation and therefore dilation of the airway with an overall increase in airflow. Salmeterol is the standard of care for daily therapy with long term disease modification, while the short acting albuterol is used as a ‘rescue’ therapy via inhaler or nebulizer for patients experiencing acute symptoms or in disease exacerbation (Sutherland & Cherniack, 2004).

Anticholinergic therapies include long acting tiotropium and short acting ipratropium which work synergistically with the β-AR agonists at the level of the airway smooth muscle to maximize muscle relaxation and thus airflow. In identical fashion to the β-AR agonists, the long acting agent is used for daily therapy, and use of ipratropium is reserved for more acute situations or at times when patients need immediate symptomatic relief.

Glucocorticosteroids are agents which act at the nuclear level to suppress the cells of the immune system and thereby reduce inflammation. Locally delivered (i.e. inhaled) corticosteroids like fluticasone are now used as daily therapy and have been shown to benefit patients by reducing disease morbidity and associated hospitalizations (“From the global strategy for the diagnosis, management and prevention of COPD, global initiative for chronic obstructive lung disease (gold)”, 2007). These agents are now considered standard for COPD maintenance therapy and are available alone or as a formulation combined with inhaled long acting β-AR agonists for daily or twice daily use. Systemic glucocorticosteroid therapy is not indicated for daily use, but is a mainstay of therapy for COPD exacerbations. The clinical use of these agents is variable and intensity of the steroid regimen is dictated by the severity of the patient's condition with oral prednisone being commonly used for outpatient treatment of a COPD exacerbation and intravenous agents like methylprednisolone or hydrocortisone used in the hospital setting.

Pathologically, COPD is characterized by the destruction of lung parenchyma and enlargement of air spaces with loss of functioning alveoli (Kobzik, 1999, Global surveillance, prevention and control of chronic respiratory diseases: a comprehensive approach, 2007). Inflammation of the bronchioles is typically neutrophilic in character, with increased release of pro-inflammatory cytokines. Furthermore, there is accumulating evidence suggesting that excessive proteolytic activity occurs which overwhelms the natural protease regulation of the lung, and results in parenchymal destruction. This model is generally referred to as the ‘protease–antiprotease hypothesis’ of disease. The inflammation associated with the disease elicits release of proteases from a variety of cell types, mainly neutrophils (polymorphonuclear leucocytes, PMN), macrophages, and epithelia resulting in tissue destruction. It is now clear that protease action induces further inflammation by generating protein fragments which can recruit leukocytes into the tissue. Multiple proteases can potentially be involved in this process of degrading the extracellular matrix (ECM) and producing emphysematous lung disease. Recently though, a few specific enzymes have been specifically implicated in the pathophysiology of COPD, including several members of the matrix metalloprotease (MMP) family, as well as some serine proteases which have ECM proteins collagen and elastin as their substrates. In the following sections of this article, we will discuss these proteases and their proposed roles in disease progression as well as the steps made thus far towards interference with this process as a therapy for this medically expensive and intractable disease.

Section snippets

Proteases implicated in chronic obstructive pulmonary disease

Proteases are enzymes involved in the digestion of a variety of proteins. Although they all catalyze the hydrolysis of the peptide (amide) bond, they have different specificities for the side chains of the scissile peptide bond. Certain proteases break down the connective tissue constituents of the lung parenchyma causing the occurrence of emphysema in the context of an apparent imbalance between proteases and endogenous antiproteases which normally protect from excessive proteolysis.

The

Constituents of the extracellular matrix (ECM) — collagen and elastin

As previously discussed, a prominent feature of proteases is the capacity to degrade the extracellular matrix. While many ECM-derived fragments are not involved in cell signaling, some such peptide fragments demonstrate the capacity to induce cellular recruitment and ongoing inflammation. Indeed, fragments of both laminin (Mydel et al., 2008) and fibronectin (Norris et al., 1982) have been shown to be chemotactic for neutrophils and monocytes, respectively, whereas hyaluronan fragments are felt

Endogenous inhibitors

The naturally occurring neutrophil elastase inhibitors are α-1 antitrypsin, SLPI, and skin derived antileukoprotease (SKALP). Of these, one human trial in alpha-1 antitrypsin repletion therapy was performed in Europe with considerable quantity of the enzyme administered (250 mg/kg every 4 weeks for at least 3 years). Ultimately, this study showed no significant benefit in spirometry performance in the treated group (Dirksen et al., 1999). However, in an α1-AT repletion study in mice, the

Chemotaxis antagonists

As detailed above, both inflammatory cell recruitment and proteolytic matrix degradation are required in the pathology of COPD (Churg et al., 2002), and the prevention of either of these mechanisms may effectively suppress disease development. Furthermore, the findings that ECM breakdown and leukocyte recruitment are intrinsically related through the production of chemoattractant peptides (Houghton et al., 2006, Weathington et al., 2006) suggests that by suppressing one of these modalities we

Summary

In COPD, the current therapeutic strategies work predominantly towards symptom management with integrated therapy focusing on a few areas of pathology. The only life extending therapies currently employed include supplemental oxygen and smoking cessation. Other approved therapies are disease modifying agents that improve dyspnea and decrease hospitalizations. β-AR agonists and anticholinergics target the airway smooth muscle and improve airflow through the larger airways without action at the

Acknowledgments

The authors would like to thank Michael P Nelson for assistance with the figures and Dr. J Edwin Blalock for reviewing the manuscript.

UVD is funded by the NIH (T32A107493). AG is funded through the UAB CIFA Award and the Cystic Fibrosis Foundation (GAGGAR07A0). NW is supported by the UAB Department of Medicine, Internal medicine residency program.

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