University of Groningen
Regenerative Pharmacology for COPD
Wu, Xinhui
DOI:
10.33612/diss.157528459
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Publication date: 2021
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Wu, X. (2021). Regenerative Pharmacology for COPD. University of Groningen. https://doi.org/10.33612/diss.157528459
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XINHUI
WU
Chapter 1
Preface
Chronic obstructive pulmonary disease (COPD) is characterized by progressive and incompletely reversible airway obstruction, in some patients associated with emphysema caused by enhanced tissue destruction in combination with defective airway and alveolar repair1–3. The main causes of COPD are tobacco smoke and air
pollution resulting in accelerated lung function decline in susceptible individuals. Current treatment options are solely reducing symptoms, but do not reverse or slow down the progression of the disease. The objective of this thesis is to unravel the mechanisms underlying cigarette smoke and air pollution-induced dysfunctional alveolar epithelial repair, thereby aiming to discover novel therapeutic targets that could be beneficial to lung repair in COPD.
COPD prevalence and perspective
Chronic obstructive pulmonary disease (COPD) is a complex and heterogenous syndrome characterized by persistent respiratory symptoms and airflow limitation that is due to airway and/or alveolar abnormalities. Globally, there are around three million deaths annually, the estimated number of COPD cases was 384 million in 2010, with a global prevalence of 11.7% and resulting in significant social as well as economic burden 4.
Pathogenesis of COPD
The growing number of COPD cases in recent years is due to the pandemic of tobacco smoking, due to environmental pollution and due to ageing of the world population5,6.
Though the pathobiological mechanisms of COPD remain incompletely understood, various contributors are suggested to play essential roles in COPD pathogenesis, such as the protease-antiprotease imbalance, the oxidant-antioxidant imbalance, cellular senescence, autoimmunity, chronic inflammation, deficient lung growth and development, metaplastic epithelial injuries, and ineffective lung repair5,7.
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Airway remodeling
Airway remodeling8–10 is one of the major pathological features of COPD (Fig.1).
It is defined as structural alterations of both small and large airways and includes epithelial abnormalities, extracellular matrix abnormalities and increased smooth muscle mass of airways. The obstruction of the small airways in COPD is associated with the airway wall thickening by means of a remodeling process related to aberrant tissue repair and a malfunction of the mucociliary clearance of the innate host defense system, which results in the accumulation of inflammation11. There is
no available therapy proven to be prevent or reverse airway remodeling, although reversibility of airway remodeling is suggested by studies in animal models12. Airway
remodeling is caused by aberrant wound healing in response to injuries by such as cigarette smoke, viruses and bacteria, and wound healing is tightly regulated by the interaction between an immune response to these stimuli and remodeling of the extracellular matrix (ECM) by myofibroblasts13,14. Structural alterations in COPD
airways occur and involve dysregulation of the epithelial-mesenchymal unit, with epithelial modification and peribronchial fibrosis15-17.
Alveolar destruction (Emphysema)
Emphysema18,19 is another important characteristic of COPD, which is characterised
by alveolar wall destruction and loss of surface area in the lung parenchyma (Fig.1) where gas exchange occurs. However, the pathogenesis of emphysema is not yet well understood, and there is no effective pharmacological treatment against it. For patients with severe emphysema20, the limited therapeutic options available
Figure 1. Overview of pulmonary pathologies of the COPD lung.
Causes of COPD
Tobacco smokingTobacco smoking is the main risk factor for COPD, which results in a rapid decline in lung function in susceptible individuals. Smoking induces epithelial injury and repeated injuries of the epithelium trigger a pathophysiologic response resulting in tissue remodeling in the conducting airways and destruction of the respiratory bronchioles and alveoli21. Furthermore, the defective epithelium increases the
risk for bacterial and viral infections and subsequent exacerbations. The current SARS-Cov-2 pandemic illustrates this as an example22. The virus has infected more
than 15 million people globally22 by July 2020 with around 650,000 deaths, and
several studies23,24 showed that cigarette smokers are more susceptible to develop
Covid-19 and have more severe disease. This is associated with consistently higher levels of the SARS-Cov-2 receptor ACE2 (angiotensin-converting enzyme 2) in their respiratory tracts, and chronic smokers may exhibit a number of co-morbidities, including emphysema, atherosclerosis, and immune dysregulations that predispose to disease severity25.
Oxidative stress has been suggested to be a key driver of accelerated pulmonary ageing and abnormal repair in COPD. Increased reactive oxygen species (ROS)
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and reactive nitrogen species (NRS) contribute to the occurrence of oxidative and nitrosative stress in the lungs26. Mitochondria are the major cellular oxidative stress
sensors that regulate metabolism, signaling, and energetics to maintain cellular and tissue homeostasis. Both cigarette smoke and air pollution are known to cause mitochondrial dysfunction contributing to the pathogenesis of chronic inflammatory lung diseases27–29.
Air pollution
Outdoor air pollution is a significantly risky factor to global morbidity and is responsible for 3.3 million premature deaths annually worldwide. Traffic-related air pollution is one of the most significant sources of outdoor air pollution exposure in many countries30,31. Emerging epidemiological studies30,32,33 demonstrate a strong
association between airborne pollutant levels and COPD exacerbations requiring hospitalization, indicating that COPD patients may be more susceptible to the harmful effects of pollutant exposure than the general population. According to World Health Organization (WHO), the major outdoor air pollution sources include vehicles, power generation, building heating systems, agriculture waste and industry4,34–39. Although ambient air pollution has well-documented adverse
effects on patients with COPD40, the evidence for air pollution causing COPD is not
conclusive41. Moreover, the emphysema seen with tobacco smoking has not been
described as being causally linked to ambient air pollution, thus, it is essential to further establish any links between air pollution and COPD.
Current treatments for COPD
As COPD is affecting around 300 million people globally and representing a leading cause of death worldwide, COPD is a clinical problem with increasing interests. The therapeutic intervention with the greatest impact on COPD is smoking cessation. Inhaled corticosteroids (ICS) and acting bronchodilators, including long-acting b-agonists (LABA) and long-long-acting muscarinic antagonists (LAMA) are the mainly used as pharmacological therapy to prevent acute exacerbations of COPD, reduce symptoms and minimize the rate of lung function decline. A combination of inhaled drugs is often required for symptomatic patients, and the combination of LAMA/LABA is proven to be more effective at reducing exacerbations than either bronchodilator alone. The combination of ICS/LABA inhaler also has been used in patients with exacerbations42. Additionally, triple inhaler therapy (ICS/LABA/LAMA)
is shown to be superior compared with dual combinations in reducing the risk of moderate or severe COPD exacerbations. At present, Budesonide43–45 is a commonly
used medication to treat lung inflammation that is believed to remain primarily in the airways, however, it is associated with side effects such as bruising easily, headache and diarrhea increase. Roflumilast, the only phosphodiesterase 4 (PDE4) inhibitor46 currently approved for the treatment of COPD has also been approved as
an alternative maintenance treatment in COPD patients47. It is an oral medication
that decreases inflammation in the lungs, but still is associated with adverse effects such as headaches and gastrointestinal side effects. In addition, GOLD (Global Initiative for Chronic Obstructive Disease) recommends as alternative option to use an antioxidant agent such as N-acetylcysteine, which is a thiol-containing drug that functions as a mucolytic and an anti-inflammatory drug and for which modest effects on reducing COPD exacerbation frequency have been reported48–51. All these
pharmacological therapies for COPD are used to reduce symptoms, the frequency and severity of exacerbations, and improve exercise tolerance and health status; however, there is no medication that effectively modifies the long-term decline in lung function currently.
Lung repair in COPD
Humans can survive only for several minutes without oxygen that is contained in the air and the lungs are the main organ of the respiratory system and function to exchange gas, taking up oxygen and removing carbon dioxide52. The respiratory
system is proximally to distally anatomically composed of the trachea, bronchi, bronchioles and alveoli, which is the main site for gas exchange and host defense functions. The upper airway epithelium consists mainly of ciliated cells, club cells, goblet cells and undifferentiated basal cells, whereas the alveolar epithelium consists mainly of a layer of alveolar epithelial type 1 and type 2 (AT1 and AT2) cells53,54. The
pulmonary anatomical and cellular composition of mice and humans share both similarities and critical differences with respect to structure and function55. Based
on the same cell specific marker genes, many cellular counterparts of the murine lung have been identified in the human lung such as AT2 cells55–57.
The lung epithelial cells are in direct contact with the outside environment. They are exposed to inhaled particles as well as pathogens and once damaged, these epithelial cells must be replaced rapidly by the resident stem or progenitor
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populations. Understanding the populations of stem cells activated under different injury conditions and understanding the role of lung resident stem or progenitor cells in the maintenance of the epithelium during lung homeostasis and repair should provide novel insights into therapeutic approaches for treating COPD. Alveolar repair
Alveolar epithelial type 2 (AT2) cells are playing essential roles in response to alveolar injuries as the main progenitors within the alveolar niche. About 95% of the alveolar surface area is covered by flat AT1 cells that die by apoptosis upon injury and the cuboidal AT2 cells are more resistant to injury. Once AT1 cells are injured, adjacent AT2 cells are triggered to self-renewal and transdifferentiate into AT1 cells58. In addition to AT2 cells, the alveolus also contains a myriad of mesenchymal
and myeloid cells that help alveolar repair. Both alveolar epithelial cells and the surrounding mesenchymal cells (Figure 2) seem to upregulate ligands and receptors in response to injury, allowing for increased, directed cell-cell interactions in the alveolar niche58–60.
Figure 2. Scheme of lung alveolar structure covered by different types of cells.
Cellular signaling pathways regulating lung repair
It is likely that activation of multiple molecular signaling pathways is required to stimulate lung regeneration61. Molecular signaling pathways, such as WNT, FGF,
TGF-b/BMP signaling pathways, have been identified to promote alveolar epithelial cell proliferation and differentiation62,63. Understanding the molecular regulation of
alveolar regeneration is of high clinical importance as it may generate strategies for drug development.
WNT signaling pathway in lung repair
WNT signaling has emerged as a fundamental growth control pathway that drives most types of tissue stem cells in adult mammals64. This signaling pathway contains
a large family of conserved secreted proteins activating intracellular signaling cascades, which is classically categorized in two types based on the downstream effectors of those proteins: 1) The canonical WNT signaling pathway (b-catenin dependent); and 2) The non-canonical WNT signaling pathway (b-catenin independent)62,65,66. WNT proteins (19 in human) comprise an important class of
glycoproteins that are essential ligands for repair activating the signaling by binding specific cell surface receptors including frizzled (FZD), and lipoprotein receptor related proteins (LRP), as well as atypical receptors, such as the receptor tyrosine kinase-like orphan receptors (ROR)67–69.
Accumulating evidence demonstrates that WNT/b-catenin signaling regulates progenitor cell fate decision during lung development as well as after lung injury62,70.
Specifically in the distal lung, WNT signaling plays a critical role in the development of both the surfactant-producing alveolar epithelial type 2 (AT2) cells and the AT1 cells that for the gas-exchange surface of the lung alveolus56,71–73. Although the WNT
signaling pathway is involved in the promotion of the proliferation and expansion of regenerative cell lineages, and is extensively indicated to be activated in response to injury, however, the regeneration of the injured lung seems to be insufficient to repulse some pulmonary pathological conditions74. The canonical
WNT/b-catenin signaling is reduced in the lung epithelium linked to COPD, and enhanced expression of non-canonical WNT-5A has been reported in experimental models of COPD and human COPD tissue specimens75. In addition to WNT-5A, WNT-5B has
been reported increased in airway epithelium of COPD patients76, and the WNT-5B
signaling induces inflammatory responses in human lung fibroblasts77. Moreover,
distal lung epithelial progenitor cells derived from the mouse model of elastase-induced emphysema exhibited reduced organoid forming capacity, whereas the activation of WNT/b-catenin signaling largely restored the reduction78.
FGF signaling pathway in lung repair
Fibroblast growth factor (FGF) signaling is another critical signaling pathway involved in a wide range of biological processes including homeostasis, repair and metabolism. Moreover, niche-derived paracrine signals modulate the behavior of AT2 cells, and FGF signaling is of particular importance. There are 23 known FGFs in both human and mouse that function in a paracrine or autocrine manner binding
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one of the FGFRs (FGFR1-4), thereby initiate the signaling pathway79. Among all
FGFs, FGF2, 7, 10 are mostly studied in respiratory studies. FGF2, also known as basic fibroblast growth factor (bFGF), which is a potent mitogen for fibroblasts that has been shown to be overexpressed in severe asthma and COPD80. Conversely,
FGF2 was decreased in the lungs of cigarette smoke-exposed mice in line with a significant decrease in COPD patients81. FGF7 tends to be upregulated in lung tissues
from COPD patients82 but has been reported together with FGF10 as growth factors
secreted from fibroblasts preventing fibrosis and promoting the differentiation and maintenance of the alveolar epithelium83. Intriguingly, it has been demonstrated
that FGF7 alone is insufficient to induce alveolar organoid formation in mesenchymal cell-free organoid culture system, implying additional factors are needed84.
TGF-β/ROCK signaling in lung repair
Aberrant tissue repair and remodeling leading to a disturbed extracellular matrix (ECM) homeostasis are features of COPD development. Lung fibroblasts are the principal cells in ECM homeostasis regulation and transforming growth factor TGF-b is the main inducer of ECM production in lung fibroblasts, which has been reported elevated levels in COPD85. TGF-b has been reported as the most potent factor for
inducing myofibroblast differentiation in fibrotic lungs86–88. Myofibroblasts express
a-smooth muscle actin (a-SMA), which is a marker of activated fibroblasts, and are capable of ECM production including collagen, laminin, and fibronectin86,87.
TGF-b not only regulates fibroblast differentiation, but also regulates epithelial differentiation as well as the epithelial-mesenchymal interactions86. TGF-b induced
myofibroblast differentiation results in WNT/b-catenin pathway skewing and impairs fibroblast ability to support epithelial repair88. Interestingly, overexpressed
TGF-b can activate the Rho/ROCK signaling pathway, which was observed in the occurrence and development of pulmonary diseases89. ROCK1 and ROCK2 are two
isoforms of the Rho-associated coiled-coil-forming protein kinase (ROCK), which play essential roles in many cellular responses to injury such as rearrangements of actin cytoskeleton. Pharmacological inhibition of ROCK using nonselective ROCK inhibitors has been shown to prevent fibrosis in animal models, however, little is known about the specific roles of each ROCK isoform90.
Pulmonary drug discovery
Many of the new pulmonary drugs that have failed in the clinical due to safety or efficacy. The absence of predictive models and tools to model lung progenitor cell function resulted in the failure of promising drug candidates from animal models to humans91. Most in vitro drug screenings were done on 2D cell-based assays,
however, although cultured cell systems can provide some information, they do not recapitulate the complex interactions between different cell types and tissues in vivo. A considerable unmet medical need remains for defective lung repair in COPD and the current approach to drug discovery is insufficient to meet the needs of the industry and patients. Recently, several new methods have been added to the portfolio of the pharmacologist and cell biologist including lung slices and the organoid, which better recapitulate the progenitor cell and its functions.
Precision-cut lung slices to model lung repair
Precision-cut lung slices (PCLS)92–94 represents an in vitro technology that is becoming
increasingly used in laboratories as an assay to study pulmonary physiology and pathogenesis. The cell types and extracellular matrix composition in the slice accurately reflect the physiological situation, and for that reason, PCLS have an advantage over cell models that are often used in drug development95. PCLS provide
emerging and exciting opportunities to advance chronic lung diseases research and to validate novel therapies, and they can be generated from explanted human lung, diseased human and animal models of disease to explore molecular and cellular interactions in disease, which also reduces the number of animal required95,96.
Crucially, PCLS contain intact alveoli rather than monolayers of one or two cells types; and cell types are present in the same ratios and with the same cell-cell and cell-matrix interactions as in vivo97. To improve translatability understanding,
human PCLS form healthy and diseased patients have been utilized as an ex vivo tool to study pulmonary diseases including asthma, COPD and Idiopathic pulmonary fibrosis (IPF)94. Recently, precision-cut lung slices imaging (PCLSi) is also widely
used, and when in combination with pharmacological treatments, more dynamic interactions between cell-cell and cell-matrix can be easily visualized97.
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Organoids model lung repair
Organoids are stem cell derived 3D structures that are supported by an extracellular matrix and contain multiple cell types whose spatial arrangement and interactions mimic those of the native organ98–101. Organoids are self-organizing units that mimic
the physiological state of the organ of origin at the morphological and molecular levels, offering a convenient means to study development, organogenesis and stem niches102–105. The stem cell niche is the microenvironment where stem cells reside,
and it assists stem cells to self-renewal and differentiation for tissue homeostasis and restoration from injury106. Moreover, organoids represent a powerful tool to
study cell-cell interactions107 within the stem cell niche and allow the modeling
of various pathological states of environmental or genetic origin that can be easily manipulated in vitro for the exploration and validation of new therapeutic approaches100,102,108,109. Organoids provide a platform to investigate the stem cell
function in vitro and have been utilized for drug screening as well103. Organoid
drug-screening assays generate reproducible high quality drug sensitivity data that positively correlate with physiological responses and achieve reproducible activity of compounds inhibiting the same target105.
In this thesis, we used a lung organoid assay88,110 of alveolar epithelial cells (EpCAM+/
CD31-/CD45-/cells) co-cultured with mesenchymal fibroblasts embedded in Matrigel based on previous publications60,111,112. The presence of mesenchymal cells
is essential not only for supporting the growth of alveolar epithelial progenitors but also determines whether the effects of added treatments on the progenitor cells behavior are direct or indirect.
Scope of the thesis
As mentioned above, current COPD medications provide insufficient inhibition of lung function decline, and a regenerative pharmacology approach with the potential to slow down lung function decline by promoting repair would be a major step forward. This thesis uses different disease models to mimic individual features of COPD pathology to investigate the mechanisms of either cigarette smoke or air pollution induced defective alveolar epithelial repair. The ultimate goal of this work is to discover novel therapeutic approaches.
Chapter 2 provides an overview of the methodology as well as the application of Precision-cut lung slices (PCLS) in mice. As PCLS can be treated ex vivo to mimic changes associated with pathological conditions, this model creates an important platform for drug screening and represents a strategy to reduce and refine animal studies.
In addition to the PCLS model, recent advances in 3D culturing technology have demonstrated that organoids represent an ideal system to study repair and regeneration56,108,109,113. Canonical WNT signaling plays a critical role in tissue
development and homeostasis, and WNT-5A signaling as well as WNT-5B signaling have been linked to COPD respecitvely75–77. Thus, in Chapter 3, we established an
organoid assay to investigate the impact of non-canonical WNT signaling mediated by WNT-5A/WNT-5B on both alveolar epithelial progenitors, on fibroblasts and on their crosstalk.
In the organoid co-culture system, we previously found88 that the role of
mesenchymal fibroblasts is essential to support alveolar epithelial growth, and multiple molecular signaling pathways are involved in the crosstalk. Accordingly, myofibroblast differentiation induced by TGF-b negatively impacts their support to epithelial cells89. In chapter 4, we examined two novel ROCK inhibitors on the
organoid model consisting of epithelial cells and TGF-b-induced myofibroblasts with the aim of reversing these negative effects of TGF-b.
Cigarette smoke (CS) has a profound impact on lung functions and therefore we aimed to establish a cigarette smoke exposed lung organoid model in chapter 5. To find novel drug targets for COPD in an unbiased manner, we utilized a transcriptomic-guided drug target strategy to identify potential novel targets that restore lung repair.
As cigarette smoke and air pollution represent the most relevant etiologic factors of COPD, we established a diesel exhaust particle (DEP) exposed organoid model in vitro in chapter 6 and aimed to uncover the mechanisms via which DEP results in defective organoid growth. Moreover, we examined the effects of two antioxidants, NAC and MitoQ on the organoid model after exposure to DEP.
Finally, in chapter 7, the findings within this thesis are summarized and discussed, thereby identify open questions and perspectives for future studies.
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