THE ROLE OF BMP-6 IN THE

PATHOGENESIS OF COPD

Wannes Van Hooste Stamnummer: 01207894

Promotor: prof. dr. Ken Bracke

Copromotor: dr. Fien Verhamme

Masterproef voorgelegd in het kader tot het behalen van de graad Master of Medicine in de Geneeskunde

Academiejaar: 2017-2018

Deze pagina is niet beschikbaar omdat ze persoonsgegevens bevat.Universiteitsbibliotheek Gent, 2021.

This page is not available because it contains personal information.Ghent University, Library, 2021.

I

Table of Contents

Samenvatting ............................................................................................................................. 1

Abstract ...................................................................................................................................... 1

1. Introduction ......................................................................................................................... 2

1.1 COPD........................................................................................................................... 2

1.1.1 Definition and burden of disease ........................................................................... 2

1.1.2 Risk Factors .......................................................................................................... 2

1.1.3 Pathophysiology .................................................................................................... 3

1.1.4 Signs, symptoms ................................................................................................... 5

1.1.5 Diagnosis .............................................................................................................. 6

1.1.6 Treatment .............................................................................................................. 7

1.1.7 Comorbidities ...................................................................................................... 10

1.1.8 Mechanism of disease ......................................................................................... 10

1.2 BMP-6 ........................................................................................................................ 12

1.2.1 The TGF-β superfamily ....................................................................................... 12

1.2.2 BMP-6 ................................................................................................................. 13

1.2.3 BAMBI ................................................................................................................. 15

2. Objectives ......................................................................................................................... 15

3. Methods ............................................................................................................................ 16

3.1 Human lung tissue...................................................................................................... 16

3.2 Emphysema quantification ......................................................................................... 17

3.3 Murine model of COPD .............................................................................................. 18

3.4 Preparation of lung homogenate ................................................................................ 19

3.5 RNA extraction and RT-qPCR .................................................................................... 20

3.6 Immunohistochemistry ............................................................................................... 21

3.7 Western Blot............................................................................................................... 21

3.8 Statistical analysis ...................................................................................................... 22

4 Results .............................................................................................................................. 23

4.1 Patients with COPD .................................................................................................... 23

4.1.1 Emphysema quantification .................................................................................. 23

4.1.2 RT-qPCR for BAMBI ........................................................................................... 23

4.1.3 RT-qPCR for BMP-6 ............................................................................................ 24

4.1.4 Correlations ......................................................................................................... 25

4.2 Murine model of COPD .............................................................................................. 26

II

4.2.1 RT-qPCR for BAMBI ........................................................................................... 26

4.2.2 RT-qPCR for BMP-6 ............................................................................................ 27

4.2.3 IHC-P .................................................................................................................. 28

4.2.4 Western Blot ....................................................................................................... 32

5 Discussion ......................................................................................................................... 35

5.1 COPD patients ........................................................................................................... 35

5.1.1 BAMBI ................................................................................................................. 35

5.1.2 BMP-6 ................................................................................................................. 35

5.2 Murine model of COPD .............................................................................................. 36

5.2.1 BAMBI ................................................................................................................. 36

5.2.2 BMP-6 ................................................................................................................. 36

5.3 Conclusion ................................................................................................................. 38

6 Acknowledgements ........................................................................................................... 39

7 Bibliography ...................................................................................................................... 40

1

Samenvatting

Chronic Obstructive Pulmonary Disease (COPD) is wereldwijd één van de belangrijkste oorzaken

van mortaliteit en morbiditeit en was in 2010 de op drie na belangrijkste doodsoorzaak wereldwijd.

Het wordt gekenmerkt door een niet reversibele obstructie van de luchtwegen, alsook door

luchtweginflammatie en emfyseem of destructie van het longparenchym. Eén van de belangrijkste

oorzaken van COPD is roken. Totnutoe is er geen curatieve therapie voor COPD voorhanden.

Daarnaast is de pathogenese van COPD onvolledig opgehelderd en is er nood aan beter begrip

van de betrokken mechanismen, hetgeen op zich aanleiding kan geven tot de ontwikkeling van

curatieve therapieën. In deze thesis onderzochten we twee leden van de TGF-β superfamilie,

namelijk BMP-6 en BAMBI omdat een Genome-Wide Association Study BMP-6 correleerde aan

Forced Vital Capacity (FVC). BMP-6 is vooral gekend als regulator van hepcidine.

Gebruikmakende van een muismodel voor COPD alsook van humane longweefselstalen

onderzochten we de mRNA expressie en lokalisatie en proteïne expressie van BMP-6 door middel

van respectivelijk RT-qPCR, immunohistochemie en western blot. We vonden een verlaagde

expressie van BMP-6 mRNA in longweefsel van humane COPD-patiënten en aan rook

blootgestelde muizen. De BMP-6 mRNA expressie correleerde significant met de Forced

Expiratory Volume in 1 second (FEV1), de Tiffeneau index (FEV1/FVC) en het aantal pakjaren.

BMP and activin membrane-bound inhibitor (BAMBI) is een pseudoreceptor die leden van de TGF-

β superfamily uit de circulatie haalt door ze als receptor te binden zonder een signaalfunctie te

hebben. De mRNA expressie van BAMBI werd onderzocht in longweefsel van aan rook

blootgestelde muizen en patiënten met COPD. Terwijl de mRNA expressie van BAMBI niet

verschilde in patiënten met en zonder COPD, was de BAMBI mRNA expressie in aan rook

blootgestelde muizen significant lager in vergelijking met gezonde muizen.

Abstract

Chronic Obstructive Pulmonary Disease is characterized by a not fully reversible airflow

limitation caused by obstruction of the small airways and emphysema. The main cause for

COPD is the smoking of tobacco. It is one of the most prominent worldwide causes of morbidity

and mortality. Up to today, there is no cure and the only action one can to take that influences

the long-term lung function decay is smoking cessation. The pathogenesis of COPD hasn’t been

entirely elucidated and only partial explanations and mechanisms have been proposed so far.

BMP-6 is a member of the TGF-β superfamily and it is best known as an inducer of hepcidin. A

Genome-Wide Association Study (GWAS) found BMP-6 to correlate to Forced Vital Capacity

(FVC) and as such became worthwhile to investigate as possible partaker in the pathogenesis of

COPD. Using both a mouse model of COPD and human lung tissue of COPD patients, we found

BMP-6 mRNA levels to be decreased in the lungs of COPD patients and smoke-exposed mice.

The BMP-6 mRNA levels correlated significantly with packyears, FEV1 and FVC. We also

investigated the location using immunohistochemistry and protein levels using western blot.

BMP and activin membrane-bound inhibitor (BAMBI) is a pseudoreceptor that binds members of

the TGF-β superfamily and thus takes it out of circulation. We investigated the BAMBI mRNA

levels in both human lung tissue and a mouse model of COPD and saw BAMBI to be

significantly decreased in the murine COPD model.

2

1. Introduction

1.1 COPD

1.1.1 Definition and burden of disease

Chronic Obstructive Pulmonary Disease (COPD) is a heterogeneous(1, 2) disease characterized

by a not fully reversible obstruction of the small airways and by emphysema which is destruction

of the lung parenchyma(3). In 2010, COPD was the fourth leading global cause of death(4) and is

estimated to rise to the third place by 2030(5). It is estimated that 384 million people had COPD

in 2010, with a global prevalence of 11,7%(6). These numbers may underestimate the burden of

COPD, as COPD often is not a primary cause of death but rather a strong contributing factor and

as such it is often excluded from the death certificate. Altogether, COPD amounts to a significant

mortality, morbidity and economic burden.

1.1.2 Risk Factors

There are several known risk factors for COPD, smoking tobacco – passive smoking included -

being the most important one(7). Other risk factors include indoor burning of biomass fuels, air

pollution, age and multiple childhood airway infections(8). Because of augmenting numbers of

daily smokers as well as the occurrence of indoor pollution due to burning biomass fuels in badly

ventilated houses, low-and middle-income countries are at risk of seeing their COPD patients

increased should this trend persist. In most high-income countries however, the prevalence of

daily cigarette smoking is estimated to decline by 2025(9).

It has been estimated that about 15-25% percent of the smokers develop COPD(10, 11). Although

there is a consensus about this percentage, some propose that most smokers will exhibit

phenomenon attributable to COPD if they only have smoked enough and long enough. While

smoking is the most important reason for an accelerated annual decline in lung function, other

factors also play a role(12). A genetic factor is thought to play a key role in individual susceptibility

since it has been observed that people suffering from severe α1-antitrypsin deficiency (AAT)

develop early-onset emphysema, especially in synergy with smoking. AAT however only counts

for 2-3% of the total COPD cases. Other genes associated with a faster decline of FEV1 (Forced

3

Expiratory Volume 1 second; this is the maximum amount of air a subject can breathe out in 1

second after maximum inhalation in a forced attempt. This is an important diagnostic parameter

for COPD; see 1.1.5) have also been proposed, as well as other host factors such as bronchial

hyperreactivity. Smokers exhibiting these traits will have a higher tendency towards developing

COPD as these traits work synergistic with the smoking(12, 13).

1.1.3 Pathophysiology

There is a decent understanding of the disease process that explains the clinical features of

COPD. All smokers have some level of airway inflammation, but COPD patients have an

exaggerated inflammatory response to toxic particles resulting in some of the key findings in

COPD. Those are emphysema, airflow limitation, gas trapping, mucus hypersecretion, pulmonary

hypertension and exacerbations.

An important feature is emphysema. Emphysema is the destruction of the lung parenchyma,

where alveolar walls and capillaries are destroyed. Destruction of alveolar walls makes the volume

of alveolar spaces larger compared to the area of the walls where gas exchange can take place.

As such it was found that the extent of emphysema correlates with diffusion capacity of the lungs

for CO (DLCO), a marker of gas exchange(14). Emphysematous destruction of lung parenchyma

is seen as a part of lung remodelling, a process in COPD where the overall architecture of the

lungs is altered as the disease progresses.

Chronic bronchitis is defined as having a chronic productive cough for at least 3 months per year

for two consecutive years. In the Rotterdam study - a population-based cohort study of subjects

aged 45 or more - it was found that COPD patients with chronic bronchitis have an increased risk

of exacerbations and respiratory mortality compared to subjects without chronic productive

cough(15). A result of chronic bronchitis is mucous overproduction due to goblet cell hyperplasia,

squamous metaplasia and an increased size of bronchial submucosal glands. In normal lungs,

ciliary movement functions as a transporter belt to clear mucous out of the lungs, which is then

coughed up and subsequently swallowed to be degraded in the stomach. This constant upwards

stream functions as a cleansing wave through the airway, expectorating pathogens and toxic

particles. Due to the overproduction of mucous in COPD as well as due to ciliary dysfunction, this

mechanism is compromised. This results in the occurrence of a chronic productive cough(16).

All these factors contribute to one of the key findings in COPD: airflow limitation due to small

airway remodelling. Airway remodelling occurs when architectural changes because of

4

proliferation of certain cells, destruction of tissue and deposition of for instance collagen or

fibronectin. The lumen of the small conducting airways is obstructed so patients experience

dyspnoea and have to put more labour into breathing. Because of the difficult exhaling, residual

air tends to stay behind in the lungs distal of the obstruction so that when exhaling, the airway

proximal of the residue collapses. This mechanism leads to hyperinflation or air trapping. Having

troubles breathing due to airway obstruction also explains why the FEV1 is used as a diagnostic

marker. Decreased exercise capacity and shortness of breath in COPD patients are correlated to

the amount of residual air in the lungs(17). Figure 1 portrays a schematic comparison between

healthy airways (left) and COPD airways (right). As can be seen, the COPD airway is clogged due

to mucous, oedema and smooth muscle cell contraction.

Fig. 1: Schematic comparison between COPD airway (left) and healthy airway (right).

On the left, features of airflow limitation are seen such as excessive mucus, contracted

smooth muscle cells and goblet cell hyperplasia. This attributes to airway and airflow

obstruction.

Another aspect of the clinical features of COPD are exacerbations. Exacerbations are defined as

an acute worsening of respiratory symptoms, resulting in the need for additional therapy or

hospitalisation(6). Frequent exacerbations are related to a decline in quality of life(18), less time

spent outdoors resulting in growing sedentarism(19) as well as being a contributing factor to the

economic burden of COPD(20, 21). Exacerbations can be caused by a variety of triggers such as

infection or change in temperature and air quality (22).

5

To summarise COPD is a chronic disease where the lungs undergo a series of changes resulting

in chronic airway inflammation, excessive mucous production, architectural changes around the

small airways and parenchymateous destruction leading to a troubled gas exchange and the

trapping of air in the distal lungs, as well as dyspnoea and coughing(6).

1.1.4 Signs, symptoms

Early symptoms of COPD include chronic cough and progressive dyspnoea. Together with

excessive mucoid production, these symptoms may vary from day to day. They can precede the

airflow limitation by years, explaining why COPD can be easily overlooked(23). The chronic cough

is often regarded by the smoking patient as an expected consequence of smoking and is as such

only a reason to seek out a physician when it affects the quality of life(24). As a result of mucous

overproduction, the cough can be productive. A large Danish longitudinal study that followed a

random population sample (n=876) found respiratory symptoms (including cough and mucus

production) to be a predictor for later hospital admission for COPD. It also predicted treatment for

obstructive lung disease(25).

Dyspnoea or shortness of breath becomes a more important problem when the disease worsens.

In early disease stages, coughing is responsible for the most loss in quality of life. In the later

stages, cough becomes less important while shortness of breath causes the most lost in quality

of life(24). The impact varies from impeding the ability to perform physical exercise to shortness

of breath when at rest.

The feeling of chest tightness has been accredited to the unpleasant awareness of the breathing

muscles contracting. Due to hyperinflation and obstructed airways, COPD patients have to put

more labour into breathing efficiently. The feeling of chest tightness and dyspnoea can induce

anxiety(26).

In the later stages of the disease, pulmonary hypertension can arise. This is a heightened pressure

in the lung circulatory system, mainly due to hypoxic vasoconstriction. In lungs, well ventilated

areas are well supplied of arterial blood. This is in contrast to other tissue in the body, where areas

low on oxygen will receive better perfusion. Due to an overall bad ventilation, the majority of lung

vessels will become constricted, an increased resistance for blood flow is formed and this

increases the afterload for the right ventricle. When this phenomenon persists, the right ventricle

will become hypertrophic(27). The presence of cor pulmonale had a negative influence on overall

6

survival(28, 29). These studies however were conducted before oxygen therapy was used. Severe

cases of cor pulmonale are less common nowadays and are mostly because of factors not related

to hypoxia(27, 30).

1.1.5 Diagnosis

In the majority of cases, a primary care physician will think about the diagnosis when seeing a

patient at risk who has a chronic cough, which can be a first sign. Physicians who are attentive for

patients at risk can rely on case-finding and will reduce the underdiagnosis of early COPD, as well

as providing an extra incentive to talk about smoking cessation with the patient(31). Another sign

that may be occasionally observed is the flattening of the diaphragm on a regular Rx thorax that

may have been taken for another reason such as pneumonia.

Spirometry is necessary for the diagnosis of COPD. Two spirometry variables are required: FEV1

and FVC. FVC or Forced Vital Capacity is the maximum amount of air a person can force himself

to blow out after maximum inhalation. For both FEV1 and FVC the reference values are adjusted

to gender, weight and height of a person. An FEV1/FVC ratio (sometimes called Tiffeneau index)

less than 70% that doesn’t entirely reverse after the use of a bronchodilator is diagnostic for

COPD(3). The inhalation of a fast-acting sympathicomimeticum is important to differentiate

between asthma, where contrarily to COPD the airflow obstruction is reversible. After diagnosis,

patients can be further stratified into 4 COPD GOLD categories based upon the value of FEV1.

This is shown in figure 2.

7

GOLD classification FEV1/FVC FEV1 (% predicted)

GOLD I <70% ≥80

GOLD II <70% 50-80

GOLD III <70% 30-50

GOLD IV <70% <30

Fig. 2: GOLD classification of COPD stages: An FEV1/FVC (Tiffeneau) index less than

70% that doesn’t reverse after bronchodilator use is diagnostic for COPD. Further

classification is possible according to the value of FEV1 compared to the predicted reference

value(6).

It should be noted that COPD is often considered a heterogeneous disease. On CT scan, different

types of findings are reported such as predominantly emphysematous patients, dominant airway

wall thickening and air trapping dominance(2). The same goes for clinical symptoms, where

different patients may display different sets of symptoms and where the subjective loss of function

does not always correlate with the objective lung function as assessed with spirometry(32). As

such, it is important to perform both the diagnostic tests as well as to assess the severity of one’s

symptoms, as these do not always correlate with GOLD stage(33). There are several validated

questionnaires available to assess symptoms. For dyspnoea, the Medical Research Council

(MRC) dyspnoea scale exists(34). As the symptomatology goes beyond only dyspnoea, other

questionnaires such as the St. George’s Respiratory Questionnaire(35) and the COPD

Assessment Test (CAT)(36) were developed. As the St. George’s counts 76 questions, the CAT

proves more practical for use in daily practice.

1.1.6 Treatment

The most important measure in the treatment of COPD is smoking cessation. To date, it is the

only measure that influences the long-term decline in lung function(37, 38). Pharmacological

interventions can reduce the severity, frequency and impact of exacerbations as well as improve

exercise tolerance and quality of life. Figure 3 shows the treatment guidelines as recommended

by the GOLD initiative(6). In this figure, patients are stratified into 4 categories based upon COPD

GOLD stage, history of exacerbations and symptom assessment. The most basic pharmaceutical

intervention is by using a fast-acting bronchodilator. Fast is the keyword rather than short-or long-

8

acting. This reliever medication is used when the patient experiences difficulty with breathing. Note

that this fast-acting β-mimetic should be available for all groups as reliever medication meaning

the patient can use it when needed. As symptoms worsen or the reliever medication proves to be

insufficient, a Long-Acting Muscarinic Antagonist (LAMA) or Long-Acting β-Agonist (LABA) can

be used. These products have to be taken once or twice daily. Recently, products with a 24-hour

duration of action have been developed. These ultra-LABA’s are more user-friendly and improve

compliance(39).

Patients stratified in group C may need a LABA and a LAMA, or a combination of LABA and ICS.

Combination of drugs proved to have better result on lung function and number of

exacerbations(39-41). For group D triple therapy (LAMA + LABA + ICS) could be an option, as

well as oxygen therapy in severe cases(42). Continuous macrolide antibiotic use has been proven

effective in reducing the number of exacerbations as well as improving the quality of life due to

immunomodulatory and anti-inflammatory effects(43). As mentioned, COPD can present itself in

different ways and the attending physician should always treat the patient and not the disease.

For group D, palliative measures should be considered.

All mentioned products but macrolides are preferably administered by inhalation. Inhalation

compared to systemic administration results in less systemic side-effects. Especially systemic

corticoids should be avoided or be kept short in time(44).

9

Treatment options in COPD C

OP

D G

OL

D s

tag

e

1

2

3

4

Group C

LAMA and LABA OR

LABA and ICS

Group D

LAMA and LABA OR

LABA and LAMA and ICS

Consider oxygen Consider macrolide

≥2 o

r ho

sp

italis

atio

n 1

0

Ex

acerb

atio

n H

isto

ry

Group A

Reliever bronchodilator (SABA)

Group B

LAMA or LABA

mMRC 0-1 CAT <10

mMRC 2+ CAT ≥10

Fig. 3 Treatment options in COPD. Based upon the GOLD 2017 report(6). Different

diagnostic and assessment tools can contribute towards making a choice in treatment

rather than only based upon the COPD GOLD stage. Left, there is the COPD GOLD

stage as determined by spirometry. Below is the patient’s score on the mMRC or CAT

scale, assessing subjective symptoms and dyspnoea. On the right is the exacerbation

history in the last year. One exacerbation leading to hospital admission counts as ≥2

exacerbations. It should be noted that while this figure acknowledges the limitations of

FEV1 as a parameter, this figure is merely a guideline and one should always treat the

patient, not the disease. Abbreviations: Long-Acting Muscarinic Antagonist (LAMA),

Long-Acting β agonist (LABA), Inhalation Corticosteroids (ICS), Short-Acting β agonist

(SABA).

Apart from the smoking cessation and pharmaceutic intervention, patient education is important,

resulting in more compliance. Crucial is the inhalation technique. A lot of mistakes are made and

face-to-face teaching proved to result in the best patient inhalator technique(45). Preventive

measures include pneumococcal and influenza vaccinations to reduce the number or severity of

exacerbations. For influenza vaccination there is evidence that it decreases the frequency of

10

exacerbations(46), for the pneumococcal vaccine there is less proof(47). Both vaccines are

recommended for all COPD patients(6).

In selected cases, surgical intervention by the means of a bullectomy may be opportune. A bulla

is a confluence of emphysematous lesions, forming a large air bulb in the lungs.

1.1.7 Comorbidities

Even when correcting for the smoking history, COPD patients are still at risk for a number of

diseases compared to healthy smokers. These include cardiovascular diseases(48, 49),

depression and anxiety(50), diabetes mellitus(51), osteoporosis (52) and possibly stroke(53). The

physician suspecting COPD in a patient should also assess the risk factors for both COPD and

comorbidities such as smoking status, inactivity and cardiovascular risk factors (54). As most

COPD patients have comorbidities and different clinical presentations, it is important to address

these and evolve to a personalised therapy(54, 55). This includes finding the therapy where both

objective lung function tests and patient satisfaction are maximised as well as working on

prevention of comorbidities.

1.1.8 Mechanism of disease

The classic triad explaining the pathogenesis of COPD consists of inflammation,

protease/antiprotease imbalance and oxidative stress(56). Genetics, autoimmunity(57) and

bacterial colonization(58) could also be implied.

Inflammation is triggered by noxious gas particles such as those in cigarette smoke. The

epithelium fulfils an important defensive role as it also secrets defensins to counter pathogens as

well as functioning as a physical barrier(59). Damage by cigarette smoke leads to necrosis and

the release of damage-associated molecular patterns (DAMPs) such as High Mobility Group Box

1 (HMGB1) (60, 61). These DAMPs bind to Pattern Recognition Receptors (PRRs), leading to the

activation and chemotaxis of the innate immune system by releasing pro-inflammatory

chemokines and cytokines such as IL-1β, IL-6 and IL-8(62). HMBG1 for example binds to TLR2,

TLR4 and RAGE(63) inducing IL-1β and the maturation of dendritic cells(64).

Cells belonging to the innate immune system are attracted to the site of injury, most importantly

neutrophils and macrophages. Main neutrophilic attractants are IL-8 as well as C5a, a member of

11

the complement system(65, 66). Monocytes and macrophages are stimulated by TNF-α, IL-1α

and IL-1β to produce IL-8(67). This leads to an accumulation of neutrophils, being a hallmark in

the genesis of COPD. Neutrophils aid in the clearance of infections, but are also potential harmful

to the lung. By releasing neutrophil proteases into the extracellular environment, neutrophils

degrade extracellular matrix components such as collagen and proteoglycans(68) and as such

attribute to emphysema(69). Neutrophil elastase is known to cleave epithelial cadherin,

compromising the epithelial monolayer integrity and cell-cell junctions(70). Collagenase-2 and

gelatinase B are also involved in breaking down the extracellular matrix(71). Neutrophilic

breakdown of collagen results in collagen fragments that act as a neutrophilic chemotaxant,

leading to chronicity and a self-sustaining cycle of neutrophilic inflammation(72).

Furthermore, the adaptive immune system is essential in the pathogenesis of COPD as well. While

asthma is regarded as a predominantly CD4+ T-cell driven disease, CD8+ T-cells are the main

lymphocyte population in COPD, as seen in CD8+ T-cell-deficient mice that did not develop

emphysema after long-term cigarette smoke exposure(73). In contrast, our research group found

that severe combined immunodeficient (SCID) mice did develop emphysema in response to

chronic cigarette smoke exposure, despite the lack of functional B-and T-cells(75). Systemic CD8+

T-cell count was found to correlate with GOLD disease stage(74). Antigen Presenting Cells

(APCs) such as Dendritic Cells (DCs) activate cells belonging to the adaptive immune system by

presenting them with antigens. CD4+ T-cells are also increased in the airways of COPD patients,

but not as much as CD8+ T-cells. Th17 cells attribute to inflammation by stimulating CD8 cells

and Th1 and Th2 cells, as well as providing a feedback loop upregulating itself via IL21(76).

A protease-antiprotease imbalance also fulfils a key role in COPD. This is nicely illustrated in AAT,

where neutrophil elastase that is unopposed by α1-antitrypsin leads to a net proteolytic activity

resulting in emphysematous lesions in the lung parenchyma(77). However, it has become clear

that the development of emphysema can’t be attributed to a single protease. While Matrix

Metalloproteinases (MMPs) and neutrophils take a prominent place, several proteases are at work

and seem to influence each other(78, 79).

Oxidative stress occurs when the antioxidant defence mechanisms fail or become overwhelmed

by oxidative agents (reactive oxygen species, ROS) (80). There are endogenous sources of ROS

such as ROS generated by mitochondrial respiration or ROS used in antimicrobial defence(81).

ROS are scavenged by antioxidants, neutralizing their potential harmful effect(82). Cigarette

smoke however is a source of ROS and smoking may tip the oxidative/antioxidative balance

favouring oxidative stress, thus leading to cell damage.

12

Inflammation, oxidative stress and protease/antiprotease imbalance are intertwined and influence

each other in their contribution to the genesis of COPD. Oxidative stress for example leads to cell

death thus leading to an increased inflammatory response. Proteolytic cleavage of collagen can

lead to molecules promoting inflammation. This may offer an explanation to why inflammation

persists even after smoking cessation.

Autoimmunity is also thought to be a contributing factor. The CD8+ T-cell repertoire in the lungs

of mice continued to expand oligoclonal, even after smoking cessation(83). Smoke components

may alter autologous proteins, creating neo-antigens. Another explanation might be the loss of

self-tolerance due to repetitive stimulation of the immune system by DAMPs. Elastin-specific

CD4+ T-cells were found, correlating with the total amount IFN-γ(84). Another study found that as

the disease worsens, natural killer cells (NK cells) are more drawn to exhibiting toxicity to

autologous structural lungs cells, suggesting autoimmunity is a late effect of COPD(85).

Tertiary lymphoid follicles arise in tissue that harbours a lot of antigens the body wants to clear.

This can be the case in sites of chronic infection or inflammation. Immunocompetent cells infiltrate

the tissue and organize themselves into a functional entity called a tertiary lymphoid follicle (TLO).

These TLOs have been found in lung tissue of severe COPD patients. The number and size of B-

cell follicles increase with CS exposure time and correlate with the enlargement of the alveolar air

space(86). Both protective as well as harmful properties have been attributed to these TLOs.

Protective because they help clearing infections, harmful because autoreactive immunoglobulins

were found, suggesting a role for autoimmunity and tissue destruction. As autoantibodies were

found to be absent in some studies, it was hypothesized they are only implicated in emphysema

predominant types(87). B-cell activating factor (BAFF) expression was found to be significantly

increased in COPD lungs. BAFF is a major activator of B-cells and plays a role in adaptive humoral

responses. Antagonising BAFF in long-term smoke-exposed mice resulted in an attenuated

inflammatory response and a lesser degree of alveolar destruction(88).

1.2 BMP-6

1.2.1 The TGF-β superfamily

The TGF-β superfamily consists of several highly conserved proteins including bone

morphogenetic proteins (BMPs), growth differentiation factors (GDFs), activins and transforming

growth factor β (TGF-β) itself. Even though the superfamily counts over 30 members, there are a

limited number of receptors. Each receptor complex consists of two type I and two type II elements,

13

with both components being Serine/Threonine kinases. Seven type I and five type II receptors are

known to exist in humans(89). While the role of TGF-β1 itself has been studied well in the context

of COPD, little is known about the other members of the TGF-β superfamily.

Each member of the TGF-β superfamily requires a set of type I and type II serine/threonine kinase

receptors. The receptor complex phosphorylates itself. Upon binding, the small mother against

decapentaplegic (Smad) is phosphorylated by the intracellular part of the receptor complex and

will oligomerise with the costimulatory Smad. The complex then translocates to the nucleus. An

inhibitory Smad works as inhibitor of the phosphorylated complex. There is an overlap between

the receptor complexes and Smads the different members of the TGF-β superfamily use.

1.2.2 BMP-6

The bone morphogenetic proteins are well-known for their role in the bone morphogenesis, having

osteoinductive properties. However, they fulfil regulatory roles in the development and

homeostasis of nearly every tissue(90).

As demonstrated in figure 4, upon binding of BMP-6 on the receptor complex consisting of two

type 1 and two type 2 receptors, Small mother against decapentaplegic 1/5/8 (Smad1/5/8) is

phosphorylated. These Smads 1, 5 or 8 are the R-Smads or receptor Smads and will be

phosphorylated by the type I receptor part. Smad4 is needed as a costimulatory molecule and will

oligomerise with the activated R-Smad. The phosphorylated Smad4-Smad1/5/8 complex then

translocates to the nucleus to regulate gene expression. There is an overlap between the different

type-1 and type-2 receptors BMP-6 can bind upon(91). Smad 6/7 functions as the inhibitory Smad.

They block the phosphorylation of the R-Smad by the receptor complex and promote ubiquitination

and degradation of the receptor complex and in doing so inhibit signalling(92).

14

BMP-6 plays a role in iron homeostasis, as BMP-6 levels were found to positively correlate with

hepcidin levels(93, 94). Increased hepcidin and lower levels of free iron are a defence mechanism

against bacterial infection. Bacterial infection is also thought to contribute to the pathogenesis of

COPD. Higher levels of iron were found in alveolar macrophages of COPD patients, correlating

with emphysema severity(95). This increased iron sequestration in macrophages could occur to

protect the lungs from iron-induced oxidative stress or as result from the bacterial ‘war on iron’. In

the same study, airflow limitation correlated with ferritin, ferritin receptor and transferrin in the

broncheoalveolar lavage fluid of COPD patients as compared to non-smokers and healthy

smokers. This suggests that iron dysregulation plays a role in the pathogenesis of COPD,

reserving a possible role for BMP-6.

There are other diseases where BMP-6 was found to play a role. Enhanced expression of BMP-6

was found to exert an antifibrotic effect, inhibiting hepatic fibrosis in non-alcoholic fatty liver

disease(96). Another display of the antifibrotic effect of BMP-6 is that BMP-6 knockout mice

displayed enhanced fibrosis after kidney injury(97). BMP-6 levels were also found to be

augmented in patients with chronic heart failure(98).

Fig. 4: BMP-6 signalling. Upon BMP-6

binding its receptor, Small Mother Against

Decapentaplegic 1, 5 or 8 (Smad 1/5/8)

will become phosphorylated with the

phosphor from the receptor complex.

Smad 4 functions as common mediator.

After complexing with Smad 4, the

phosphorylated Smad 4 – Smad 1/5/8

complex translocates to the nucleus.

15

As of late, BMP-6 was found in a genome-wide association study (GWAS) to be associated with

forced vital capacity (FVC)(99). Little is known about BMP-6 in relation to COPD. There is some

evidence suggesting BMP’s are involved in airway inflammation in asthma, where BMP-2, -4 and

-6 were found to be inducted and BMP-5 and -7 where downregulated in bronchial epithelial cells

during airway inflammation(100).

1.2.3 BAMBI

BMP and activin membrane-bound inhibitor (BAMBI) is a pseudoreceptor able to inhibit signalling

of TGF-β members. It is a highly reserved transmembrane glycoprotein that resembles type I TGF-

β receptors. However, it lacks a functional intracellular domain, taking TGF-β members out of

circulation by binding them(101). It was found that after infection with non-typeable Haemophilus

influenza, BAMBI was upregulated in COPD patients(102). It was also found that in in vitro

experiments on lung tissue, BAMBI protein levels on CD4+ T-cells and plasma mRNA levels were

elevated in COPD patients compared to healthy smokers and healthy non-smokers. The

enhanced BAMBI levels correlated positively with increased TGF-β1 levels and with an increased

Th17/Treg ratio(103). The hypothesis is that by binding TGF-β and thus taking it out of circulation,

BAMBI drives the lung tissue towards inflammation and compromises tissue repair. This again

supports the theory that BAMBI has a role in the inflammatory status in COPD patients, as Th17

secretes pro-inflammatory cytokines and Treg CD4+ cells secrete TGF-β.

2. Objectives

This study will focus on both BMP-6 and BAMBI. First, we will study the expression and location

of BMP-6 in healthy murine lung tissue and in patients with COPD. We will correlate the BMP-6

levels with lung function parameters. Secondly, we aim to confirm the findings in a cigarette

smoke-induced mouse model of COPD. A similar approach has been used for BAMBI

16

3. Methods

3.1 Human lung tissue

The lung tissue originates from patients who underwent a (partial) lung resection. In the majority

of the cases this is following the diagnosis of a solid lung tumour. In the more severe COPD cases

(COPD GOLD IV), tissue was obtained after lung transplantation. All patients were categorized

using the criteria supplied by the Global Initiative for Chronic Obstructive Lung Disease (GOLD).

All subjects provided their written informed consent according to protocols approved by the Ghent

University Hospital Ethical Committee. The pathologist harvested tissue as far as possible from

the tumoral lesion and without signs of pneumonia. Table 1 and table 2 show the characteristics

of the patient populations used for respectively the RT-qPCR for BAMBI and the RT-qPCR for

BMP-6.

Table 1: Characteristics of the human study population used for the RT-qPCR for BAMBI (n=92)

never-smokers healthy smokers COPD GOLD

II COPD GOLD IV

Number 18 26 34 14

Gender ratio (male/female) 6/12 # 19/7 # 31/3 # 8/6 #

Age (years) 65 (56-70) 63 (55-70) 66 (58-69) ç 56 (54-60)* ç ²

Current- / ex-smoker - 16/10 22/12 0/14

Smoking history (PY) 0 (0-0) 28 (15-45)* 45 (40-60)* ç 30 (25-30)*²

FEV1 post (L) 2,7 (2,3-3,2) 2,7 (2,3-3,3) 2,0 (1,8-2,4)* ç 0,7 (0,7-0,9)* ç ²

FEV1 post (% predicted) 102 (92-116) 95 (93-112) 68 (61-75)* ç 26 (20-32)* ç ²

FEV1 / FVC post (%) 78 (75-83) 75 (71-79)* 56 (53-60)* ç 32 (27-35)* ç ²

DLCO (% predicted) 90 (80-105) 80 (61-102) 67 (51-87)* 35 (33-41)* ç ²

KCO (% predicted) 103 (88-123) 91 (68-107)* 87 (62-108)* 59 (50-65)* ç ²

ICS (yes/no) 0/18 # 1/25 # 15/19 # 13/1 #

17

Table 2: Characteristics of the human study population used for the RT-qPCR for BMP-6 (n=84)

never-smokers healthy mokers

COPD II COPD IV

Number 16 24 30 14

Age (years) 64 (51-71) 65 (55-71) 65 (59-69) 56 (54-60) * ç ¶

Gender (m/f) 3/13# 19/5# 29/1# 8/6#

Current-smoker/Ex-smoker NA 12/12# 17/13# 0/14#

Pack-years NA 33 (15-50) † 45 (40-60) † ç 30 (25-30) †¶

FEV1 (% predicted) 110 (92-118) 96 (92-113) 69 (64-74) †§ 23 (20-33) †§¶

FEV1/FVC (%) 78 (75-83) 76 (73-78) 56 (53-60) †§ 32 (27-35) †§¶

DLCO (% predicted) 88 (107-82) 85 (105-74) 73 (87-54) 35 (41-33) ²†§

KCO (% predicted) 95 (116-86) 95 (104-81) 94 (108-69) 56 (65-50) ²† ç

ICS (yes/no) 0/16# 1/23# 13/17# 13/1#

Table legend:

Smoking history expressed as Packyears (PY). This is the equivalent of smoking 1 pack of cigarettes a day

during a year. FEV1 post (L) is the Forced Expiratory Volume in 1 second post bronchodilator, expressed in

liter. FEV1 post (%predicted) is the FEV1 post bronchodilator expressed as percentage reached as

compared to the age-, weight- and gender adjusted reference value. FEV1/FVC post % or Tiffeneau. FVC is

the Forced Vital Capacity or maximum volume one can exhale after bronchodilation and maximum

inhalation. Dlco (% predicted) or Diffusing Lung Capacity for CO is a measure for gas exchange between

lung and blood, expressed as percentage reached of one’s reference value. Kco (% predicted) or CO

transfer coefficient is a measure of the efficiency of alveolar transfer, expressed as percentage reached of

one’s reference value. ICS (yes/no) refers to whether a patient takes Inhalation Corticosteroids.

Indices:

#: Fisher’s exact test, P < 0.001

*: Mann-Whitney-U test, P < 0.05 versus never smokers ç: Mann-Whitney-U test, P < 0.05 versus smokers without COPD

²: Mann-Whitney-U test, P< 0.05 versus COPD GOLD II

†: Mann-Whitney-U test, P<0.001 versus never smokers

§: Mann-Whitney-U test, P<0.001 versus smokers without COPD ¶: Mann-Whitney-U test, P<0.001 versus COPD GOLD II

Data are presented as median (IQR)

3.2 Emphysema quantification

Preoperative CT-scans of patients were scored for their degree of emphysema. The extent of

emphysema was graded into five categories: 0%, 1-5%, 6-25%, 26-50% and >75%. The main

reason for this stratification is because of the heterogeneity of COPD patients. Apart from the

18

Tiffeneau index, different CT phenotypes have been described, all presenting with different

subsets of symptoms(2). Different components of COPD influence different parameters.

Emphysematous prominent patients may mostly have a low DLCO, while those with a lot of air

trapping may have the worst FEV1(104). For the scoring of the preoperative CT scans, a workshop

on visual evaluation of COPD was used as guidelines(105). They compared the visual assessment

with the objective quantitative CT scoring. Figure 5 shows two of the reference images they used.

It was found that the visual grading of the extent of emphysema correlates well to the software-

driven quantitative CT measurements(106).

1-5% emphysema 6-25% emphysema

Figure 5: two of the reference images used to visually grade the extent of emphysema. Note

the emphysematous lesions seen as black attenuated zones on both pictures. Taken from

Lynch et al.(105). The red arrows indicate two of the many emphysematous lesions.

3.3 Murine model of COPD

Animal models function as a link between in vitro studies and studies in humans. They allow the

research of in vivo mechanisms in an ethically acceptable way. The murine genome has been

entirely sequenced and offers the possibility of altering their genetic constitution, allowing for a

thorough research of gene and gene function. While being good as an animal model and having

19

many commercial scientific supplies available, it should be remarked that every model has its

drawbacks and is never totally representative for real-life human systems.

Male BMP-6 knockout mice (C57BL/6Jx129Sv) were mated with C57BL/6J female mice. The

offspring was genotyped(107) and male KO and WT offspring were used. Jackson Laboratory

(Bar Harbor, ME, USA) provided the C57BL/6J mice. All mice had access to ad libitum food and

chlorinated tap water. They were housed in sterilised cages in an artificial 12 hour diurnal cycle.

All in vivo manipulations where approved by the local Ethics Committee for animal experimentation

of the Faculty of Medicine and Health Sciences of Ghent University.

The mice are placed in a smoking chamber connected to a smoking apparatus where they are

exposed to mainstream CS of 5 simultaneously lit cigarettes at a rate of 4 times per day for 5 days

per week with 30 minute smoke-free intervals between the smoking sessions(108). The used

cigarettes are standardised (Kentucky Reference Cigarette 3R4F without filter; University of

Kentucky, Lexington, KY, USA). An optimal smoke/air ratio of 1/6 is used. Control mice were

subjected only to air. The duration of exposure was 4 weeks (subacute) or 24 weeks (chronic).

Mice exposed for 4 weeks showed pulmonary inflammation, whereas chronic exposure resulted

in mice exhibiting pulmonary inflammation and hallmarks of COPD such as emphysema,

peribronchial lymphoid follicles and airway remodelling(75, 108).

After sacrificing the mice by means of an intraperitoneal overdose pentobarbital, lung tissue was

collected for studying. For the lung homogenate, the middle lobe of the right lung is used for

homogenisation. The tissue is snap-frozen using liquid nitrogen to prevent water from forming

crystals when freezing and damaging the sample. It was then stored at -80°C until further analysis.

The small lobe of the right lung is frozen in RNA-later (Qiagen). The left lobe is used for

immunohistochemistry.

3.4 Preparation of lung homogenate

For homogenisation of lungs, the tissue samples were transferred to tubes containing 1 mL RIPA

buffer (Cell Signalling Technology, Danvers, USA) which also contained Halt™ Protease Inhibitor

Cocktail Kit (Thermo Scientific, Waltham, MA, USA) to inhibit endogenous protease activity.

TissueRuptor (Qiagen, Hilden, Germany) was used for tissue disruption and homogenisation.

Samples were next sonicated for enhanced detection of nuclear proteins (4 times for 5 seconds)

and centrifuged (14000 rpm for 10 minutes at 4°C). Subsequently the middle layer was transferred

20

to microcentrifuge tubes. All steps were performed on ice. Total protein concentration was

measured using the Pierce™ BCA Protein Assay Kit (Thermo Scientific). This is an assay to

measure the protein concentration compared to a standard.

3.5 RNA extraction and RT-qPCR

The miRNeasy Mini kit from Qiagen was used to extract RNA from both human and mice lung.

First, tissue stored in RNA-later was added to QIAzol Lysis Reagent (Qiagen, Hilden, Germmany).

This is a phenol/guanidine thiocyanate used to facilitate lysis of tissue, to inhibit RNase and to

extract most of the DNA and proteins. Next, the TissueRuptor (Qiagen, Hilden, Germany) was

used to homogenise the samples for 30 seconds each. After 5 minutes incubation to dissociate

nucleoprotein complexes, chloroform was added. The samples underwent centrifugation at

12000g for 15 minutes at 4°C. This separates the homogenate into aqueous and organic phases.

The upper aqueous phase holds the RNA while DNA partitions to the middle phase and proteins

are in the organic lower phase or interphase. The upper phase is extracted and brought on the

RNeasy Mini spin column along with 100% ethanol. Here, RNA binds to the column. After 15s

centrifugation at 8000g, the contaminants are washed away and thrown away. RWT and RPE

buffers are used to further wash the column, with 15s centrifugation between each step. In the

end, RNA is eluted with RNase-free water. cDNA is obtained using the Transcriptor Universal

cDNA Master Kit (Roche, Basel, Switzerland). Reverse transcriptase is used to create

complemental DNA from the isolated mRNA. The solutions include the necessary nucleotides to

build in, a buffer and random hexamer primers.

To investigate the RNA levels, RT-PCR for BAMBI and BMP-6 on human and murine samples

was performed. Samples from different subjects were used including never smokers, healthy (ex-

)smokers, COPD GOLD II patient and COPD GOLD IV patient (see table 1 and table 2).

Housekeeping genes GAPDH, HPRT1 and SDH were used as loading control on human samples,

GAPHD, HRPRT1 and TFRC were used on the murine samples.

The expression of BAMBI and BMP-6 relative to the three mentioned household genes is

measured using TaqMan Gene Expression assays which includes specific primers and

fluoregenic mix (Applied Biosystems, Halle, Belgium). First there is a 10 minutes denaturation

phase at 95°C, followed by 50 cycles of 10 seconds at 95°C and 15 seconds at 60°C. Serial

dilutions of a mixture of all samples are used to create a standard curve. The cycles are performed

by a Lightcycler 96 detection system (Roche).

21

3.6 Immunohistochemistry

The localisation of BMP-6 in lung tissue was studied using immunohistochemistry on paraffin (IHC-

P). First, paraffin-embedded sections mouse lungs were dewaxed using xylene followed by

rehydrations. Next, antigen retrieval was performed using a citrate buffer. After blocking for

endogenous peroxidase activity because HRP conjugated antibodies were used, an additional

block (1109616001 Roche Blocking Agent, Sigma-Aldrich) was applied. Next, the specimens were

incubated during 24 hours with either anti-BMP-6 (see table 3 for used antibodies) or isotype rabbit

monoclonal IgG (see table 3). After this, sections were rinsed in PBS with 0,3% Triton (Triton

X100, Sigma) and incubated during 30 minutes with polymer HRP anti rabbit (Klinipath). After

rinsing again with 0,3% Triton PBS, diaminobenzidine substrate (DAB+, DAKO) was applied,

followed by counterstaining with Mayer’s haematoxyline (Sigma-Aldrich), dehydrating and

mounting on a medium (DPX, Prosan).

Table 3: antibodies

Target Company Code Technique Dilution

BMP-6 Abcam ab155963 IHC-P/WB WB 1:1000/IHC-P

1:4000

BMP-6 Biorbyt orb155848 IHC-P/WB WB 1:250/IHC-P

1:500

BMP-6 Acris AP20577U-N WB WB 1:750

β-actin Sigma-Aldrich A2066 WB 1:1000

GAPDH-HRP Cell Signalling

Technologies

14C10 WB 1:1000

Rabbit isotype Abcam ab125938 IHC-P (with Abcam

ab155963))

1:250

Rabbit isotype Abcam Ab27478 IHC-P (with Biorbyt

orb155848)

1:250

Table 3: the different antibodies used for Western Blot and/or immunohistochemistry

3.7 Western Blot

After preparation of the lung homogenate as stated in 3.4, western blot for BMP-6 was performed

on murine samples. Specific quantities of samples with known protein concentrations were

calculated and were added to NuPAGE LDS Sample buffer (Thermo Fisher) and were denatured

for 10 minutes at 70°C. Each sample contained 30 µg of protein. NuPage Reducing Agent was

added to cleave disulphide bonds and ensure no tertiary or quaternary structure remained.

NuPage SDS Running buffer was used to run the electrophoresis using the Xcell II (Thermo

Fisher) with NuPage Bis-Tris 10% protein gels. Apart from the samples, SeeBlue™ Plus2 Pre-

22

Stained Protein Standard was loaded into a lane as molecular weight reference. During blotting,

proteins were transferred to PVDF membrane (Novex PVDF Membrane Filter Paper Sandwich

0,2 µm pore size) using the same Xcell II. NuPage Transfer Buffer with NuPage Antioxidant was

used to run the blotting and keep proteins in a reduced state.

For immunodetection, the WesternBreeze™ Chemiluminescent kit (Thermo Fisher) was used. A

blocking solution was used to reduce nonspecific binding and background noise

(Westernbreeze™ Blocker/Diluent A and B). Membranes were incubated with the primary

antibody overnight (see table 3 for used antibodies). After primary antibody incubation, wash steps

(Westernbreeze™ Wash Solution) were performed. Next, the membranes were incubated with

the secondary anti-rabbit antibody for half an hour followed by wash steps. After being treated with

a chemiluminescent substrate (alkaline phosphatase substrate) or HRP, results were visualised

using the Chemidoc system (Bio-Rad, Hercules, USA). Image Lab software was used to analyse

the blot pictures (Bio-Rad, Hercules, USA). The quantitative value of the lanes was normalised

against β-actin or GAPDH (see table 3) in the same blot, resulting in relative quantities and

allowing statistical analysis.

3.8 Statistical analysis

Statistical analysis was performed with SPSS (IBM Corporation) using Mann-Whitney-U test,

Spearman correlation and Kruskal-Wallis test. P-values <0.05 were considered significant (*).

23

4 Results

4.1 Patients with COPD

4.1.1 Emphysema quantification

Figure 6.A: correlation of COPD classification

with the emphysema scoring. Each dot

represents several patients. n=28. P=0.004.

Figure 6.B: correlation of DLCO with

emphysema scoring. n=26. P>0.05.

Figures 6.A and 6.B represent correlations for the emphysema scoring with respectively COPD

classification and DLCO. The correlation with COPD classification was significant. Sadly, no

emphysema scoring of severe COPD (GOLD III-IV) was could be obtained.

4.1.2 RT-qPCR for BAMBI

The mRNA expression of BAMBI in human lung tissue was analysed by RT-qPCR in 92 subjects

(patient characteristics in table 1). The mRNA levels of patients stratified into four groups were

compared. No statistical significant differences between the groups were found (P>0.05). The

results are displayed in figure 7.

24

Figure 7: RT-qPCR for BAMBI on human

lung. RT-qPCR results of patients stratified

into four categories (never smokers, healthy

(ex-)smokers, COPD GOLD II patients and

COPD GOLD IV patients). No significant

difference between the different groups was

found (P=0.961; Kruskal-Wallis).

4.1.3 RT-qPCR for BMP-6

To investigate the BMP-6 mRNA levels in human lung tissue, a RT-qPCR was performed on lung

tissue (n=84), harvested as stated above (see 3.1). See table 2 for patient characteristics

(appendix)

Figure 8: RT-qPCR for BMP-6 on human

lung tissue. The x-axis shows four groups:

never smokers, current smokers without

COPD, COPD GOLD II ex-smokers and

COPD GOLD IV ex-smokers. *p<0.05,

**p<0.01, ***p<0.001

Quantification of BMP-6 mRNA levels in human lung tissue in figure 8 showed that the BMP-6

mRNA levels in never smokers were significantly higher compared to smokers, COPD GOLD II

and IV patients. The mRNA levels in lung tissue of healthy smokers were significantly higher

25

compared to COPD GOLD IV patients. Importantly, patients with very severe COPD have the

lowest levels of BMP-6.

4.1.4 Correlations

Using the BMP-6 mRNA levels we found, we performed some correlations to investigate the

relation of BMP-6 levels to lung function parameters and packyears (PY). Figures 9.A-9.F show

the results. Significant results were found for the correlation of BMP-6 mRNA levels with FEV1,

FEV1/FVC and PY.

Figure 9.A: r= 0,4099 (P=0,0002). n=78 Figure 9.B: r= 0,438 (P<0,0001). n=77

Figure 9.C: r= 0,01945 (P=0,8879). n=55 Figure 9.D: r= 0,2177 (P=0,1069). n=56

26

Figure 9.E: r= -0,1648 (P=0,4020). n=28

emphysema score: 0=0%, 1=1-5%, 2=6-25%,

3=26-50%, 4=>75% emphysema

Figure 9.F: r= -0,4403 (P<0,0001). n=76

4.2 Murine model of COPD

4.2.1 RT-qPCR for BAMBI

Figure 10: RT-qPCR for BAMBI on cigarette

smoke exposed mice for 1 month exposure

and 6 months exposure. There was a

significant (p<0,05) decrease of BAMBI

mRNA levels from both smoke exposure

mice groups compared to their air exposed

equivalents.

Figure 10 shows the RT-qPCR for BAMBI in mice. Two pairs of categories were compared: 1

month air exposure and 1 month smoke exposure, 6 months air exposure and 6 month smoke

27

exposure. In both pairs a significant difference was found, with BAMBI being decreased in smoke-

exposed mice.

4.2.2 RT-qPCR for BMP-6

Figure 11: RT-qPCR for BMP-6 on murine

lungs. Two sets were compared: one

where the mice were exposed to air or

smoke for 1 month and one where they

were exposed for 6 months. **p<0.01

Figure 11 shows the RT-qPCR for BAMBI on air-exposed mice versus smoke-exposed mice in

two groups. In the group where the mice were exposed for 1 months, no significant difference in

BMP-6 mRNA levels was found between air-exposed and smoke-exposed mice. In mice that were

exposed for 6 months, a significant decrease (p=0.0047) in BMP-6 mRNA levels was found in

smoke-exposed mice compared to their air-exposed counterparts.

28

Figure 12: RT-qPCR for BMP-6 on BMP-

6 knockout mice. ****p<0.0001

To determine the BMP-6 deficiency of BMP-6 knockout mice, we also performed a RT-qPCR on

the knockout mice. The results are displayed in figure 12. There was nearly no expression

measured of BMP-6 mRNA in the knockout mice (p<0.0001).

4.2.3 IHC-P

BMP-6 expression and localisation was studied in mouse lungs using immunohistochemistry

staining. In order to validate the antibody’s specificity for BMP-6, we performed knockout validation

for three antibodies (see table 3). Each antibody was tested on wild type mouse and on BMP-6

knockouts. For each there also is an isotype control. BMP-6 was mainly detected in the smooth

muscles around bronchioles and blood vessels. Figure 13 and figure 14 show the results for

respectively the Abcam anti-BMP-6 antibody and the Biorbyt anti-BMP-6 antibody (see table 3 for

antibodies). As seen in figure 13.B and 14.B, the BMP-6 knockout mice show a colouring pattern

similar to the wild types in 13.A and 14.A.

29

Figure 13: BMP-6 immunohistochemistry with antibody from Abcam

13.A: wild type with detail

13.B: BMP-6 knockout with detail

A

B

30

13.C: wild type isotype control 13.D: knockout mouse isotype control

Representative photomicrographs of immunohistochemical stainings for BMP-6 on lung tissue

with close-up of a wild type (fig. 13.A) and BMP-6 knockout mouse (fig. 13.B) with their

corresponding isotype controls. In 13.C and 13.D, isotype controls are displayed.

C D

31

Figure 14: BMP-6 immunohistochemistry with antibody from Biorbyt

14.A: wild type mouse with detail

14.B: BMP-6 knockout with detail

A

B

32

14.C: wild type mouse with isotype 14.D: knockout mouse with isotype

Representative photomicrographs of immunohistochemical stainings for BMP-6 on lung tissue

of a wild type (fig. 14.A) and BMP-6 knockout mouse (fig. 14.B) with their corresponding close-

ups. In 14.C and 14.D, isotype controls are displayed.

4.2.4 Western Blot

To investigate the BMP-6 protein expression, western blots for BMP-6 were performed on mouse

lung tissue samples. We assessed the specificity of 3 anti-BMP-6 antibodies (see table 3) by using

lung homogenates of wild type and BMP-6 knockout mice. Figures 15 and 16 also show a

household protein blot (β-actin and GADPH respectively) for loading control. This allows for a

quantitative analysis of the BMP-6 expression.

Figure 15: Western Blot for BMP-6 using

the Abcam ab155963 monoclonal anti-

BMP-6 antibody. The first 4 lanes were

loaded with wild type mice samples while

the last 4 lanes were loaded with samples

from BMP-6 knockout mice. The bands are

at the predicted 42 kDa.

33

Figure 15 shows the Western Blot for the Abcam ab155963 antibody. As can be seen on the blot,

both the wild type and knockout mouse show intense bands. No loading control household protein

was determined.

Figure 16.A: Western Blot for BMP-6 using

the Biorbyt orb155848 polyclonal anti-BMP-6

antibody. The first 4 lanes were loaded with

wild type mice samples while the last 4 lanes

were loaded with samples from BMP-6

knockout mice. The arrow indicates BMP-6 at

56 kDa.

Figure 16.B: Loading control on the same

western blot, using Sigma-Aldrich A2066 anti-

actin antibody as housekeeping protein. The

β-actin is at 42 kDa.

Figure 16.C: Quantification of the Biorbyt

orb155848 analysis. P<0.05.

Figure 16 shows the result for the Biorbyt orb155848 antibody. The differences in protein

expression for BMP-6 between wild type and knockout mice were found to be statistically

significant (P<0.05), with the expression of BMP-6 in knockout mice being less than in in wild type

mice. The P-value stayed <0.05 when the outlier in the wild type section was taken out of the

calculation (not shown).

34

Figure 17.A: Western Blot for BMP-6 using

the Acris AP20577PU-N polyclonal anti-

BMP-6 antibody. The first 4 lanes were

loaded with wild type mice samples while the

last 4 lanes were loaded with samples from

BMP-6 knockout mice. The bands are at the

predicted 57 kDa.

Figure 17.B: Loading control on the same

western blot, using Cell Signalling

Technology 14C10 anti-GADPH HRP-

conjugated antibody as housekeeping

protein. The bands are at the predicted 37

kDa.

Figure 17.C: Quantification of the Acris

AP20577PU-N analysis. P=0.2.

Figure 17 shows the results for the Acris AP20577PU-N antibody. The protein levels for BMP-6,

as measured by western blot analysis, were not statistically different between wild type and BMP-

6 knockout mice (P=0.2).

In conclusion, all the tested antibodies reacted on both the wild type and knockout mice.

35

5 Discussion

5.1 COPD patients

5.1.1 BAMBI

We attempted to investigate the potential role of BMP and activin membrane-bound inhibitor

(BAMBI) in the pathogenesis of COPD. The hypothesis is that the pseudoreceptor BAMBI takes

TGF-β out of the circulation and drives the system towards inflammation(103). Zhang et al. found

BAMBI to be upregulated in relation to the COPD severity and to correlate with the T17/Treg cell

ratio(76, 103). Drömann et al. found BAMBI to be strongly expressed in COPD lungs and to be

influenced by infection(102). In human lung tissue, we did not find a significant difference between

the BAMBI mRNA levels of never smokers, current healthy (ex-)smokers, COPD GOLD II patients

and COPD GOLD IV patients.

5.1.2 BMP-6

BMP-6 became a suspect because of a GWAS study where it was found to correlate to FVC(99).

However, FVC is not a disease marker of obstructive lung disease but the FEV1/FVC ratio or

Tiffeneau index is. While the FVC can be altered in COPD(109), it is also a marker for restrictive

lung diseases such as lung fibrosis. Little is known about BMP-6 in COPD. It is best described as

a major regulator of hepcidin(94). Iron dysregulation is suspected to play a role in COPD and an

increased iron sequestration in alveolar macrophages of COPD patients has been found(95). We

found BMP-6 mRNA levels to be significantly less in COPD patients compared to never smokers,

reserving a potential role for BMP-6 in the pathogenesis of COPD. It has to be determined if BMP-

6 is an innocent bystander or whether it is a main driver for iron-induced damage in COPD patients.

Our hypothesis is that because of decreased BMP-6 levels in correlation with COPD severity,

more iron-induced reactive oxygen species form. It has already been shown that BMP-6 deficient

mice develop massive iron overload in the liver(110).

Using the results from the RT-qPCR for BMP-6 on human tissue, we performed correlations for

FEV1, FEV1/FVC, emphysema score, packyears, KCO and DLCO. We found a significant result

(P<0,05) for FEV1, FEV1/FVC and packyears. This suggests a correlation to lung function tests

as well as to the main risk factor of COPD being cigarette smoking. However, it is again not clear

whether this is a causal relation or coincidence. A possible explanation why no significant results

36

were found for KCO and DLCO could be because these lung function parameters rely on the gas

exchange capacity. As already stated, different COPD patients will exhibit different symptoms as

well as different CT phenotypes(2). KCO and DLCO are not diagnostic parameters but FEV1/FVC

is, so every diagnosed COPD patient will have an obligate FEV1/FVC ratio lower than 0,7. This

could also explain why the BMP-6 mRNA levels did not correlate with the emphysema score, as

not all COPD patients are dominant emphysematous(2, 104). Another reason may be that the

emphysema scoring only succeeded in 28 patients, resulting in a rather small group that was

available for analysis, lacking sufficient power.

5.2 Murine model of COPD

5.2.1 BAMBI

In contrast to the study by Zhang et al. (103), our results show BAMBI mRNA levels to be

significantly lower in smoke-exposed mice compared to their air exposed counterparts. There

are several reasons as to why our results differ from those in the literature. First, there may be

different conditions involved in the study setup that influences the outcomes. Second, as a

pseudoreceptor, BAMBI may have an ambivalent role, enhancing or attenuating inflammation

depending on the trigger or environment. More research on the role of BAMBI is needed to

assess its role.

5.2.2 BMP-6

We attempted to study the localisation of BMP-6 on the protein level. Here we encountered a

problem as both the wild type and the BMP-6 knockout mice showed a strong signal in the smooth

muscle cells around bronchioles and a weaker sign in the vascular walls and macrophages. Three

different antibodies were used, but the BMP-6 knockout mice never failed to show an equal

reaction as the wild types.

When investigating the protein levels of BMP-6 using western blot, we encountered the same

problem. Only for the Biorbyt orb155848 anti-BMP-6 antibody, a statistical significant difference

between the wild type and knockout mice on western blot was found but this needs to be confirmed

in an independent experiment.

37

There are several possible reasons as to why the knockout mice showed reaction. First of all,

there may have been technical flaws. However, this is unlikely as the procedures are all

standardised, have been previously validated and have been done more than once. This leaves

the mice or the antibodies as potential suspects.

The mice were designed by Solloway et al.(107) by replacing the second exon of the BMP-6 gene

by a Acc1-gene targeting vector carrying a neomycin resistance gene. The offspring mice were

controlled using RNA probes for the mature section of the BMP-6 gene, downstream of the

replaced section. The homozygote knockout mice did not show any reaction to the probes and as

such were deemed full BMP-6 knockout mice. It is however never specified how this results in a

loss-of-function of BMP-6 and one should note that knockout of a gene isn’t the same as deletion.

This may be due to the degrading of the altered mRNA, altering a splice site or by causing a

premature stopcodon (the one in the used Acc1 gene for example). While this results in a

phenotypic knockout, it is possible that the part between the BMP-6 promotor region and the

beginning of the altered exon 2 escapes nonsense mediated decay and gets translated. For the

Abcam anti-BMP-6 antibody, it is known from the datasheet that this antibody has specificity for a

region in the pro-peptide that is later processed. If the other used antibodies (their immunogen

was not included in the datasheet) also have their specificity for a sequence that is included in

exon 1, this may be the reason why the western blot showed equal signal in the wild type and

knockout lanes. For this theory to be true, the BMP-6 exon 1 of the knockout gene should still be

transcribed and translated, providing the epitopes for the antibodies to bind upon. The mice are

still true knockouts, as they don’t have BMP-6 function and exhibit the BMP-6 knockout phenotype.

While western blot uses denatured proteins, immunohistochemistry does not. For the antibodies

to bind the secreted part of the BMP-6 exon 1, it should also have a 3D structure that enables the

antibody to recognise its epitope. The fact that the bands in the knockout lanes on WB were at the

same height as the wild type bands does not agree with our theory that a partial protein is

synthesized. Additionally, we determined by RT-qPCR that the BMP-6 mRNA expression in the

BMP-6 knockout mice is almost absent using a probe against sequences downstream from exon

3. For further analysis, it would be recommended to check the knockout mouse with an antibody

against sequences coded for by the deleted part of exon 2 or further downstream as well a different

antibody towards sequences coded for by exon 1. A more sensitive technique would be the use

of a one-step RT-qPCR with probes against exon 1 compared to probes against exon 3-7. This

would allow to confirm whether exon 1 gets transcribed and translated. It would also explain why

there was a significant difference measured using the Biorbyt antibody. If the used immunogen for

this polyclonal antibody contains both sequences derived from exon 1 and other sequences

38

derived from other exons of BMP-6, the signal from the knockout mice we found could represent

those antibodies reacting to epitopes coded for by exon 1.

Another option is that the antibodies are not specific enough. BMP-6 belongs to the TGF-β

superfamily and shares a strong similarity to other members of its superfamily. Especially BMP-5

and BMP-7 are strongly similar with BMP-6. It is possible that the antibodies aren’t specific enough

and react with other TGF-β members. Studies have found that a lot of commercial antibodies lack

sufficient specificity(111). The percentage of aspecific or polluted antibodies was found to be up

to 30%(112, 113). The lack of sufficient specificity of some commercial antibodies may offer an

explanation as to why the BMP-6 knockout mice showed reaction to anti-BMP-6 antibodies despite

not having BMP-6 protein.

We also studied the mRNA expression in smoke-exposed mice. We found no significant difference

between 1 month smoke-and air-exposed mice. For the 6 months exposed mice, we found that

the BMP-6 mRNA levels in smoke-exposed mice were significantly lower compared to their air-

exposed counterparts. The negative result from the 1 month exposure test can be explained by

the fact that 1 month smoke-exposure only yield airway inflammation but not COPD, while 6

months smoke-exposed mice exhibit hallmarks of COPD.

5.3 Conclusion

We demonstrated a potential role for BMP-6 in the pathogenesis of COPD. An RT-qPCR

performed on human lung tissue from never smokers, smokers, COPD GOLD II patients and

COPD GOLD IV patients showed a statistical significant difference between never smokers and

ex-smokers, GOLD II and GOLD IV. A difference was also found between smokers and GOLD IV

patients. This suggests that BMP-6 is decreased in COPD. In mice, we found a similar result where

smoke-exposed mice showed significantly lower BMP-6 mRNA levels. Correlations between

human BMP-6 mRNA levels and FEV1, FEV1/FVC and packyears were significant. Further

studies should elucidate whether BMP-6 is an active driver of COPD, or only plays a passive role.

While BAMBI has been a suspect after it was found elevated in COPD patients(102, 103), we did

not found significant different BAMBI mRNA levels in humane COPD lung tissue. We did find

significant decreased BAMBI mRNA levels in smoke-exposed mice. Further research should

elucidate the exact role of BAMBI and BMP-6 in the pathogenesis of COPD.

39

6 Acknowledgements

I would like to thank prof. dr. Ken Bracke for his excellent guidance, feedback and for sharing his

ideas. Furthermore, I would like to thank everyone of the laboratory for Translational Research on

Obstructive Pulmonary Diseases for their advice, technical assistance (and availability of coffee).

I would also like to acknowledge prof. dr. Guy Brusselle for his advice on COPD and the pulmonary

CT scans.

In particular I would like to thank dr. Fien Verhamme for her guidance, most helpful feedback, help

with interpretation and patience.

The University of Ghent and UZ Ghent are acknowledged for offering the use of their facilities.

40

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