University of Ulm

Institute of General Physiology

Head Prof. Dr. Paul Dietl

The role of TRPV4 in membrane barrier integrity and

inhibition in stretch-induced pathological lung cellular

responses during mechanical ventilation

Dissertation

submitted to obtain the doctoral degree of Human Biology

of the Medical Faculty of Ulm University

Nicolas Pairet

Castres, Frankreich

2018

II

Amtierender Dekan: Prof. Dr. Thomas Wirth

Erstgutachter: Prof. Dr. Paul Dietl

Zweitgutachter: PD. Dr. Jürgen Schymeinsky

Tag der Promotion: 01.02.2019

Index

III

Index:

List of abbreviations ....................................................................................................................... V

1 Introduction ............................................................................................................................. 1

1.1 Transient receptor potential (TRP) channels an overview ....................................................... 1

1.2 Transient receptor potential cation channel subfamily V member 4 (TRPV4) ...................... 5

1.2.1 TRPV4 gene and structure ......................................................................................................... 5

1.2.2 Protein interaction and regulation of TRPV4 ............................................................................ 8

1.2.3 Chemical activation and inhibition of TRPV4......................................................................... 11

1.2.4 TRPV4 function and physiological activation ......................................................................... 13

1.3 Ventilator induced lung injury (VILI)...................................................................................... 19

1.4 Acute respiratory distress syndrome (ARDS) .......................................................................... 21

1.5 The role of TRPV4 in ARDS and VILI .................................................................................... 23

1.6 The aim of the thesis ................................................................................................................... 26

2 Methods .................................................................................................................................. 27

2.1 In vitro studies ............................................................................................................................. 27

2.1.1 TER measurement ................................................................................................................... 27

2.1.2 Vascular Permeability Assay ................................................................................................... 28

2.1.3 Calcium 6 assay on the FLIPRTETRA

........................................................................................ 29

2.1.4 TRPV4 agonism effect on LDH release .................................................................................. 30

2.1.5 RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay .................................................. 30

2.1.6 Cell-IQ®

................................................................................................................................... 31

2.1.7 TRPV4 agonism effect on cytokine release ............................................................................. 31

2.1.8 Uniaxial cell strain and microscopy......................................................................................... 32

2.1.9 Equibiaxial cell strain .............................................................................................................. 33

2.1.10 Cells ......................................................................................................................................... 33

2.2 In vivo studies .............................................................................................................................. 35

2.2.1 Effect of TRPV4 activation on vascular permeability ............................................................. 35

2.2.2 Murine mechanical ventilation model ..................................................................................... 36

2.3 Molecular biology assays ........................................................................................................... 37

2.3.1 Pierce™ BCA Protein Assay Kit ............................................................................................. 37

2.3.2 ELISA/MSD ............................................................................................................................ 38

2.3.3 Phospho/Total ERK1/2 assay .................................................................................................. 38

2.3.4 ATP release measurement ....................................................................................................... 39

2.3.5 LDH release ............................................................................................................................. 39

2.3.6 Human cAMP / Calcium Signaling PathwayFinder ................................................................ 40

2.4 Compounds ................................................................................................................................. 44

2.5 Calculations & Statistics ............................................................................................................ 44

2.6 Ethics statement .......................................................................................................................... 45

Index

IV

3 Results ..................................................................................................................................... 46

3.1 Results: Role of TRPV4 in regulating endothelial membrane integrity ................................ 46

3.1.1 TRPV4 mediated calcium influx ............................................................................................. 46

3.1.2 TER measurement in HUVECs ............................................................................................... 47

3.1.3 Effect of TRPV4 agonism on TER .......................................................................................... 48

3.1.4 TRPV4 agonism effect on vascular permeability assay with FITC-Dextran in HUVECs ...... 50

3.1.5 Effect of TRPV4 agonism and antagonism on TER ................................................................ 51

3.1.6 Effect of TRPV4 activation on vascular permeability in vivo ................................................. 53

3.1.7 Effect of TRPV4 activation on lung vascular permeability in Balb/c mice ............................. 54

3.1.8 Effect of TRPV4 activation and inhibition on lung vascular permeability in vivo .................. 56

3.1.9 TRPV4 antagonist reverses the effect of TRPV4 agonism ...................................................... 56

3.1.10 TRPV4 mediated cytotoxicity .................................................................................................. 58

3.1.11 Time point of TRPV4 induced cytotoxicity and calcium dependent TRPV4 induced

LDH release ........................................................................................................................ 61

3.1.12 Life cell imaging of HUVECs exposed to the TRPV4 agonist GSK1016790A ...................... 62

3.1.13 TRPV4 activation in the RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay ........... 64

3.2 Results: Role of TRPV4 in stretch induced pathological cellular response ........................... 66

3.2.1 Effect of TRPV4 agonism on cells Ca2+

influx........................................................................ 66

3.2.2 Effect of stretch on cells Ca2+

influx ........................................................................................ 67

3.2.3 Effect of TRPV4-agonist on cell cytokine release ................................................................... 69

3.2.4 Effect of TRPV4 antagonism on stretch induced cytokine release .......................................... 70

3.2.5 TRPV4 mediated regulation of genes in the Human cAMP / Calcium Signaling

PathwayFinder ......................................................................................................................... 72

3.2.6 Effect of stretch on macrophages cytokine release .................................................................. 76

3.2.7 TRPV4 antagonist effect on mechanical ventilation induced cytokine release

and permeability increase in vivo............................................................................................ 77

4 Discussion ............................................................................................................................... 80

4.1 Role of TRPV4 in regulating endothelial membrane integrity ............................................... 80

4.2 Role of TRPV4 in stretch induced pathological cellular response ......................................... 86

4.3 Summary and clinical relevance ............................................................................................... 92

4.4 Next steps .................................................................................................................................... 95

5 Abstract .................................................................................................................................. 98

6 References ............................................................................................................................ 100

Acknowledgement ........................................................................................................................ 119

List of abbreviations

V

List of abbreviations

4α-PDD 4α-phorbol 12,13 didecanoate

5´,6´-EET 5´,6´-epoxyeicosatrienoic acid

aa amino acid

AA arachidonic acid

AC Voltage

AECC American-European Consensus Conference

AIP4 Ubiquitin ligase Atrophin-1-interacting protein 4

Ag Agonist

AKAP79 A kinase anchoring Protein 79

ALI Acute lung injury

ANK Ankyrin repeats

Ant Antagonist

AQP2 Aquaporin 2

AQP5 Aquaporin 5

ARDS Acute respiratory distress syndrome

ATP Adenosine triphosphate

BAA Bisandrographolide A

BALF Bronchoalveolar lavage fluid

BCA Bicinchoninic acid

BKCa Ca2+

-sensitive large-conductance K+ channels

CaM Calmodulin

CCL Capacitance

CHO Chinese hamster ovary cells

CIRB Calmodulin/inositol 1,4,5-trisphophate receptor binding domain

COPD Chronic obstructive pulmonary disease

CT Computed tomography

Ctrl Control

DMAPP dimethylallyl pyrophosphate

DPBS Dulbecco's Phosphate-Buffered Saline

DRG Dorsal root ganglia

ECMO Extracorporeal membrane oxygenation

EET Epoxyeicosatrienoic acids

ER Endoplasmatic reticulum

ERK Extracellular signal Regulated Kinases

List of abbreviations

VI

EthD-III Ethidium homodimer III

FITC Fluorescein isothiocyanate

GM-CSF Granulocyte-Macrophage Colony Stimulating Factor

HBSS Hank´s Balanced Salt Solution

HCL Hydrochlorid acid

HUVEC Human umbilical vein endothelial cell

ICU Intensive Care Units

IL Interleukin

INT Iodonitrotetrazolium

IP3 Inositol 1,4,5-trisphophate

i.p. Intraperitoneal

i.t. Intratracheal

i.v. Intravenous

KC Keratinocyte chemoattractant chemokine

KCa2.3 Calcium-activated potassium channels

KO / -/- Knockout

LDH Lactate dehydrogenase

LPS Lipopolysaccharide

M1 Macrophage phenotype M1

M2 Macrophage phenotype M2

MACS Magnetic activated cell sorting

MAP7 Microtubule-associated protein 7 domain

MCP-1 Monocyte Chemoattractant Protein-1

M-CSF Macrophage Colony Stimulating Factor

MMP Matrix metalloproteinase

MSOF Multiple-system organ failure

MV Mechanical ventilation

NCI-H292 Human lung epithelial cells

NGF Nerve growth factor

NO Nitric oxide

OS-9 Osteosarcoma amplified 9 protein

OTRPC4 Osmosensitive transient receptor potential channel 4

P Pore domain

PACSIN-3 Protein kinace C and casein kinase substrate in neurons protein 3

PBMC Peripheral blood mononuclear cell

PBS Phosphate-Buffered Saline

PDE5 Phosphodiesterase 5

List of abbreviations

VII

PEEP Positive end-expiratory pressure

PIBS Phosphoinositide-binding site

PIP2 Phosphatidylinositol 4,5-biphosphate

PKA Protein kinases A

PKC Protein kinases C

PLA2 Phospholipase A2

p.o. Per-oral

PRD Proline-rich domain

PS Phosphatidylserine

RANTES Regulated upon activation, normal T cell expressed and secreted

chemokine

RR Ruthenium red

RVD Regulatory volume decreases

S Transmembrane spanning domain

SACs Stretch-activated ion channels

SAEC Small airway epithelial cell

SGK1 Serum glucocorticoid-induced protein kinase 1

SR Sarcoplasmatic reticulum

STIM1 Stromal interaction molecule 1

SU Subunit

TER Transepithelial/transendothelial electrical resistance

TM Transmembrane domain

TNF-α Tumor necrosis factor α

TRP Transient receptor potential channel

TRPA Transient receptor potential ankyrin channel

TRPC Transient receptor potential canonical channel

TRPM Transient receptor potential melastin channel

TRPML Transient receptor potential mucolipin channel

TRPP Transient receptor potential polycystin channel

TRPN / NOMPC No mechanoreceptor potential C channel

TRPV Transient receptor potential vanilloid channel

TRPV4 Transient receptor potential vanilloid type 4 channel

TRP12 Transient receptor potential channel 12

VEGF Vascular endothelial growth factor

Veh Vehicle

VILI Ventilation induced lung injury

VRL-2 Vanilloid receptor-like channel 2

List of abbreviations

VIII

VR-OAC Vanilloid receptor-related osmotically activated channel

WT Wild-type

Z Impedance

Introduction

1

1 Introduction

1.1 Transient receptor potential (TRP) channels an overview

Transient receptor potential (TRP) channels form an ion channel superfamily that is

involved in sensing and transmission of a plethora of external and internal stimuli (Yin and

Kuebler 2010). The first TRP channel was described in the Drosophila photoreceptor,

where a deletion of the trp gene led only to a transient response in the presence of

continuous light instead of a substained retinal depolarization (Minke 1977, Montell et al.

1985). Further investigations identified about 70 TRP channels in both invertebrates and

vertebrates (60 in zebrafish, 24 in nematodes, 16 in fruit flies and one in yeast) (Montell

2005). In mammals, 33 different TRP channels have been found so far (Montell 2001,

Clapham 2003). Based on amino acid homologies, the TRP channel superfamily can be

differentiated into seven main subfamilies: TRPA (ankyrin), TRPC (canonical), TRPV

(vanilloid), TRPM (melastin), TRPML (mucolipin), TRPP (polycystin) and TRPN (no

mechanoreceptor potential C, or NOMPC) (Clapham 2003). In humans and mice 28 TRP

channels have been identified with one member of TRPA, seven members of TRPC (with

TRPC2 as a pseudogene in humans), six members of TRPV, eight members of TRPM,

three members of TRPML and three members of TRPP (Venkatachalam and Montell

2007). TRPN is the only TRP subfamily not represented in mammals and have only been

found in worms, Drosophila and zebra fish (Clapham 2003, Montell 2005).

For all TRP channels the predicted subunit structure consists of six helix transmembrane

(TM) spanning domains (S1-S6) with a loop between the fifth (S5) and sixth (S6) TMs

(Figure 1A) forming a pore domain (P) (Clapham 2003, Hoenderop et al. 2003). The NH2

and COOH termini are located intracellularly in the cytoplasm and differ depending on the

TRP families with N-termini containing a various number of ankyrin repeats, a putative

caveolin-binding site and a predicted coiled coil region. The C-terminal comprises of a

TRP domain of about 23-25 amino acids that is loosely conserved in all TRP mammalian

subfamilies and encompass a highly conserved 6-amino acid TRP box1 (EWKFAR in

TRPCs) and a proline rich domain that has been referred as TRP box2. Depending on the

TRP subfamily the C-terminus can contain a calmodulin/inositol 1,4,5-trisphophate (IP3)

receptor binding (CIRB) domain, a coiled coil region, an enzyme domain and an PDZ

binding domain for protein-protein interactions (Montell 2005, Ramsey et al. 2006,

Introduction

2

Venkatachalam and Montell 2007, Yin and Kuebler 2010). TRP channels are likely

composed of four subunits that coassemble to form a 24-helix functional homo- or

heterotetramers (Clapham 2003, García-Sanz et al. 2004, Cheng et al. 2010, Liao et al.

2013, Hellmich and Gaudet 2014, Moran 2018). There is relative low sequence homology

between the family members and the channel structure can diverse significantly (Figure

1B) (Liao et al. 2013, Paulsen et al. 2015, Moran 2018).

Figure 1: TRP channel structure organization. A Simplified structure sketches of TRP channel subunit and tetramer

organization. The following domains are indicated: transmembrane segments of one TRP channel subunit (S1-6) and the

pore loop (PL). Four TRP subunits (SU) coassemble to a tetramer. B Comparison of recently elucidated transient receptor

potential (TRP) channel structures determined by electron cryo-microscopy with each of the four subunits colour-coded.

View from the top through the channel. Simplified structure sketch of TRP channel subunit derived from Yin and

Kuebler (2010). TRPV1 and TRPA1 structures were taken from EMPIAR, the Electron Microscopy Public Image

Archive (Iudin et al. 2016) based on published results (Liao et al. 2013, Paulsen et al. 2015).

Introduction

3

TRP channels are widely distributed throughout the body and are expressed in a large

number of tissues and excitable and nonexcitable cell types, including immune cells. They

are particularly expressed in sensory organs and receptor cells, pointing to their critical role

as cellular sensors for diverse signal sensation and transduction (Clapham 2003). The

majority of TRP channels are Ca2+

permeable non-selective cation channels (Montell 2001,

Owsianik et al. 2006) and are exceptional in the sense that they are polymodal and

activated by many types of different stimuli, such as temperature, pH, osmotic and

mechanical stress, pheromones, chemicals, intra- and extracellular messengers and

probably by the filling state of intracellular Ca2+

stores (Clapham 2003, Pedersen et al.

2005).

TRP channels are permeable for cations and except for only two TRP channels (TRPM4,

TRPM5) that are impermeable for calcium, all other TRP channels are Ca2+

permeable.

The permeability ratios between these channels vary significantly with PCa/PNa selectivity

ranging from 0.3 to ˃ 100 with TRPV5 and TRPV6 showing the highest Ca2+

permeability

(Pedersen et al. 2005, Owsianik et al. 2006). The Ca2+

concentration in extracellular

biologic fluids ranges from 1.6 to 2 mM. In contrast the cytosolic free Ca2+

concentration

is maintained by cells around 100 nM, meaning that for a cell at rest the [Ca2+

] is ~ 20.000

times lower in the cytoplasm than outside the cell, producing a high electrochemical

gradient of about 180 mV between the extracellular and intracellular (cytoplasmic) space

(Berridge et al. 2003, Clapham 2003, Bootman 2012). TRP channels modulate the cations

flux through the plasma membrane down an electrochemical gradient, thereby playing an

important role in raising the free intracellular Ca2+

concentration. In addition to their role

as plasmalemmal Ca2+

channels a number of studies also indicate that in some cases TRP

channels could also function as calcium release channels from organelles acting as

intracellular calcium store such as the endoplasmatic (ER) and sarcoplasmatic reticulum

(SR) (Pedersen et al. 2005, Bootman 2012). Changes in cytosolic free calcium

concentration has a fundamental role in cellular process and calcium entry through plasma

membrane channels is recognized as a cellular signalling event per se. Changes in

transmembrane voltage leads to central cellular events such as neurotransmitter release,

neuronal action potential propagation and muscle contraction, but Ca2+

entry also plays a

crucial role in nonexcitable cells by gating other voltage-dependent channels and affecting

effector proteins sensitive to elevated intracellular calcium concentrations and so

Introduction

4

controlling a plethora of cellular processes such as transcriptional regulation, proliferation,

cell death and migration (Berridge et al. 2003, Ramsey et al. 2006, Bootman 2012).

There is also considerable evidence that TRP channels are regulated by post-translational

mechanisms such as multimerization of TRP subunit to heterometric complexes,

translocation and interaction with membrane proteins may dramatically modulate TRP

channel function (Yin and Kuebler 2010) and that these regulatory processes may be

triggered via chemical or physical stimulation, demonstrated in the mechanical shear stress

induced translocation of TRPV4 and TRPM7 to the plasma membrane (Bezzerides et al.

2004, Oancea et al. 2006, Loot et al. 2008, Yin and Kuebler 2010). The gating mechanism

of TRP channels is poorly understood and it remains unclear whether they are directly

activated by a stimulus or indirectly via second messengers and serve rather as transducers

that are functionally activated by an upstream stimulus (Clapham 2003, Ramsey et al.

2006, Christensen and Corey 2007, Yin and Kuebler 2010).

TRP channels are involved in numerous fundamental cell functions, diverse physiological

processes and act as sensors for external irritants and inflammation products (Nilius et al.

2005). Because of their properties it is not surprising that an increasing number of

pathophysiological conditions and diseases are now been linked to TRP channels

(Pedersen et al. 2005). The importance of these channels is emphasized by the broad

number of genetic diseases caused by aberrant TRP functions leading to skeletal, skin,

sensory, cardiac, ocular and neuronal disturbance (Moran 2018). Other indications for the

role of TRP channels implication in diseases is shown by their correlation between the

level of channel expression and the disease symptoms, e.g. the abundance of TRPV1 is

higher in patients with gastrointestinal diseases such as inflammatory bowel disease,

Crohn´s disease and ulcerative colitis (Yiangou et al. 2001, Geppetti and Trevisani 2004,

Nilius et al. 2005) and TRPV1 expression is considerably increased in the airway nerves of

patients exhibiting chronic cough (Groneberg et al. 2004). Furthermore phenotypes of TRP

knockout mice and other transgenic models also point to the potential role of this channel

in diseases and allow a degree of extrapolation to their impact in human diseases, e.g.

TRPV4 knockout mice display a blunting of inflammation induced thermal hyperalgesia

(Todaka et al. 2004, Nilius et al. 2005, Nilius et al. 2007) and in a mouse model of chronic

itch, scratching evoked by impaired skin barrier was shown to be abolished in TRPA1-

deficient animals (Wilson et al. 2013). Compounds modulating TRP channels have been

studied in preclinical experiments targeting indications such as pain, atopic dermatitis, itch,

Introduction

5

disorders of the central nervous system and cardiovascular disorders with some of those

already entered clinical trials, e.g. a TRPV4 inhibitor from GlaxoSmithKline entered Phase

2 clinical trials as a potential treatment for pulmonary edema and reduced pulmonary gas

transfer in patients with heart failure, as well as TRPA1 antagonist entered Phase 1 trials,

where a significant reduction in pain scores was reported after treatment with the

antagonist GRC 17536 in patients with painful diabetic neuropathy who have intact

neuronal function (Moran 2018).

1.2 Transient receptor potential cation channel subfamily V member 4 (TRPV4)

The transient receptor potential vanilloid 4 (TRPV4) ion channel is a Ca2+

-permeable

nonselective cation channel (Yin and Kuebler 2010). It has a higher permeability to Ca2+

than to Ba2+

, Sr2+

or Mg2+

and in absence of divalent ions it is also permeate by

monovalent cations, such as K+, Cs

+, Rb

+, Na

+ and Li

+ and discriminates poorly between

them (Nilius et al. 2001, Voets et al. 2002). It is distributed widely throughout the body

and participates in the transduction of both chemical stimuli and physical stimuli such as

heat, pH, osmotic and mechanical stimuli (reviewed in Garcia-Elias et al. 2014). TRPV4

was first described in 2000 as a channel with a role in osmosensation and was initially

given different names: osmosensitive transient receptor potential channel 4 (OTRPC4),

vanilloid receptor-related osmotically activated channel (VR-OAC), vanilloid receptor-like

channel 2 (VRL-2), and transient receptor potential channel 12 (TRP12). Finally in 2002

the current nomenclature TRPV4 was accepted (Liedtke et al. 2000, Strotmann et al. 2000,

Wissenbach et al. 2000, Delany et al. 2001, Nilius et al. 2001, Garcia-Elias et al. 2014,

White et al. 2016).

1.2.1 TRPV4 gene and structure

The human TRPV4 gene is found in chromosome 12 at q23-q24.1 and has 15 exons with

five splice variants (TRPV4-A-E) (Arniges et al. 2006, Garcia-Elias et al. 2014).

Progesterone has been shown to reduce expression of TRPV4 in epithelial and vascular

smooth muscle cells (Jung et al. 2009). Other factors have been identified that increase

TRPV4 expression such as interleukin 1β and interleukin 17 in dorsal root ganglia (DRG)

neurons (Segond von Banchet et al. 2013) and nerve growth factor (NGF) in the

Introduction

6

urothelium (Girard et al. 2013). Increased TRPV4 expression has also been reported in

pulmonary arterial smooth muscle cells and astrocytes of mice exposed to

hypoxia/ischemia (Butenko et al. 2012, Xia et al. 2013). But there is a poor knowledge

about the regulation of TRPV4 transcription (Garcia-Elias et al. 2014, White et al. 2016).

Figure 2: Schematic TRPV4 monomer structure. The TRPV4 protein consists of six transmembrane domains (S1-6) with

a pore loop (PL) between S5 and S6. Two key amino acids, D672 and D682, for the regulation of TRPV4 permeability

are highlighted in the pore region. Both the N- and C-termini are situated in the cytosol. The N-terminus includes a series

of ankyrin repeats (ANK), a proline-rich domain (PRD) and a phosphoinositide-binding site (PIBS). The C-terminus

contains a putative TRP box, a microtubule-associated protein 7 (MAP7) domain, a calmodulin (CaM) domain and a

PDZ-like domain for protein-protein interaction (derived from Yin and Kuebler 2010, White et al. 2016).

The TRPV4 protein consists of 871 amino acids (aa) and has a predicted relative molecular

mass of 98 kDa. The TRPV4 protein possesses six transmembrane (TM) domains and an

N- and C-terminal tails localized in the cytoplasm. Like other TRPs the pore of the channel

(aa 663–686) is situated in the loop between TM5 and TM6. Two key amino acids

localized in the pore region, D672 and D682 have been shown to regulate TRPV4

Introduction

7

permeability (Figure 2). Neutralization of these two negatively charged residues decreases

the permeability for calcium and D682 has also been shown to participate in Ruthenium

red block (Voets et al. 2002, Garcia-Elias et al. 2014).

The N-terminal tail is the longest part of the TRPV4 protein and represents more than 50%

of the total protein and is suggested to play an important role in gating of the channel

(Phelps et al. 2010). The N-terminus houses up to six ankyrin repeats (ANK), depending

on the splice variant of TRPV4, which are involved in protein-protein interactions, channel

oligomerization and are necessary for the channel function (Arniges et al. 2006). The N-

terminal tail also contains a phosphoinositide-binding site (PIBS) which enables to bind to

phosphatidylinositol 4,5-biphosphate in the plasma membrane and that is required for

channel activation by physiological stimuli such as heat and hypotonicity (Garcia-Elias et

al. 2013). In addition a proline-rich domain (PRD) is localized in the NH2 terminus,

playing an important role in the regulation of TRPV4. The PRD is used to bind to kinases

like protein kinace C and casein kinase substrate in neurons protein 3 (PACSIN-3)

(Cuajungco et al. 2006) and is complete deletion renders the channel insensitive to all

stimuli, including the synthetic small molecule agonist 4α-phorbol 12,13 didecanoate (4α-

PDD) (Garcia-Elias et al. 2008, Garcia-Elias et al. 2014).

The C-terminal tail contains a putative TRP box, proposed for TRPV1 and suggested for

TRPV4 because of the similar structure predicted for TRPV1 and TRPV4 (Garcia-Sanz et

al. 2007, Garcia-Elias et al. 2014). This region, localized adjacent to the channel gate is

essential for the tetramerization of the channel subunits into functional channels (Garcia-

Sanz et al. 2007). Channel protein folding, maturation and trafficking are dependent on the

component of TRPV4 COOH-terminal and the COOH terminus interacts with the

microtubule-associated protein 7 (MAP7) (Suzuki, Hirao et al. 2003). MAP7 in the C-

terminal tail of TRPV4 has also been suggested to interact with the cytoskeleton and a

single mutation at E797 in the MAP7 domain results in constitutive opening of the channel

(Watanabe, Vriens et al. 2002). TRPV4 N-terminus houses a calmodulin domain for the

binding of calcium-calmodulin to TRPV4 promote to lead to conformational change

followed by channel opening and calcium influx (Strotmann et al. 2003). In the final four

amino acid residues of the TRPV4 C-terminus a PDZ-like domain is found for further

protein-protein interaction (Garcia-Elias et al. 2008, Garcia-Elias et al. 2014).

Introduction

8

The monomer structure normally coassembles to a homotetrameric functional TRPV4 ion

channel and is suggested to have the same tetramer structure as TRPV1 (Figure 1B)

(Shigematsu et al. 2010). TRPV4 has been reported to heterotetramize with e.g. TRPC1,

TRPP2 and also to form TRPV4-TRPC1-TRPP2 complexes (Stewart et al. 2010, Ma et al.

2011, Du et al. 2014). This heteromerization alter the properties of TRPV4 and give these

channels additional functions to its already large array of tasks (Du et al. 2014).

1.2.2 Protein interaction and regulation of TRPV4

In addition to the activation of TRPV4 by a wide range of stimuli, TRPV4 activity in the

plasma membrane is modulated at different levels: modifying TRPV4 localization and

expression on the plasma membrane, interaction with signal molecules and proteins,

cytoskeletal protein interaction and interaction with other ion channel proteins (reviewed in

Garcia-Elias et al. 2014, White et al. 2016).

Proteins modulating TRPV4 expression and location on the plasma membrane

Like other integral membrane proteins TRPV4 is synthesized in the endoplasmatic

reticulum (ER) and targeted to the plasma membrane. In the ER, TRPV4 has been shown

to interact with osteosarcoma amplified 9 (OS-9), a ubiquitous protein on the cytoplasmic

side of the ER playing a role in selecting substrates for degradation. OS-9 interacts with all

TRPV4 splice variants and this interaction is strongest with those lacking full ankyrin

repeats. It interacts with the N-tail of TRPV4 monomers and so reduces the amount of

channels in the membrane and protects TRPV4 monomers from ubiquitination and

degradation allowing formation of mature tetramers to occur (Wang et al. 2007).

Expression of TRPV4 at the plasma membrane is a net consequence of endocytosis and

exocytosis and expression of TRPV4 at the cell membrane has been shown to be regulated

by Protein kinase C casein kinase substrate in neuron protein 3 (PACSIN-3) (Cuajungco et

al. 2006). Binding of PACSIN 3 to TRPV4 decreases endocytosis resulting in an increased

TRPV4 plasma membrane expression and an overexpression in PASCIN-3 has been

associated with an increase in plasma membrane associated TRPV4. Interestingly, TRPV4

bound to PACSIN 3 is no longer activated by cellular swelling or heat but remain sensitive

Introduction

9

to the TRPV4 agonist 4α-Phorbol 12,13-didecanoate (4α-PDD) (D'Hoedt et al. 2008),

suggesting that TRPV4 is activated by different non-overlapping mechanism.

Conversely, activation of TRPV4 can be associated with its downregulation at the cell

membrane. Ubiquitin ligase Atrophin-1-interacting protein 4 (AIP4) and the protein β-

arrestin, which serves as an adaptor between TRPV4 and AIP4, binds TRPV4 in the

presence of angiotensin and lead to monoubiquitination of TRPV4 and its subsequent

endocytosis (Wegierski et al. 2006, Shukla et al. 2010).

Finally, the precise location of TRPV4 in the plasma membrane also seems to have

important functional consequences and appear to be tightly regulated (Goldenberg et al.

2015). TRPV4 has been shown to associate with caveolin-1, a primary structure

component of the caveolae, which are plasma membrane microdomains rich in proteins as

well as lipids and have several functions in signal transduction, such as mechanosensation

(Cuajungco et al. 2006, Saliez et al. 2008). This placement associates TRPV4 in close

proximity of proteins with critical importance in vascular biology. In the lung, caveolae are

key sites of nitric oxide (NO) production and interestingly other TRP channels have been

shown to translocate to caveolae in response to acute hypoxia (Tabeling et al. 2015),

making such translocation critical to the function of TRP channels. The stromal interaction

molecule 1 (STIM1) has also been proposed to complex with the COOH-terminal tail of

TRPV4 for guiding TRPV4 from the ER to the cell membrane and for its proper function

(Shin et al. 2015).

Cytoskeletal proteins interacting with TRPV4

TRPV4´s C-tail interacts with the microtube-associated protein 7 (MAP7) and it has been

described that TRPV4 interacts with the cytoskeleton via F-actin and tubulin which

compete for the binding to the COOH terminus (Suzuki, Hirao et al. 2003). The interaction

between TRPV4 and F-actin support channel activation following cell swelling (Becker et

al. 2009). Additionally MAP7 has been promoted to enhance TRPV4 presence at the

plasma membrane, thereby indirectly increasing its activity (Suzuki, Hirao et al. 2003).

TRPV4 has also been shown to interact with key molecules that connect the cytoskeleton

with structures that maintain the barrier function in epithelia. β-catenin and E-cadherin,

major components of the tight junctions in keratinocytes, interacts with the N-tail of

Introduction

10

TRPV4 to maintain the integrity of skin barrier (Sokabe et al. 2010, Sokabe and Tominaga

2010). Additionally mechanical forces applied to β1 integrin are activating TRPV4

(Matthews et al. 2010).

Proteins and signal molecules modulating TRPV4

It has been observed that several enzymes affect the activity of TRPV4. Activation of

TRPV4 is enhanced by the phosphorylation of specific sites in the N- and C-tail of TRPV4

by protein kinases C (PKC) (Xu et al. 2003) as well as by protein kinases A (PKA)

(Alessandri-Haber et al. 2006) and TRPV4 phosphorylation by PKA and PKC has been

shown to be dependent on interaction with A kinase anchoring Protein 79 (AKAP79) (Fan

et al. 2009). Phosphorylation of TRPV4 by Serum glucocorticoid-induced protein kinase 1

(SGK1), amplifies the TRPV4 response to appropriate stimuli and enable TRPV4 binding

to F-actin (Shin et al. 2012). An important nonprotein modulator of TRPV4 is the

membrane phospholipid, phosphatidylinositol 4,5-biphosphate (PIP2), localized on the

inner leaflet of the plasma membrane (Garcia-Elias et al. 2013). The TRPV4 N-terminal

proline-rich domain (PRD) has been shown to interact with plasma membrane PIP2 and is

thought to stabilize the intracellular tail of TRPV4 in an open conformation. Depletion of

PIP2 makes the channel unresponsive to heat or osmotic stimuli, but maintain activation by

epoxyeicosatrienoic acids EETs or 4α-PDD (Garcia-Elias et al. 2013). Adenosine

triphosphate (ATP) also interacts with these sites and is a positive modulator of TRPV4

channel activity (Lorenzo et al. 2008). Nitric oxide (NO) has been shown to cause S-

nitrosylation of TRPV4 in a residue of the C-tail and reduces activation of TRPV4 (Lee et

al. 2011). Calmodulin (CaM) has been identified to bind to TRPV4 within the second ANK

domain of the N-tail and at the C-tail (Phelps et al. 2010). The reported effects of CaM

binding on TRPV4 range from a positive modulation (Strotmann et al. 2003) to an

inhibitory effect (Phelps et al. 2010). In a heterologous expression system, increasing

intracellular calcium has been shown to inhibit TRPV4 channel function (Phelps et al.

2010). However another group demonstrated that TRPV4 is activated by increasing

intracellular Ca2+

through direct binding to TRPV4 calmodulin (Strotmann et al. 2003).

Introduction

11

Channel proteins interacting with TRPV4

As already mentioned heteromeric channels are formed by TRPV4 interacting with TRPP2

resulting in a mechano- and thermosensitive sensor in the cilium (Kottgen et al. 2008).

TRPV4-TRPC1-TRPP2 channel complexes found in TRPV4, TRPC1, and TRPP2

cotransfected cells of the vascular endothelium, are activated by flow to mediate calcium

influx (Du et al. 2014). TRPV4 and aquaporin 5 (AQP5) cell membrane expression is

increased by hypotonicity, and in this system AQ5 is essential for gating TRPV4 (Liu et al.

2006). TRPV4 and Aquaporin 2 (AQ2) are suggested to assemble in response to

anisosmotic conditions (Galizia et al. 2012). TRPV4 may also interact indirectly with other

calcium-sensitive proteins and channels located close to TRPV4 channels (White et al.

2016). TRPV4 functions in conjunction with Ca2+

-sensitive large-conductance K+ channels

(BKCa) in the bronchial epithelium and vascular smooth muscle (Earley et al. 2005,

Fernandez-Fernandez et al. 2008). Activation of Ca2+

-sensitive K+ channels via as few as

three TRPV4 channels mediating a localized Ca2+

influx (sparklets) has been shown for

intermediate- and small-conductance K+ channels (Sonkusare et al. 2012). Similar

observations were made for calcium-activated potassium channels (KCa2.3) that have been

shown to interact with TRPV4 inducing vascular relaxation (Ma et al. 2013).

1.2.3 Chemical activation and inhibition of TRPV4

TRPV4 is activated by a wide array of chemicals. The relevant ones will be described and

compared in this section. Furthermore an overview of relevant chemical antagonists of

TRPV4 will be given.

Activators of TRPV4:

Endogenous arachidonic acid (AA) and its metabolites epoxyeicosatrienoic acids (EETs)

and dimethylallyl pyrophosphate (DMAPP) are TRPV4 activators and thought to be

downstream effectors of other stimuli affecting TRPV4, including endocannabinoid

anandamide and cellular swelling (Watanabe et al. 2003, Vriens et al. 2004, Bang et al.

2012). The natural 5´,6´-EET gates TRPV4 by a direct action on a site formed by residues

from the S2-S3, S4 and S4-S5 transmembrane domains (Berna-Erro et al. 2017).

Introduction

12

Plant derived non-selective agonists of TRPV4 are e.g. Bisandrographolide A (BAA) with

an EC50 of about 800 nM (Smith et al. 2006), Apigenin (EC50 ~ 10 µM) (Ma et al. 2012)

and several plant cannabinoids (De Petrocellis et al. 2012). Phorbol is an organic

compound of croton plants and its derivatives are also agonists of TRPV4 (Watanabe,

Davis et al. 2002).

One of the more specific agonists of TRPV4 and a widely used synthetic activator of

TRPV4 is the ester 4α-Phorbol 12,13-didecanoate (4α-PDD) activating TRPV4 in the

micromolar range by binding between the transmembrane domain 3 and 4 (S3, S4), which

is not mediated by PKC enzymes (Vriens et al. 2007, Klausen et al. 2009). Although its

exclusivity for TRPV4 has been put in question, by the fact that it can activates mouse

DRG neurons independently of TRPV4 (Alexander et al. 2013).

Finally a potent and selective small molecule created by GlaxoSmithKline, GSK106790A,

is a useful TRPV4 activator with an EC50 in the low nanomolar range (Thorneloe et al.

2008). Treatment with this compound in vivo was shown to cause serious vascular effects,

leading to disruption of endothelial barrier, particularly in the lung and widespread

vascular leakage (Willette et al. 2008).

Inhibitors of TRPV4:

Lanthanium and gadolinium are non-selective TRP channel blockers, and gadolinium was

one of the earliest inhibitor used to address TRPV4 (Nilius et al. 2004). Gadolinium was

identified as an inhibitor of stretch-activated ion channels and is viewed these days as a

nonselective inhibitor of extracellular Ca2+

entry (Goldenberg et al. 2015).

A commonly used but nonspecific compound for studying TRPV4, is the cationic dye

Ruthenium red (RR). RR is unfortunately nonspecific and blocks most TRPV channels,

and also members of the TRPM and TRPA subfamily (Guler et al. 2002, Goldenberg et al.

2015).

Newer compounds have appeared with more specificity and affinity to TRPV4. One of the

most selective TRPV4 inhibitor used to date is the antagonist HC-067047 with an IC50

ranging from 17 to 133 nM (Everaerts et al. 2010). At doses, that block TRPV4 function, it

also displays no adverse cross signs of sickness in mice, but his clinical safety profile

Introduction

13

remains untested. HC-0670747 is a powerful tool for studying TRPV4, although a study in

pulmonary vasculature in mice lacking TRPV4 shown vasodilatation caused by HC-

0670747 [30 µM] indicating that this inhibitor may have off-target effects at high

concentrations and has also been shown to inhibit TRPM8 at submicromolar

concentrations (Everaerts et al. 2010, Xia et al. 2013, Goldenberg et al. 2015).

One of the largest efforts in TRPV4 inhibitor design has been conducted by

GlaxoSmithKline (Darby et al. 2016). The newer antagonist GSK2193874 displayed

remarkable specificity for rodent and human TRPV4, demonstrated by a screen against

approximately 200 other channel proteins, including other TRPV subfamily members

(Thorneloe et al. 2012). A key advantage of this compound lies also in its oral activity and

can so potentially be dosed repeatedly. GSK2193874 has been shown to prevent

pulmonary edema in a mouse model of heart failure and in isolated human lung tissues and

appeared safe for potential use in human trials (Thorneloe et al. 2012). This compound and

newer version of it, may present an important tool for a variety of pulmonary disease

states.

Finally another strategy for clinical inhibition of TRPV4 is by blocking phosphodiesterase

5 (PDE5) a downstream effector of TRPV4 (Goldenberg et al. 2015). PDE5 inhibitor,

sildenafil has been shown to attenuate TRPV4 mediated endothelial calcium entry and

pulmonary edema formation in ex vivo and in vivo models of congestive heart failure (Yin

et al. 2008) and indicate a potential indirect route for preventing adverse physiologic

effects of TRPV4 activation.

1.2.4 TRPV4 function and physiological activation

TRPV4 is a polymodal ion channel activated by a wide range of diverse stimuli and

simultaneous stimuli of different natures may interact. E.g. TRPV4 activation by 4α-PDD

or hypotonic solutions induces minor channel activation compared to all stimuli at 37°C

(Gao et al. 2003). TRPV4 is a mechano-, osmo- and thermosensitive Ca2+

channel that is

involved in multiple physiological functions such as hearing, renal function, skeletal

development, nociception, vascular tone and blood pressure, endothelial and epithelial

barrier function, and has also been related to several motor sensory neuropathies and has

been shown to play a role in regulatory volume decreases (RVD) of cells (reviewed in

Introduction

14

Darby et al. 2016, White et al. 2016). This section will concentrate on TRPV4 functions

relevant for this thesis.

TRPV4 and heat:

Non-noxious heat was one of the earliest physiological activator of TRPV4 described and

TRPV channels in general are activated by specific non-overlapping temperature ranges.

TRPV4 is activated at temperature between 24 and 38°C, TRPV1 is activated at

temperature greater than 43°C, while TRPV2 is activated by temperature greater than 52°C

(Watanabe, Vriens et al. 2002, Clapham 2003). Therefore TRPV4 has been suggested to

play a role in normal thermoregulation (Guler et al. 2002, Watanabe, Vriens et al. 2002).

However, there is no agreement for TRPV4 in the detection of noxious temperature in vivo

(Garcia-Elias et al. 2014, Darby et al. 2016, White et al. 2016), e.g. TRPV4-/- mice show

normal escape latencies from hot plates and conversely TRPV4-/- exhibit reduced sensory

nerve discharge frequency in response to noxious temperature during electrophysiological

studies (Todaka et al. 2004). Additionally, heat (37°C) increases the efficacy of other

stimuli in activating TRPV4 (Gao et al. 2003).

TRPV4 and pH:

TRPV4 has been reported to be activated by low pH or citrate in Chinese hamster ovary

(CHO) cells expressing TRPV4 in vitro. Mice lacking TRPV4 have been shown to exhibit

a diminished response to acids (Suzuki, Mizuno et al. 2003). Further studies implicate

TRPV4 in acid induced lung injury, where it has been demonstrated to mediate the lung

injury response in mice exposed to hydrochlorid acid (HCL), assessed by lung

permeability increase, inflammatory cell influx and pro-inflammatory cytokine levels

increase (Balakrishna et al. 2014, Yin et al. 2016, Scheraga et al. 2017). Protection from

acute lung injury response to HCL was observed in TRPV4-KO mice or in mice treated

with different small molecule TRPV4 inhibitors (Balakrishna et al. 2014, Yin et al. 2016,

Scheraga et al. 2017), which will be further described below.

Introduction

15

TRPV4 in epithelial and endothelial barrier function:

Endothelial and epithelial barriers are characterized by intercellular cell junctions

consisting of tight junctions and adherens junctions (Mullin et al. 2005, Bazzoni 2006).

Adherent junctions containing VE-cadherins interconnect cells to a width of approximately

3 nm and tight junctions prevent extravasation of much smaller molecule (˂ 1 k Da) (Curry

2005, Mehta and Malik 2006). These junctions play an important role in barrier function

by restricting the paracellular passage of fluid and proteins across tissue membranes.

Pathophysiological states such as inflammation can disrupt barrier integrity and increased

endothelial permeability can be triggered by endothelial Ca2+

influx, resulting in

cytoskeletal reorganization and loss of interendothelial junction proteins (Tiruppathi et al.

2006, Darby et al. 2016). TRPV4 activation has been shown to result in epithelial and

endothelial permeability increase from in vitro and in vivo studies (Darby et al. 2016). For

example, TRPV4 has been shown to function in linking cell-to-cell junctions in skin

keratinocytes with the actin cytoskeleton to ensure the development of a tight barrier

(Sokabe et al. 2010). Activation of TRPV4 was observed to reduce the level of filamentous

actin and to disintegrate cell junctions between epithelial cells of the brain ventricles

(Narita et al. 2015). In the lung TRPV4 activation activates matrix metalloproteinases

(MMPs) MMP2 and MMP9, that contributes to lung injury by degrading components of

the basement membrane as well as non-matrix components such as integrins and

intercellular structure like E-cadherin (Villalta et al. 2014). TRPV4 has been shown to

regulate vascular permeability most notably within the lungs (Willette et al. 2008) and its

activation, whether via physical stimuli such as mechanical ventilation, pulmonary venous

hypertension or with pharmacological tools leads to an increased endothelial permeability

in an intracellular calcium-influx dependent manner (Hamanaka et al. 2007, Jian et al.

2008). TRPV4 regulates the integrity of the alveolar barrier and its activation has been

shown to causes endothelial detachment from the basement membrane, leading to

disruption of the pulmonary endothelial barrier, resulting in pulmonary edema formation

and alveolar flooding (Alvarez et al. 2006, Jian et al. 2008). TRPV4 also has been shown

to initiate the acute endothelial calcium-dependent permeability increase during ventilator-

induced lung injury in isolated mouse lungs (Hamanaka et al. 2007), which will be further

discussed below.

Introduction

16

TRPV4 in osmoregulation and response to mechanical deformation:

In cell-based assays TRPV4 respond to osmotic changes in the cell environment,

decreasing its activity in hypertonic solutions and increasing its activity in hypotonic

solutions and so contributing to cellular homeostasis (Strotmann et al. 2000). TRPV4-KO

mice have been shown impaired osmotic regulation, supporting a role of TRPV4 in

osmosensation (Liedtke and Friedman 2003, Mizuno et al. 2003). Changes in osmolarity

causes cell swelling or shrinkage that deform the plasma membrane and may therefore

involve aspects of mechanosensation (Darby et al. 2016, White et al. 2016). Cell

deformation and lipid bilayer tension can affect further cellular processes (Hoffmann et al.

2009). TRPV4 has been implicated in the control of regulatory volume decrease (RVD), a

regulatory response to cell swelling after exposure to a hypotonic solution that is normally

associated with changes in intracellular calcium concentration (Arniges et al. 2004).

TRPV4 has been shown to provide the Ca2+

signal, required to activate further Ca2+

potassium channel and the subsequent RVD in epithelial cells and also interacts with

aquaporins to control RVD in astrocytes (Arniges et al. 2004, Benfenati et al. 2011, Jo et

al. 2015), an important observation, suggesting that disruption of cell volume regulation

may have crucial consequences for cell signalling, barrier integrity and cell viability

(Benfenati et al. 2011).

Whether mechanical forces are generated by hypotonicity, trauma, pressure, shear stress

evoked by flow or direct cell stretch, it typically results in the deformation of the cell

membrane and it is now clear that TRPV4 responds to the application of mechanical forces

to the cell membrane and therefore it is correct to describe TRPV4 as mechanosensitive

(White et al. 2016). Thus cell stretch evoked increase in intracellular Ca2+

applied to

urothelial cells is significantly reduced in cells from TRPV4-KO mice compared to

wildtype mice (Mochizuki et al. 2009). Furthermore TRPV4 has been shown to be

activated when cyclically stretch in capillary endothelial cells (Thodeti et al. 2009). Flow

evoked shear stress activates TRPV4 leading to an increased intracellular calcium

concentration in vascular endothelial cells and HEK293 cells (Mendoza et al. 2010,

Baratchi et al. 2014). Mechanical activation of TRPV4 has also been reported to trigger

ATP release from different epithelial cells (Seminario-Vidal et al. 2011, Ueda et al. 2011).

Shear stress has also been shown to cause TRPV4 to traffic from cytoplasmic vesicles to

the plasma membrane (Baratchi et al. 2016). TRPV4 is expressed in the bladder urothelium

where it has been show to participate in sensing of intravesical mechanical pressure during

Introduction

17

bladder filling and ATP release (Birder et al. 2007, Everaerts et al. 2010), additionally

TRPV4-/- mice manifest an incontinent phenotype (Gevaert et al. 2007). Furthermore

TRPV4 in the lung has been shown to initiate the acute calcium-dependent permeability

increase during mechanical ventilation with high tidal volumes leading to ventilator-

induced lung injury in isolated mouse lungs (Hamanaka et al. 2007) that will be further

discussed below.

Gating of TRPV4 by mechanical stimuli:

The question remains however, whether TRPV4 is directly or indirectly gated by

mechanical stimuli and there are several possible mechanism of TRPV4 activation in this

purpose (Darby et al. 2016):

First TRPV4 may respond directly to the effect of mechanical deformation of the

membrane, whether secondary to hypotonicity or to a direct mechanical pressure

impinging on the cell membrane. This concept includes the direct gating of TRPV4 by

mechanical forces to the cell membrane, which induces a conformational change within the

ion channel and results in channel gating because of energy differences between the open

and closed conformation (Brohawn et al. 2014) and in this context the lipid-bilayer directly

effects TRPV4 gating (Liedtke 2005). But the direct response of TRPV4 to the effect of

mechanical deformation also includes an alternative theory, by which mechanical forces

applied to cell membrane structures, attached or tethered to the ion channel, leads to its

opening (Christensen and Corey 2007, White et al. 2016). Such structures include

accessory proteins, the cytoskeleton or even the extracellular matrix and mechanical forces

are transmitted via these structures to effect a conformational change of the channel

resulting in gating (Kung 2005, Christensen and Corey 2007, Pedersen and Nilius 2007).

The concept of direct gating of TRPV4 by mechanical forces is supported by a study on rat

TRPV4 expressed in Xenopus oocytes by repeatedly examining excised patches in a simple

buffer (Loukin et al. 2010). In this system TRPV4 could be activated by pipette suction

even in the presence of relevant enzyme inhibitors to eliminate any enzyme effects. The

evidence that TRPV4 interacts with the cytoskeleton also supports the concept that

mechanical deformation of the cell membrane per se is capable of activating TRPV4. This

is also supported by a study showing that forces applied to β1-integrins resulted in ultra-

rapid activation of Ca2+

influx through TRPV4 within 4 msec and that TRPV4 is rather

Introduction

18

activated by mechanical stretch in the cytoskeletal backbone than by deformation of the

lipid bilayer (Matthews et al. 2010).

A second putative mechanism of TRPV4 activation, is consistent with TRPV4 as

mechanosensitive rather than mechanically gated, explaining an indirect gating of TRPV4

by a force-sensing protein, that might be more distant to TRPV4 and communicate with the

channel by generating a secondary signal such as a diffusible second messenger molecule

or activation of a kinase (White et al. 2016). In this view osmotic and mechanical

sensitivity of TRPV4 has been claimed to be dependent of phospholipase A2 (PLA2).

Activation of TRPV4 by cell swelling has been described to depend on formation of

arachidonic acid (AA) and its subsequent metabolization to 5´,6´-epoxyeicosatrienoic acid

(5´,6´-EET) by cytochrome P450 epoxygenase (Watanabe et al. 2003, Vriens et al. 2004,

Fernandes et al. 2008), whereby 5´,6´-EET has then been recently shown to directly bind to

TRPV4 resulting in its gating (Berna-Erro et al. 2017). AA has also been claimed a direct

and potent activation of TRPV4 (Zheng et al. 2013). Both viscous loading and

hypotonicity have been suggested to employ a PLA2 dependent mechanism and the

production of EET to gate TRPV4 in ciliated epithelial cells (Fernandes et al. 2008).

Furthermore when limited activation of PLA2 is possible, these stimuli employ

extracellular ATP-mediated activation of inositol trisphosphate (IP3) to gate TRPV4,

thereby IP3 do not act as an agonist of TRPV4 but sensitise TRPV4 to EET in which an

interaction of TRPV4 with IP3 receptor 3 appears to occur (Fernandes et al. 2008),

requiring the binding of IP3 to a domain in the TRPV4 COOH-terminal and so leading to a

IP3-mediated sensitization of TRPV4 to these stimuli (Garcia-Elias et al. 2008).

Interestingly heat and 4α-PDD are suggested to activate TRPV4 independently of PLA2

and P-450 epoxygenase (Vriens et al. 2004) in turn pointing to the possibility that TRPV4

may be activated by more than one mechanism.

A third possible mechanism is that alterations in extracellular tonicity per se, e.g. induced

by mechanical pressure, activates intracellular proteins independent of plasma membrane

deformation, which in turn gates TRPV4 (White et al. 2016). In doing so, osmotic

stimulation results in activation of various intracellular phosphorylation/dephosphorylation

signalling processes. Given its range of activators it is not unexpected that TRPV4 has, as

already mentioned, more than a single mechanism of activation (Brewster et al. 1993,

Liedtke 2005, White et al. 2016).

Introduction

19

1.3 Ventilator induced lung injury (VILI)

Mechanical ventilation (MV) is an important tool in intensive care units (ICU) for the

treatment of respiratory failure. Despite its lifesaving effects, mechanical ventilation has

been demonstrated to induce lung damage by itself. It may aggravate lung conditions in

previously diseased lungs as well as induce serious tissue damage in previously healthy

lungs, a process named ventilator-induced lung injury (Halbertsma et al. 2005, Sutherasan

et al. 2014, Carrasco Loza et al. 2015). Several experimental studies postulate that a

previous inflammation, also named first inflammatory hit of the lung, is crucial for the

development of VILI (Carrasco Loza et al. 2015).

During mechanical ventilation, lung strain is poorly defined, especially in humans and

difficult to estimate because of the heterogeneous local lung susceptibility during MV

(Protti et al. 2014, Carrasco et al. 2015). During MV injured regions of the lung will

receive smaller fractions of the total tidal volume from the inspired tidal volume, e.g. due

to alveolar collapse and fluid extravasation, therefore other lung areas will receive the

majority of the tidal volume leading to massive overdistension of this areas and local

damage perhaps even with protective ventilation strategies (Carrasco Loza et al. 2015,

Bellani et al. 2016). In turn areas that receive the higher tidal volume, may promote a local

inflammatory response, that might trigger a subsequent generalized inflammatory response

in the lung tissue (Carrasco Loza et al. 2015, Beitler et al. 2016).

Ventilator induced lung injury (VILI) is characterized by a reduction of the alveolar epi-

and endothelial barrier function resulting in pulmonary oedema formation, inflammation

and alveolar flooding (Webb and Tierney 1974). Two main forces act on the lung tissues

and cells during mechanical ventilation, excessive volumes and/or pressures, leading to

volu- or barotrauma that causes rupture of the lung parenchyma (Dreyfuss and Saumon

1993, Dreyfuss and Saumon 1998). Studies revealed that the end-inspiratory volume

responsible for the volutrauma was the main determinant of VILI rather than a barotrauma

induced by an end-inspiratory pressure (Halbertsma et al. 2005). Another process termed

Atelectrauma, describes the cyclical opening and collapse of the alveoli in response to

mechanical ventilation, resulting in increasing stretch and shear forces in other regions

leading to lung damage and surfactant dysfunction. This effect can be attenuated by an

increased positive end-expiratory pressure (PEEP), to prevent the collapse of the alveoli,

but requires elevated inspiratory pressures (Dreyfuss and Saumon 1993, Halbertsma et al.

Introduction

20

2005). In the lungs, cytokines are produced by alveolar macrophages but also by bronchial,

bronchiolar and alveolar epithelial cells (Pugin et al. 1998, Vlahakis et al. 1999, Carrasco

Loza et al. 2015). Previous studies have demonstrated that most alveolar cells are capable

of producing pro-inflammatory mediators such as tumor necrosis factor (TNF)-α,

interleukin (IL) -6, IL-8 and IL-1β when stretched in vitro or when ventilated in ex vivo

experiments (nicely reviewed in Halbertsma et al. 2005). High level of mechanical stretch

is also associated with an increased epithelial cell necrosis and a reduction of apoptosis

(Lionetti et al. 2005, Carrasco Loza et al. 2015). A mechanism of injury, termed biotrauma,

has been elaborated postulating that the stress produced by mechanical ventilation through

overdistension of lung units not only exacerbate, but also initiate an inflammatory response

in form of an upregulation of pulmonary cytokine production due to the MV (Tremblay

and Slutsky 1998, Lionetti et al. 2005). Loss of the alveolar-capillary barrier due to the

mechanical forces may result in losing the compartmentalization of the local pulmonary

response and releasing pro-inflammatory mediators into the systemic circulation leading to

multiple-system organ failure (MSOF) (Slutsky and Tremblay 1998, Frank and Matthay

2002). Ranieri et al. (1999) support this concept by demonstrating that the concentration of

pro-inflammatory cytokines in both bronchoalveolar lavage fluid (BALF) and serum could

be decreased with a lung-protective ventilation strategy. This concept may also explain the

observation that most ARDS patients die from MSOF rather than from respiratory failure

(Montgomery et al. 1985, Halbertsma et al. 2005).

How mechanical stimuli is converted into a biochemical response (mechanotransduction)

such as cytokine release when lung cells are stretched during mechanical ventilation

remains to be clarified. Mechanical ventilation causes the expansion of the plasma

membrane and transmembrane receptors such as integrins, stretch-activated ion channels

and also the cytoskeleton by itself have been identified as key structures in

mechanosensing this physical stimuli, that then induces various cellular processes (Pugin

2003, Vlahakis and Hubmayr 2003, Halbertsma et al. 2005).

The potential involvement of cation channels in mediating the response generated in the

lung after mechanical stress has been demonstrated in isolated rat lungs in which the

increase in microvascular permeability was abolished by gadolinium (inhibitor of stretch-

activated nonselective cation channels) and concluded that stretch-activated cation

channels may initiate the increase in permeability induced by mechanical ventilation

through an increase in intracellular Ca2+

concentration (Parker et al. 1998). From the TRP

Introduction

21

channels known to be implicated in mechanotransduction such as TRPA1, TRPC1,

TRPC3, TRPC6, TRPM4, TRPM7, TRPP2 , TRPV1, TRPV2 and TRPV4 (Yin and

Kuebler 2010), TRPV4 has received specific attention as potential new molecular target

for the treatment of mechanical stress induced pathological conditions of the lung such as

ventilator-induced lung injury (Hamanaka et al. 2007, Yin and Kuebler 2010, Hamanaka et

al. 2010). The force-sensitive ion channel TRPV4 (Yin and Kuebler 2010) that is also

expressed in many cells of the lung (Alvarez et al. 2006, Hamanaka et al. 2010), has been

suggested to initiate the acute calcium-dependent permeability increase during ventilator-

induced lung injury in isolated mouse lungs (Hamanaka et al. 2007).

1.4 Acute respiratory distress syndrome (ARDS)

Acute respiratory distress syndrome (ARDS) is a rapidly progressive form of acute

respiratory failure characterized by severe hypoxemia and noncardiogenic pulmonary

edema (Ashbaugh et al. 2005) contributing to systemic inflammation and frequently

resulting in death (Silversides and Ferguson 2013).

Because ARDS is not a disease, but a syndrome composed of a multifaceted means of

diagnosis and is determined by different causes with many different clinical histories, an

entirely satisfactory definition of ARDS remains an elusive goal (Rezoagli et al. 2017).

The first common definition of ARDS was achieved in 1994 during the American-

European Consensus Conference (AECC) on ARDS (Umbrello, Formenti et al. 2017).

However, it had numerous limitations across the diagnostic criteria and a new definition

emerged in 2012. This most recent revisited definition of ARDS, known as the Berlin

definition of ARDS was proposed by an expert panel endorsed by the European Society of

Intensive Care Medicine (Ranieri et al. 2012, Rezoagli et al. 2017). The Berlin criteria

provided a small but significant improvement in the predictive ability for mortality when

compared to the AECC criteria (Umbrello et al. 2017). The Berlin definition of ARDS is

based on four variables including timing (1), chest imaging (2), origin of edema (3) and

oxygenation (4) and is defined by the following criteria: (1) onset within 1 week of a

known clinical insult or new/worsening respiratory symptoms; (2) presence of bilateral

opacities in radiograph (X-ray) or computed tomography (CT) scan on the chest that are

not fully explained by effusion, lobar/lung collapse or nodules; (3) diagnosis of respiratory

failure not fully explained by cardiac failure or fluid overload; (4) presence of hypoxemia,

Introduction

22

as defined by a specific threshold of the arterial partial pressure of oxygen to fraction of

inspired oxygen ratio (PaO2/FiO2) measured with a minimum of required positive end-

expiratory pressure (PEEP) ≥ 5 cm H2O, thus able to identify three categories of severity

based on the degree of hypoxemia: mild (200 millimeters of mercury (mm) Hg <

PaO2/FiO2 ≤ 300mmHg), moderate (100 mmHg < PaO2/FiO2 ≤ 200 mmHg), severe

(PaO2/FiO2 ≤ 100 mmHg) (Ashbaugh et al. 2005, Ranieri et al. 2012, Umbrello et al.

2017).

Approximately 5% of hospitalized patients with the need for mechanical ventilation meet

the diagnostic criteria for ARDS and it has been shown that only 25% of these patients

have a mild form of ARDS, while the remaining 75% display a moderate to severe form

(Rubenfeld et al. 2005, Esteban et al. 2008, Umbrello et al. 2017). Based on various

population studies the incidence of ARDS varies from about 10 to 80 per 100.000 person

per year with a relevant geographic diversity (in Europe 17.9, in USA 78.8 per 100.000

person per year) (Rezoagli et al. 2017). In the US alone more than 200.000 cases per year

are affected by this clinical syndrome (Rubenfeld et al. 2005) and this number could even

be significantly higher according to the LUNG SAFE study, an international multicentre

prospective cohort study conducted in intensive care units (ICU) in 50 countries based on

the current Berlin definition, showing that clinicians, even trained on ARDS diagnosis,

missed almost 40% of ARDS diagnosis (Bellani et al. 2016, Rezoagli et al. 2017). This

study also pointed to the fact, that ARDS occurrence in intensive care units was estimated

to be 10.4% of the admissions and more than doubled (23.4%) when patients had to be

mechanically ventilated (Rezoagli et al. 2017).

One of the main hallmarks of ARDS is an increased pulmonary capillary permeability

leading to accumulation of protein-rich fluid inside the alveoli. This results in damage to

the capillary endo- and alveolar epithelium, causing the release of cytokines further

producing diffuse alveolar damage (Martin 1999, Umbrello et al. 2017). The pathological

features of ARDS have been described by three overlapping phases: an inflammatory

phase, a proliferative phase and a fibrotic phase. However these sequences may be

complicated by other variables such as ventilator induced lung injury (VILI) (Umbrello et

al. 2017). ARDS remains a syndrome with an elevated incidence and is associated with a

mortality ranging from 40% to 60% and a significant long-term morbidity (Phua et al.

2009, Herridge 2011, Umbrello et al. 2017). A high dead space fraction in the lung,

restricting the proportion of the lungs capable of participating in gas exchange, was

Introduction

23

correlated to an increase in mortality for patients with ARDS (Nuckton et al. 2002,

Rezoagli et al. 2017). The ultimate cause of death is often through multiple-system organ

failure (MSOF) due to systemic inflammation rather than hypoxia and is not fully

understood (Montgomery et al. 1985, Meduri et al. 2009, Umbrello et al. 2017).

ARDS can be caused by several factors like pneumonia, sepsis, gastric content aspiration,

trauma, pancreatitis, inhalation injury, burns, non-cardiogenic shock, drug overdose, near

drowning, acute lung injury, smoking and also by mechanical ventilation (Ferguson et al.

2012, Rezoagli et al. 2017, Umbrello et al. 2017). It is noteworthy that ARDS does not

develop in the majority of patients with clinical risk factors for this syndrome, suggesting

that genetic or epigenetic susceptibility may also play an important role in the pathogenesis

of this disorder. In fact one third of all patients with ARDS have a hyper-inflammatory

subphenotype with elevated plasma concentrations of interleukin-6 (IL-6), interleukin-8

(IL-8), and tumor necrosis factor α (TNF-α) (Thompson et al. 2017).

Currently, no effective pharmacological treatments exist for ARDS (Thompson et al. 2017)

and the primary target for the treatment of ARDS is to ensure gas exchange while

minimizing the risk of VILI. The patient management strategies remain to date largely only

supportive (Umbrello et al. 2017) and consists of prone positioning patients, fluid

management, extracorporeal membrane oxygenation (ECMO), inhaled vasodilators,

corticosteroids and a protective mechanical ventilation with low tidal volumes (Matthay et

al. 2012, Umbrello et al. 2017). Despite the fact that mechanical ventilation is an important

tool for life support of ARDS patients, it also has the potential to exert pathological

mechanical forces on different lung cells leading to Ventilator-Induced Lung Injury (VILI)

(Slutsky and Imai 2003).

1.5 The role of TRPV4 in ARDS and VILI

In the lungs TRPV4 is expressed in different pulmonary cell types, such as bronchiolar and

alveolar epithelial cells, alveolar macrophages, neutrophils, smooth muscle cells and

endothelial cells (Jia et al. 2004, Alvarez et al. 2006, Hamanaka et al. 2010, Nayak et al.

2015, Yin et al. 2016) and has been suggested to play a role in pulmonary diseases and

diseases conditions including pulmonary hypertension, cough, asthma, cystic fibrosis,

edema formation, ciliary beat dysfunction, chronic obstructive pulmonary disease (COPD),

Introduction

24

acute lung injury (ALI) and ARDS (reviewed in Goldenberg et al. 2015, Darby et al. 2016,

Scheraga et al. 2017). ARDS can be induced by acid inhalation or by ventilation with high

tidal volumes leading to ventilator induced lung injury (VILI) (Goldenberg et al. 2015) and

this section will focus on the role of TRPV4 in ARDS and VILI.

In this concept TRPV4 has been shown to mediate the acute lung injury response to a

sterile stimulus in a murine model of acid inhalation. It has been demonstrated to mediate

the lung injury response in mice exposed to hydrochlorid acid (HCL), assessed by lung

vascular permeability increase, inflammatory cell influx and pro-inflammatory cytokine

levels (e.g. IL- IL-6, KC, IL-1β, MCP-1, RANTES) (Balakrishna et al. 2014). Protection

from acute lung injury in response to HCL was observed in TRPV4-KO mice or in mice

treated with different small molecule TRPV4 inhibitors showing significantly lower levels

of chemokines/cytokines and permeability increase compared to wildtype mice

(Balakrishna et al. 2014).

Another sterile cause with the potential to result in lung injury is mechanical ventilation

(Goldenberg et al. 2015). The potential involvement of cation channels in mediating the

response generated in the lung after mechanical stress has been demonstrated in isolated rat

lungs in which the increase in microvascular permeability was abolished by gadolinium

(inhibitor of stretch-activated nonselective cation channels) and concluded that stretch-

activated cation channels may initiated the increase in permeability induced by mechanical

ventilation through an increase in intracellular Ca2+

concentration (Parker et al. 1998).

TRPV4 has been shown to be a particularly promising candidate for the initiation of the

acute calcium-dependent permeability increase during ventilation in isolated mouse lungs

(Hamanaka et al. 2007). In this study pretreatment with inhibitors of TRPV4 (Ruthenium

red), arachidonic acid production (methanandamide), or P-450 epoxygenases (miconazole)

prevented the increases in lung permeability in isolated perfused mice lungs during

mechanical ventilation, an effect that was also absent in TRPV4-KO mice compared to

untreated WT mice. Furthermore lung distention caused calcium entry in the isolated mice

lungs which was absent in TRPV4-KO and Ruthenium red treated lungs (Hamanaka et al.

2007). Pharmacological activation of TRPV4 with 4α-PDD also showed an increase in

endothelial permeability in isolated rat lungs and this effect was reversed by Ruthenium

red administration (Alvarez et al. 2006). Prevention of ventilator induced-lung edema

formation was also demonstrated by inhalation of nanoparticles releasing Ruthenium red in

an murine isolated perfused lung model of ventilation (Jurek et al. 2014).

Introduction

25

TRPV4 has also been suggested to play a prominent role in mediating the mechanical

activation of macrophages suggesting to initiate this pathological response during

ventilation (Hamanaka et al. 2010). An important role for alveolar macrophages in

mechanical ventilation models has been demonstrated by depletion of macrophages in rat

lungs using clodronate-filled liposomes resulting in an attenuation of ventilator-induced

lung injury, where high volume ventilation resulted not only in an activation-associated

adhesion of alveolar macrophages but also in an increased alveolar protein leakage and

lung edema formation that was attenuate by depletion of macrophages (Frank et al. 2006,

Eyal et al. 2007). A more recent investigation linked TRPV4 channels and macrophages in

the role of modulating VILI. In this study the ventilator-induced lung injury was markedly

attenuated in TRPV4-KO mice, whereas reintroduction of TRPV4-WT macrophages in

TRPV4-KO mice reconstituted the lung injury response to mechanical ventilation, showing

that TRPV4 activation in macrophages plays a crucial role in initiating this injury

(Hamanaka et al. 2010). Additionally macrophages isolated from WT mice exhibited an

increase in intracellular Ca2+

and produced reactive oxygen species in response to 4α-PDD

that was not seen in TRPV4-KO cells (Hamanaka et al. 2010). Macrophages TRPV4 has

also been shown to regulate cytokine secretion (Scheraga et al. 2016, Scheraga et al. 2017).

Previous studies have demonstrated that most alveolar cells are capable of producing pro-

inflammatory mediators such as tumor necrosis factor (TNF)-α, interleukin (IL) -6, IL-8

and IL-1β when stretched in vitro or when ventilated in ex vivo experiments (nicely

reviewed in Halbertsma et al. 2005). TRPV4 activation has also been promoted to induce

inflammatory pathways in immune cells and to induce pro-inflammatory

cytokines/chemokines secretion in response to lipopolysaccharide (LPS) in epithelial cells

(Henry et al. 2016, Scheraga et al. 2017). A recent study in fetal mouse distal lung

epithelial cells linked cell stretch and an inflammatory response to TRPV4 in vitro and

demonstrated that TRPV4 may also play an important role in the transduction of

mechanical signals in the lung epithelium during ventilation by modulating the stretch-

induced release of pro-inflammatory cytokines (Nayak et al. 2015). Taken together, these

data demonstrated the potential of TRPV4 inhibition for the prevention of ARDS in

response to mechanical ventilation.

Introduction

26

1.6 The aim of the thesis

To better understand TRPV4 biology and its role in the regulation of membrane barrier

integrity, the purpose was firstly to establish in vitro and in vivo models of permeability

and to investigate on the role of TRPV4 in modulating membrane barrier integrity with

pharmacological tools. We used two reported selective activators of TRPV4,

GSK1016790A and 4α-PDD and the potent and selective TRPV4 blocker GSK2193874,

promoted as an excellent in vitro and in vivo tool for the study of TRPV4. We also

questioned the link between pharmacological activation of TRPV4 and the corresponding

functional observations on calcium influx and barrier integrity when there is no classical

signal transduction pathway affirmed that can be followed to substantiate such a link. We

therefore hypothesized that such effects may also be caused by selective TRPV4 mediated

cytotoxicity.

The second part of this thesis focusses on the effect of lung cell stretch due to over-

distention of lung region during mechanical ventilation and the role of TRPV4 in

mediating a pathological cellular response to these physical stimuli. Therefore the purpose

was to establish cell stretch experiments in vitro to investigate firstly the cellular calcium

response of lung cells to a mechanical stimulus and secondly the effect of TRPV4

inhibition in this system. The next aim was to find other possible stretch induced cellular

responses in lung cells that might play a role in VILI and ARDS such as inflammatory

mediators and the potential of TRPV4 inhibition in such a system. The final aim was to

establish a disease related murine model of ventilation hypothesizing that a selective orally

active inhibitor of TRPV4 could improve cell stretch induced pathological cellular

response during mechanical ventilation also in vivo, such as lung permeability increase and

inflammatory mediator release.

Methods

27

2 Methods

2.1 In vitro studies

2.1.1 TER measurement

The CellZscope Automated Cell Monitoring System (nanoAnalytics GmbH; Münster,

Germany) was used for continuous measurement of transepithelial/transendothelial

electrical resistance (TER). A direct correlation between the permeability of a cell layer

and its transepithelial/-endothelial electric resistance exists. Therefore a cell layer cultured

till confluence on a permeable membrane forms the interface between two medium-filled

compartments while a voltage (AC) is applied across the electrodes and TER and

capacitance (CCL) of the layer is measured over time by recording the frequency-dependent

impedance (Z) and using an electrical equivalent circuit to analyze the data. Human

umbilical vein endothelial cells (HUVECs, EndoGRO™, SCCE001, Merck Millipore,

USA) were seeded at a density of 3 x 104 cells per transwell filters (Corning #3470; 0.4 μm

Pores; Polystyrene; 24 wp) in 100 µl EndoGRO-LS Complete Culture Media Kit

(SCME001, Millipore, Billerica, MA, USA) and incubated at 37°C in humidified air for 24

h. Afterwards cells on Transwell filters were incubated in humidified air for another 24 h

in an Invivo2 300 Hypoxia Chamber (Ruskinn Technology, Pencoed, UK) at 1% O2, 5%

CO2 at 37°C. The 24 wells in the CellZscope were filled with 810 µL of medium and

warmed up in the incubator. Afterwards cells on transwell filters were transferred in the

machine and another 160 µl of medium was given on the apical side of the transwell filters.

To maintain optimal culture conditions, the CellZscope was placed in a tissue culture

incubator (37°C, 5% CO2) and TER measurement was initiated. Cells were preincubated in

the presence or absence of different concentrations of the TRPV4 antagonist GSK2193874

for 1h or more and afterwards different concentrations of the TRPV4 agonists 4α-PDD or

GSK1016790A were added from a 10-fold concentrate in medium on the apical side of the

transwell filters and TER was measured continually for up to 24 h. In this system the effect

of the cytokines IL-1β and TNF-α on TER were also investigated. For better illustration,

TER was normalized (0% was defined as the TER level of the no cell control and 100%

was defined as the largest mean in each data set) with GraphPad Prism Software and

group mean only was shown as percentage.

To investigate whether the effect of TRPV4 activation can not only be prevented but also

reversed by TRPV4 inhibition, HUVECs were treated firstly with the TRPV4 agonist

Methods

28

GSK1016790A and afterward with the TRPV4 antagonist GSK2193874 during TER

measurement.

TER measurement was also performed in small airway epithelial cells (SAEC)

differentiated on transwells and cultured in air-liquid interface (ALI), as described in

section 2.1.10. Therefore ALI cultures on transwell filters were placed in the cellZscope

and medium was added basolaterally and apically to enable impedance measurement as

previously described. The cellZscope was placed in an incubator at 37°C, 5% CO2 in

humidified air and TER measurement was initiated. Cells were preincubated in presence or

absence of different concentrations of the TRPV4 antagonist GSK2193874 for 1 h or more

and afterwards were treated with the TRPV4 agonist 4α-PDD or GSK1016790A and TER

measurement was performed continuously for up to 24 h.

2.1.2 Vascular Permeability Assay

HUVECs were seeded on HTS-Transwell-96 Well Plates (Corning # 3391; 0.4 µm Pores;

Polycarbonate Membrane, NY, USA) with a density of 25 x 103 cells/well in 100 µl

medium. The reservoir plate was filled with 25 ml medium and cells were incubated at

37°C and 5% CO2 in humidified air for 24 h. Afterwards cells on Transwell filters were

incubated in humidified air for another 24 h in an Invivo2 300 Hypoxia Chamber (Ruskinn

Technology, Pencoed, UK) at 1% O2, 5% CO2 at 37°C. The HTS-Transwell-96 well plate

was then transferred on a receiver plate which was filled on the basal side with 225 µl

medium/well and different concentrations of the stimuli. The apical medium was

exchanged by 100 µl medium with the same concentration of the stimuli as the basal

medium and cells were incubated at 37°C and 5% CO2 in humidified air up to 20 h.

Fluorescein isothiocyanate-dextran (FITC-Dextran, 2000 kDa, #FD2000S, Merck KGaA,

Darmstadt, Germany) solved in H2O (25 mg/ml) was diluted 1:100 in medium. Afterwards

20 µl of the FITC-Dextran dilution was given on the apical side of each transwells and the

transwell plate was incubated protected from light at RT for 1 h. Then 100 µl medium from

each well of the basal side of the receiver plate was transferred to a black 96-well plate

(96F Nunclon Delta Black Microwell SI, Nunc, Langenselbold, Germany) and

fluorescence was measured in a SpectrMax M5 plate reader (Molecular Devices,

Sunnyvale, CA). In this system the effect of the cytokines IL-1β and TNF-α on membrane

permeability were also investigated.

Methods

29

2.1.3 Calcium 6 assay on the FLIPRTETRA

Pharmacological activation and inhibition of TRPV4 was analyzed using the FLIPR

Calcium 6 Assay kit (molecular devices #R8191 bulk kit) and was performed according to

the manufacturer’s instructions. Briefly cells were seeded with a density of 1 x 104

(HUVECs) or 3 x 104 (NCI-H292) cells/well in appropriated medium with 25 μL

medium/well on assay plates (384 well Poly-D-Lysin black/clear bottom, Biocoat #4663)

and incubated for 24 h. Cells were incubated for 2 h with the calcium 6 dye solution

(calcium 6 dye in assay buffer (HBSS [+ CaCl2/MgCl2] + 20 mM Hepes + 0.1% BSA; pH

7,4) according to the manufacturer´s instruction at 37°C in 5% CO2, humidified air. For

fluorescence measurement, cells were transferred to the FLIPR and buffer or compounds

were given in 10 μl/well with different concentrations and cells were preincubated in the

presence or absence of different concentrations of the TRPV4 antagonist GSK2193874 for

15 min during measurement (FLIPRTETRA

, Molecular Devices, excitation 470-495 nm,

emission 515-575 nm, with 2 read intervals, first read interval with 1 read per second for

10 s before compound dispersion and 1 read per second after first compound addition for

50 s and a second read interval with 1 read every 10 s for 84 times) before second

compound addition. For EC 50 or IC 50 measurement cells were afterward stimulated with

10 μl/well of different concentrations of the TRPV4 agonist 4α-PDD or GSK1016790A

during read out (FLIPRTETRA

, Molecular Devices, excitation 470-495 nm, emission 515-

575 nm, with 2 read intervals, first read interval with 1 read per second for 10 s with 1 read

before and 9 reads after agonist dispersion and a second read interval with 1 read every 3 s

for 210 times) and the concentration-dependent inhibition or activation of calcium influx

was determined. To investigate the different effects of the TRPV4 agonist 4α-PDD or

GSK1016790A on calcium-influx over time the readout after the second compound

addition was also extended up to ~ 5 h (FLIPRTETRA

, Molecular Devices, excitation 470-

495 nm, emission 515-575 nm, with 2 read intervals, first read interval with 1 read per

second for 10 s with 1 read before and 59 reads after second compound dispersion and a

second read interval with 1 read every 30 s for 596 times). To investigate whether the

effect of TRPV4 activation can also be reversed by TRPV4 inhibition, cells were also

treated firstly with the TRPV4 agonist GSK1016790A and afterward with the TRPV4

antagonist GSK2193874 in intracellular calcium concentration measurements.

Methods

30

2.1.4 TRPV4 agonism effect on LDH release

Cells were seeded (25 x 103 cells/well for HUVECs; 5 x 10

4 cells/well for NCI-H292) in

appropriate medium on 96 well culture plates (NunclonTM Delta Surface, Thermo

scientific) and incubated for 24 h. Afterwards cells were preincubated for 1 h in the

presence or absence of the TRPV4 antagonist GSK2193874 in 100 μL medium. Medium

was removed one more time and cells were incubated at 37°C in 5% CO2, humidified air

for up to 12 h in 100 μL medium in presence or absence of different concentrations of the

TRPV4 agonist GSK1016790A or 4α-Phorbol 12, 13-didecanoate (4α-PDD). Then

supernatant was collected at different time points and lactate dehydrogenase (LDH) release

was detected using a CytoTox96® Non-Radioactive Cytotoxicity Assay kit (Promega,

Madison, WI) following manufacturers instruction (see section 2.3.5).

Experiments were also performed with Hank´s Balanced Salt Solution (HBSS, Gibco, Life

technologies, Grand Island, NY) or Dulbecco's Phosphate-Buffered Saline (DPBS, Gibco,

Life technologies, Grand Island, NY) in the presence or absence of CaCl2 and MgCl2.

2.1.5 RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay

The RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay (Promega, Madison,

USA) is a live-cell real-time assay that measures the exposure of phosphatidylserine (PS)

on the outer leaflet of the cell membrane during the apoptotic process and is detected by

annexin V binding with a simple luminescence signal. The assay also includes a cell-

impermeant, profluorescent DNA dye, which detects necrosis. In the assay, time-dependent

increases in luminescence that occur before increases in fluorescence reflect the apoptotic

process. A significant time delay between the emergences of PS, indicated by Annexin V

binding, leading to a luminescence signal and the loss of membrane integrity visualized by

fluorescence signal, indicate an apoptotic phenotype leading to secondary necrosis.

Increases in fluorescence or increase in both luminescence and fluorescence concurrently

consist with necrosis or other non-apoptotic mechanisms.

The RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay were performed as

prescribed by the manufacturer. Briefly HUVECs were seeded with a density of 25 x 103

cells/well on 96 well white plates (96F Nunclon™ Delta White Microwell SI, Thermo

Fisher Scientific, Roskilde, Denmark) in 50 µl medium (SCME001, Millipore, Billerica,

Methods

31

MA, USA) and incubated at 37°C in 5% CO2, humidified air for 24 h. Afterwards cells

were preincubated for 1 h in the presence or absence of the TRPV4 antagonist

GSK2193874. Cells were then treated with different concentration of the TRPV4 agonists

GSK1016790A or 4α-PDD and directly equal volume of detection reagent was added, the

plate was covered with an imaging seal (4titude 4ti-0516/96, LabSource, Switzerland) and

a kinetic mode (1 read every 30 sec for up to 20 h) using a multimode instrument with

temperature control was initiated for assay signal detection.

2.1.6 Cell-IQ®

The Cell-IQ® (Chip-Man Technologies, Tampere, Finland) is a fully integrated live cell

imaging and comprises a temperature controlled incubator, a tailored gaseous environment

and the use of light emitting diodes for both phase and fluorescence imaging allowing

biological cellular responses to be monitored in real time.

HUVECs were seeded (25 x 103 cells/well) in medium (SCME001, Millipore, Billerica,

MA, USA) on 96 well culture plates (NunclonTM

Delta Surface, Thermo scientific) and

incubated at 37°C in 5% CO2, humidified air for 24 h. Afterwards cells were preincubated

for 1 h in the presence or absence of the TRPV4 antagonist GSK2193874. Cells were then

incubated at 37°C in 5% CO2, humidified air for up to 4 h in 100 μL medium in presence

or absence of different concentrations of the TRPV4 agonist GSK1016790A and live cell

imaging was recorded in a Cell-IQ®

.

2.1.7 TRPV4 agonism effect on cytokine release

NCI-H292 cells were seeded with a density of 5 x 104 cells/well with 200 μL RPMI-1640

medium (Gibco, Grand Island, N.Y.) containing 10% heat-inactivated fetal bovine serum

(FBS) on 96 well culture plates (NunclonTM Delta Surface, Thermo scientific) and

incubated at 37° C in 5% CO2, humidified air for 24 h. Afterwards medium was removed

and cells were preincubated for 1 h in the presence or absence of the TRPV4 antagonist

GSK2193874 [1 µM] in 200 μL medium. Medium was removed one more time and cells

were incubated at 37°C in 5% CO2, humidified air for 24 h in 200 μL medium in presence

or absence the TRPV4 agonist GSK1016790A or 4α-PDD. Then supernatant was collected

Methods

32

and stored at −80°C for later analyses or cytokine measurement was performed using

multiplexing technology from Meso Scale Discovery (see section 2.3.2).

2.1.8 Uniaxial cell strain and microscopy

Uniaxial cell strain was performed on the Stretch/compression device (University Ulm,

Ulm, Germany), a device for simultaneous live cell imaging during uniaxial mechanical

strain or compression (Gerstmair et al. 2009). An elastic silicon membrane (Specialty

Manufacturing, Saginaw, MI 48603-3440 USA) was cut into a rectangular piece (9 x 2 cm)

and clamped into the membrane holders that shape the membrane into a chamber

(Gerstmair et al. 2009), autoclaved and coated overnight at 4°C with fibronectin (5 µg/ml

in PBS, both from Sigma-Aldrich, Steinheim, Germany). Human lung epithelial cells

(NCI-H292) were seeded in the elastic silicon chamber (4 x 105 cells/membrane) and

cultivated in medium at 37°C in 5% CO2, humidified air for 24h. Prior to imaging, the cells

were pre-incubated in medium at 37°C, 5% CO2 with 2 µM of the fluorescent Ca2+

dye

fluo-4 and 0.2% Pluronic F127 (Molecular Probes, Karlsruhe, Germany), protected from

light with or without the TRPV4 antagonist GSK2193874 [1 μM] for 30 min and another

30 min at RT. For cell stretch, the medium was replaced with bath solution (pH 7.4; 140

mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose, and 10 mM HEPES; all

from Sigma-Aldrich). Then the membranes were fastened onto the stretch apparatus

(Gerstmair et al. 2009), mounted on a Zeiss Axiovert 200 (Carl Zeiss, Oberkochen,

Germany) with a 20X plan Neofluar Zeiss objective. Images were acquired with a

CoolSnap EZ CCD camera and Metamorph software (exposure time of 30 ms and an

acquisition rate of 0.5 frames per second) and an EGFPfilter cube (excitation 470/20 nm,

emission 525/25 nm, dichroic 490 nm). The membranes were stretched at RT with a

triangular waveform one single time from 0% to 80% length increase and back to 0%

within 800 ms. Prior to this stretch protocol other stretch protocols with different stretch

amplitudes and frequencies were performed.

The average grey values in the image sequence were determined with ImageJ (Abràmoff et

al. 2004) by drawing a region of interest that comprised the adherence area of a single cell.

To compensate for the slight sideward shift of the cell after the strain, the region of interest

was manually repositioned. Data were transferred to MS-excel and after background

subtraction, the average fluorescence values of each cell before and 10 s after the strain

Methods

33

were determined. The strain-induced change after stimulation was expressed as the percent

change in intensity compared to the baseline signal before stretch.

2.1.9 Equibiaxial cell strain

The Flexcell FX-5000 Tension System (FX5K®; Flexcell International Corp,

Hillsborough, NC) was used to apply mechanical cyclic tensile stretch on lung epithelial

cells (NCI-H292) and human monocyte derived macrophages (see section 2.1.10). The

FX5K® is a computer-based system that uses a vacuum to strain cells adhered to flexible

silicon membranes (BioFlex® plates; Flexcell International Corp) arranged in a format of

six wells per plate with a total growth area of 9.62 cm2 per well. The deformation of the

flexible membrane also causes the attached cells to deform. NCI-H292 cells were seeded

onto Collagen Type I-coated BioFlex® plates at a density of 1 × 106 cells/well,

macrophages at a density of 2 x 106 cells/well. Cells remained untreated or were pretreated

with compounds and exposed to continuous mechanical stimulation with an equibiaxial

half sinusoidal waveform with an elongation from 8% to 30% and a frequency of 1.25 Hz

for up to 48 h at 37°C in 5% CO2, humidified air. Control cultures were grown under the

same conditions but without the strain protocol. Then supernatant was collected and stored

at −80°C for later analyses. Prior to this stretch protocol other stretch protocols with

different stretch amplitudes and frequencies had to be performed.

2.1.10 Cells

HUVECs

Human umbilical vein endothelial cells (HUVECs, EndoGRO™, SCCE001, Merck

Millipore, USA) were cultured in EndoGRO-LS Complete Culture Media Kit (SCME001,

Millipore, Billerica, MA, USA). Washing steps were performed with Dulbecco's

Phosphate-Buffered Saline (DPBS, Gibco, Life technologies, Grand Island, NY) and

TrypLE Express (GIBCO, Life technologies, Grand Island, NY) was used for cell

detachment.

Methods

34

NCI-H292

Lung epithelial cells NCI-H292 (Cat. No. CRL-1848TM

from the American Type Culture

Collection ATCC, Manassas, VA) were cultured in RPMI-1640 medium (Gibco, Grand

Island, N.Y., containing 10% heat-inactivated fetal bovine serum). Washing steps were

performed with DPBS (Gibco, Life technologies, Grand Island, NY) and TrypLE Express

(GIBCO, Life technologies, Grand Island, NY) was used for cell detachment.

SAEC

Small airway epithelial cells (SAECs, Lonza, Donor # 408031, Verviers, Belgium) were

cultured and differentiated following the Lonza CloneticsTM

S-ALITM

air-liquid interface

medium protocol. Briefly SAECs were seeded into cell culture flask (T175 NUNC flask,

178883, Thermo Fischer) on day - 8 in Clonetics S-ALI growth medium. On day - 4 cells

were trypsinised and seeded with a density of 22 x 103 cells/well on Corning Transwell

filters (Corning #3470; 0.4 μm Poren; Polystyrene; 24 wp). On day 0 airlift of the cells was

performed by removing the apical medium and substituting the basolateral growth medium

with S-ALI Differentiation Medium (Clonetics S-ALI differentiation medium). On the

apical side cells were washed to remove growth factors. Afterwards SAECs were

differentiated in air-liquid interface (ALI) for at least 4 weeks with basolateral medium

changes 3 times a week with apical washing step ones a week.

Human Monocyte Derived Macrophages

Human whole blood was obtained from anonymised healthy volunteers. Blood was

donated by internal donors at the centre for occupational health at Boehringer Ingelheim in

Biberach. The donors provided signed informed consent that allows use for scientific

purposes. Peripheral blood mononuclear cells (PBMCs) were isolated by means of density

gradient centrifugation using Ficoll-Paque™ and a Leucosep Tube (Greiner Bio-One

GmbH) according to manufacturer’s instructions. CD14 positive monocyte purification

was performed by magnetic activated cell sorting (MACS) according to the manufacturer’s

instructions (Monocyte Isolation Kit II, Miltenyi Biotec) and seeded 2 x 106 cells/well in

XVIVO-10 medium (Lonza) on Collagen Type I-coated BioFlex®

plates (BioFlex® plates;

Methods

35

Flexcell International Corp). Medium was supplemented with either 100 ng/mL

Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) to induce an M1

phenotype or Macrophage Colony Stimulating Factor (M-CSF) to induce an M2 phenotype

for 7 days.

2.2 In vivo studies

2.2.1 Effect of TRPV4 activation on vascular permeability

TRPV4 induced vascular permeability increase was investigated in male Balb/c or

C57BL/6J mice (Charles River, Sulzfeld, Germany) with weights ranging from 20 - 25 g.

Mice were held at 55% relative humidity, 22°C with a day-night-cycle of 12 h. For

experimental procedure the dye Evans blue was used to monitor vascular leakage by

measurement of Evans blue in formamide extracts of mice tissue after protein leakage

induction.

Experimental procedure:

time point - 2 h : application of TRPV4 antagonist (p.o.)

time point - 10 min: narcosis (i.p.)

time point - 1 min: Evans blue application (i.v.)

time point 0 h: application of TRPV4 agonist (i.v. or i.t.)

time point + x min: euthanasia and samples collection

Two hours before agonist addition mice were pretreated with different concentrations of

the TRPV4 antagonist GSK2193874 (solved in 0.5% Natrosol, Merck, # 8.22068.0500)

given orally (p.o.) with a volume of 10 ml/kg. Afterwards mice were anesthetized with

Medetomidin (Zoetis, # 07725752) /Midazolam (Roche, # 03085793) /Fentanyl (Janssen, #

4795545) (0.5 mg/kg + 5 mg/kg + 0.05 mg/kg; application volume 150 μl/animal) given

intraperitoneal (i.p.) at time point -10 min and were placed on thermostatically-controlled

heat mats to preserve body temperature during experimental procedure. At time point -1

min, 2% Evans blue (Sigma; # E2129) and 33 U/ml Heparin (Ratiopharm; # 3029843)

solved in 0.9% NaCl was applicated with a volume of 100 µl per mouse intravenously (i.v.)

on the tail vein. At time point 0 h different concentrations of the TRPV4 agonist

Methods

36

GSK1016790A (solved in 0.9% NaCl, Fresenius) were given intratracheally (i.t.; 50

µl/mouse) or i.v. (100 µl/mouse) through the tail vein. At the desired time points

experiment was stopped. Mice were euthanized by application of an overdose of narcoren

(Merial; # 6088986.00.00) given i.p. (400 mg/ml; ~ 400 µl/mouse). Afterwards mice were

perfused with 10 ml buffer/animal (25 000 IE Heparin, Ratiopharm, # 196621, solved in

500 ml NaCl). Therefor mice chest were opened and a small cut was performed in the left

heart ventricle, where a cannula (23G, 0.6 x 25 mm, Luer Lock, Braun, # 105107) was

inserted to inject the perfusion-buffer. Afterwards organs were excised and washed in PBS

(Lonza, # BE17-516F). Lungs were further prepared and separated in bronchus and

parenchyma. Organs were then added to 750 µl formamide (Sigma, # F7503) in Safe-Lock

reaction tubes (2 ml, Eppendorf, # 0030120094) and incubated overnight at 65°C. On the

next day samples were collected and 250 µl formamide supernatant were given to a 96-

well plate and absorbance was measured at 620 nm in a spectrometer.

2.2.2 Murine mechanical ventilation model

Experiments were performed in female Balb/c mice (n=82; Charles River, Sulzfeld,

Germany) aged 10 - 12 weeks, with weights ranging from 23 to 28 g and were held in the

same conditions as previously describe. Mice were mechanically ventilated and the effect

of ventilation on lung permeability increase, function and inflammatory response was

investigated with or without pretreatment with the orally active TRPV4 inhibitor

GSK2193874.

Experimental procedure:

time point – 1 h: application of the TRPV4 antagonist (p.o.)

time point 0 h: narcosis (i.p.)

time point + 1 h: start of mechanical ventilation

time point + 4 h: euthanasia and samples collection

The TRPV4 antagonist GSK2193874 [90 mg/kg] or the solvent (0.5% Natrosol with

0.015% Tween80) were administered orally by gavage 2 h before ventilation (time point -1

h). At time point 0 h mice received intraperitoneal narcoren [60 mg/kg] and rompun [2.5

Methods

37

mg/kg] solved in 0.9% NaCl (Fresenius) with additional dosing as needed to maintain

appropriate anesthesia. A cannula (Fa. Harvard, USA, Art.-Nr.: NP73-2836) was inserted

into the trachea, sutured, and coupled to a flexiVent (SCIREQ, Montreal, Quebec, Canada)

small animal ventilator and controlled with the Software FlexiWare (Fa. EMKA

Technologies, Paris, France). Mechanical ventilation was performed with different

ventilation protocols, with tidal volumes of 20 ml/kg (control n=5; treated n=4), 30 ml/kg

(control and treated n=8) and 40 ml/kg (control and treated n=4) with a frequency of

75/min and 2 cm H2O PEEP and a control group ventilated with a normal tidal volume of

6.5 ml/kg (n=4) and a frequency of 150/min and 3 cm H2O PEEP for 3 h and an additional

unventilated control group (n=6). During the experiments mice were placed on

thermostatically-controlled heat mats to preserve body temperature in a supine position.

During the 3 h of ventilation on the FlexiVent, parameters such as lung resistance,

compliance and elastance were recorded every 15 min. With the ending of the ventilation

protocol, mice were euthanized with an overdose of narcoren (500 mg/ml, ~ 0.5 ml/ animal

given i.p.) remaining therefor on the ventilator until cardiac arrest. Bronchoalveolar lavage

(BAL) was performed using 2 times 0.8 ml Hanks Salt Solution (Fa. Biochrom AG) and

0.6 mM EDTA (Fa.: Promega). The bronchoalveolar lavage fluid (BALF) was centrifuged

at 1500 rpm at 4°C for 10 minutes and the supernatant was stored at -80°C for later

analyses such as BCA protein assay and cytokine measurements.

2.3 Molecular biology assays

2.3.1 Pierce™ BCA Protein Assay Kit

For total protein concentration measurement in BALF supernatant the Pierce™

BCA Protein Assay Kit (Thermo Scientific, Rockford, USA) was used. The assay is a

formulation based on bicinchoninic acid (BCA) for colorimetric detection and quantitation

of total protein and combines the reduction of Cu+2

to Cu1+

by protein in an alkaline

medium with a selective colorimetric detection of Cu1+

using a reagent containing BCA.

Assays were performed according to the manufacturer’s instructions and absorbance at 562

nm was measured in a spectrophotometer (SpectrMax M5 plate reader, Molecular Devices,

Sunnyvale, CA).

Methods

38

2.3.2 ELISA/MSD

For cytokine measurement in human cell culture supernatants and murine BALF

supernatants the Meso Scale Discovery V- and U-PLEX multiplexing technology (MESO

SCALE DISCOVERY®, Rockville, USA) was used. The assays are sandwich

immunoassays for measuring the levels of protein targets within a single sample. Samples

and detection antibodies conjugated with electrochemiluminescent labels (MSD SULFO-

TAGTM

) are added to plates coated with capture antibodies on independent and well-

defined spots. Analytes in the samples bind to the capture antibodies on the working

electrode surface and later bounding of the detection antibodies to the analytes completes

the sandwich. Plates were loaded in the MSD instrument (MESOTM

SECTOR S 600,

MESO SCALE DISCOVERY, Rockville, USA), where a voltage applied to the plates

electrodes causes the captured labels to emit light, which intensity is measured to provide a

quantitative measure of analytes in the sample. V-PLEX plates were used in the human

Chemokine Panel 1 (Eotaxin, MIP-1β, Eotaxin-3, TARC, IP-10, MIP-1α, IL-8, MCP-1,

MDC and MCP-4), Cytokine Panel 1 (GM-CSF, IL-1α, IL-5, IL-7, IL-12/IL-23p40, IL-15,

IL-16, IL-17A, TNF-β and VEGF) and Pro-inflammatory Panel 1 (IFN-γ, IL-1β, IL-2, IL-

4, IL-6, IL-8, IL-10, IL-12p70, IL-13 and TNF-α) configuration or U-PLEX plates were

individually spotted with the antibody pairs against the desired analytes. For cytokine

measurement in mouse BALF the V-PLEX Pro-inflammatory Panel 1 Mouse Kit (IFN-γ,

IL-1β, IL-2, IL-4, IL-5, IL-6, KC/GRO, IL-10, IL-12p70, and TNF-α.) was used. Assays

were performed according to the manufacturer’s instructions.

2.3.3 Phospho/Total ERK1/2 assay

For the measurement of Extracellular signal Regulated Kinases (ERK) 1 and 2 in cell

lysate the Meso Scale Discovery phosphoprotein assay was used. The Phospho

(Thr202/Tyr204; Thr185/Tyr187)/Total ERK1/2 Assay (MESO SCALE DISCOVERY®,

Gaithersburg, USA) is a sandwich immunoassay and provides a plate pre-coated with

capture antibodies for phosphorylated ERK1/2 ((Thr202/Tyr204; Thr185/Tyr187) and total

ERK1/2 on spatial distinct spots. As for the cytokine measurement, analytes in the samples

binds to the capture antibody on the working electrode and afterwards conjugated detection

antibody bound to the analytes completing the sandwich. Plates were loaded in the MSD

Methods

39

instrument for analysis. Assays were performed according to the manufacturer’s

instructions.

2.3.4 ATP release measurement

For quantifying cell ATP release in supernatant, the ATP-GloTM

Bioluminometric Cell

Viability Assay Kit (Biotium, Hayward, CA) was performed. ATP is an indicator of

metabolically active cells and therefore the number of viable cells can be assessed based on

the amount of ATP available. Furthermore the assay was used for the detection of ATP

release from cells exposed to mechanical stretch. The ATP detection kit takes advantage of

firefly luciferase’s use of ATP to oxidize D-Luciferin and the resulting production of light

to detect the amount of ATP available and was measured in a luminometer. Assays were

performed according to the manufacturer’s instructions.

2.3.5 LDH release

Lactate dehydrogenase (LDH) release was detected using a CytoTox96® Non-Radioactive

Cytotoxicity Assay kit (Promega, Madison, WI) following manufacturers instruction.

Briefly released LDH in culture supernatants is measured with a 30-minute coupled

enzymatic assay, which results in the conversion of a tetrazolium salt (iodonitrotetrazolium

violet; INT) into a red formazan product. The amount of color formed is proportional to the

number of lysed cells. Briefly the release of LDH from damaged cells is measured by

supplying lactate, NAD+ and INT as substrates in the presence of diaphorase. Generation

of a red formazan product in the presence of LDH is proportional to the amount of LDH

released from lysed cells. The conversion of tetrazolium salt to a red formazan product in

the presence of LDH is detectable by measurement of the absorbance at 490 nm using a

SpectrMax M5 plate reader (Molecular Devices, Sunnyvale, CA). Percent cytotoxicity is

calculated by the following formula: 100 x Experimental LDH release divided by the

maximum LDH release (= lysate).

Methods

40

2.3.6 Human cAMP / Calcium Signaling PathwayFinder

NCI-H292 seeded in 1 ml medium with a density of 3 x 105 cells/well on a 12 well plate

(Thermo Fisher Scientific, NunclonTM

Delta Surface, #150628, Roskilde, Denmark), were

incubated at 37°C in 5% CO2, humidified air for 24 h. Afterwards cells were preincubated

for 2 h in the presence or absence of the TRPV4 antagonist GSK2193874 [1 µM]. Medium

was removed and cells were stimulated either with 0.1 % DMSO, 1 µM GSK2193874, 10

µM 4α-PDD or 10 µM 4α-PDD and 1 µM GSK2193874 in 1 ml medium and incubated at

37°C in 5% CO2, humidified air for 24 h. For purification of total RNA from cells the

RNeasy®

Mini Kit (Qiagen, #7410, Hilden, Germany) was used.

Cells were washed twice with Dulbecco's Phosphate-Buffered Saline (DPBS, Gibco, Life

technologies, Grand Island, NY) without Ca2+

and Mg2+

and 350 μL RLT buffer

(QIAGEN, #79216) + 1% β-Mercaptoethanol was given to each well and incubated for 5

min on ice. Samples were pooled from 3 wells each group to 1 sample. Afterwards samples

were transferred on a QIAshredder tubes (QUIAGEN, #79656) and centrifuge at 10.000

rpm for 2 min. Afterwards 350 µl 70% cold EtOH was given to each flow-through and

samples were transferred to an RNeasy spin column placed in a 2 ml collection tube and

centrifuge for 15 s at 10.000 rpm. The flow-through was discarded and the collection tube

was used for the next step. 350 μL Buffer RW1 (QIAGEN, Hilden, Germany) was given to

the RNeasy spin column and centrifuge for 15 s at 10.000 rpm to wash the spin column

membrane. The flow-through was discarded and the collection tube was used for the next

step. Afterwards 80 µl of DNase I incubation mix (10 µl DNase I stock solution and 70 µl

buffer RDD) was added to each column and incubated at RT for 15 min. Afterwards 350

μL Buffer RW1 were given to the RNeasy spin column and centrifuge for 15 s at 10.000

rpm to wash the spin column membrane. Flow-through was discarded and collection tubes

were used in the next step. 500 µl Buffer RPE (QIAGEN, Hilden, Germany) was given to

the RNeasy spin column and centrifuge for 15 s at 10.000 rpm. Flow-through was

discarded and another 500 µl Buffer RPE was given to each RNeasy spin column and

centrifuge for 2 min at 10.000 rpm. Afterwards the RNeasy spin column was removed

carefully from the collection tube and placed in a new 2 mL collection tube and centrifuge

at ≥ 10.000 rpm for 1 min. The RNeasy spin column was then placed in a new 1.5 mL

collection tube and 50 µl RNase-free water (QIAGEN, Hilden, Germany) was added

directly to the spin columns membrane and centrifuge for 1 min at 10.000 rpm to elute the

Methods

41

RNA. RNA concentration of each sample was determined by measuring the absorbance

(260 nm) in a NanoDrop 8000 (Thermo Fisher Scientific).

For cDNA synthesis from purified RNA in prior to real-time PCR the RT2 First Strand Kit

12 (Qiagen, #330401, Hilden, Germany) was used and performed according to the

manufacturer’s instructions. Briefly the RNA samples were treated with DNA elimination

mix according to the RNA concentration of each samples (25 ng - 5 µg RNA sample + 2 µl

Buffer GE + x µl RNase-free water to a total volume of 10 µl) and incubated for 5 min at

42°C and then placed immediately on ice for at least 1 min. 10 µl of reverse-transcription

mix was added to each tube containing 10 µl DNA elimination mix and incubated at 42°C

for exactly 15 min. The reaction was then immediately stopped by incubating at 95°C for 5

min. 91 µl RNase-free water was added to each reaction and cDNA samples were placed

on ice or stored at -20°C before proceeding with PCR.

Afterwards the Human cAMP / Calcium Signaling PathwayFinder RT2 Profiler PCR Array

(Qiagen, # 330231, Hilden, Germany) for 384-well (4 x 96 PCR arrays) was performed

according to the manufacturer’s instructions. Briefly the PCR components mix was

prepared by adding 548 µl of RNase-free water and 650 µl RT2 SYBR Green ROX qPCR

Mastermix 2 (SABiosciences, Qiagen, #330520, Hilden, Germany) to 102 µl of each

cDNA samples. Afterwards the PCR components mix from the 4 samples was dispensed

into the cAMP / Calcium Signaling PathwayFinder RT2 Profiler PCR Array. The array was

sealed with an optical adhesive film and placed in the real-time PCR cycler system ViiA 7

(Applied Biosystems by Life Technologies, Carlsbad, USA) with 1 cycle at 95°C for 10

min to HotStart DNA Taq Polymerase and further 40 cycles (1 min each) at 60°C

performing fluorescence data collection. Afterwards the CT values for all wells were

exported to a blank Excel® spreadsheet for use with the SABiosciences PCR Array Data

Analysis Template Excel and Web-based software

(www.SABiosciences.com/pcrarraydataanalysis.php.).

Table 1: Gene table of the Human cAMP / Calcium Signaling PathwayFinder RT2 Profiler PCR Array

Position Unigene Refseq Symbol Description

A01 Hs.99913 NM_000684 ADRB1 Adrenergic, beta-1-, receptor

A02 Hs.171189 NM_001621 AHR Aryl hydrocarbon receptor

Methods

42

A03 Hs.159118 NM_001634 AMD1 Adenosylmethionine decarboxylase 1

A04 Hs.645475 NM_001657 AREG Amphiregulin

A05 Hs.460 NM_001674 ATF3 Activating transcription factor 3

A06 Hs.150749 NM_000633 BCL2 B-cell CLL/lymphoma 2

A07 Hs.502182 NM_001709 BDNF Brain-derived neurotrophic factor

A08 Hs.194143 NM_007294 BRCA1 Breast cancer 1, early onset

A09 Hs.65425 NM_004929 CALB1 Calbindin 1, 28kDa

A10 Hs.106857 NM_001740 CALB2 Calbindin 2

A11 Hs.282410 NM_006888 CALM1 Calmodulin 1 (phosphorylase kinase, delta)

A12 Hs.515162 NM_004343 CALR Calreticulin

B01 Hs.417050 NM_003914 CCNA1 Cyclin A1

B02 Hs.523852 NM_053056 CCND1 Cyclin D1

B03 Hs.647078 NM_004935 CDK5 Cyclin-dependent kinase 5

B04 Hs.72901 NM_004936 CDKN2B Cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4)

B05 Hs.119689 NM_000735 CGA Glycoprotein hormones, alpha polypeptide

B06 Hs.150793 NM_001275 CHGA Chromogranin A (parathyroid secretory protein 1)

B07 Hs.465929 NM_001299 CNN1 Calponin 1, basic, smooth muscle

B08 Hs.516646 NM_004379 CREB1 CAMP responsive element binding protein 1

B09 Hs.200250 NM_183011 CREM CAMP responsive element modulator

B10 Hs.483811 NM_001330 CTF1 Cardiotrophin 1

B11 Hs.8867 NM_001554 CYR61 Cysteine-rich, angiogenic inducer, 61

B12 Hs.505777 NM_004083 DDIT3 DNA-damage-inducible transcript 3

C01 Hs.171695 NM_004417 DUSP1 Dual specificity phosphatase 1

C02 Hs.326035 NM_001964 EGR1 Early growth response 1

C03 Hs.1395 NM_000399 EGR2 Early growth response 2

C04 Hs.511915 NM_001975 ENO2 Enolase 2 (gamma, neuronal)

C05 Hs.166015 NM_020996 FGF6 Fibroblast growth factor 6

C06 Hs.25647 NM_005252 FOS FBJ murine osteosarcoma viral oncogene homolog

C07 Hs.590958 NM_006732 FOSB FBJ murine osteosarcoma viral oncogene homolog B

C08 Hs.516494 NM_002054 GCG Glucagon

C09 Hs.654463 NM_005261 GEM GTP binding protein overexpressed in skeletal muscle

C10 Hs.251412 NM_000164 GIPR Gastric inhibitory polypeptide receptor

C11 Hs.406266 NM_000189 HK2 Hexokinase 2

C12 Hs.90093 NM_002154 HSPA4 Heat shock 70kDa protein 4

D01 Hs.743241 NM_005347 HSPA5 Heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa)

D02 Hs.89679 NM_000586 IL2 Interleukin 2

D03 Hs.654458 NM_000600 IL6 Interleukin 6 (interferon, beta 2)

D04 Hs.583348 NM_002192 INHBA Inhibin, beta A

D05 Hs.25292 NM_002229 JUNB Jun B proto-oncogene

D06 Hs.2780 NM_005354 JUND Jun D proto-oncogene

D07 Hs.150208 NM_002234 KCNA5 Potassium voltage-gated channel, shaker-related subfamily,

member 5

D08 Hs.2795 NM_005566 LDHA Lactate dehydrogenase A

D09 Hs.134859 NM_005360 MAF V-maf musculoaponeurotic fibrosarcoma oncogene homolog

(avian)

D10 Hs.407995 NM_002415 MIF Macrophage migration inhibitory factor (glycosylation-inhibiting

factor)

D11 Hs.503878 NM_000615 NCAM1 Neural cell adhesion molecule 1

D12 Hs.113577 NM_000267 NF1 Neurofibromin 1

Methods

43

E01 Hs.709191 NM_000625 NOS2 Nitric oxide synthase 2, inducible

E02 Hs.1832 NM_000905 NPY Neuropeptide Y

E03 Hs.563344 NM_006186 NR4A2 Nuclear receptor subfamily 4, group A, member 2

E04 Hs.75812 NM_004563 PCK2 Phosphoenolpyruvate carboxykinase 2 (mitochondrial)

E05 Hs.147433 NM_182649 PCNA Proliferating cell nuclear antigen

E06 Hs.339831 NM_006211 PENK Proenkephalin

E07 Hs.445534 NM_002616 PER1 Period homolog 1 (Drosophila)

E08 Hs.491582 NM_000930 PLAT Plasminogen activator, tissue

E09 Hs.170839 NM_002667 PLN Phospholamban

E10 Hs.96 NM_021127 PMAIP1 Phorbol-12-myristate-13-acetate-induced protein 1

E11 Hs.591654 NM_000306 POU1F1 POU class 1 homeobox 1

E12 Hs.654525 NM_006235 POU2AF1 POU class 2 associating factor 1

F01 Hs.631593 NM_014330 PPP1R15A Protein phosphatase 1, regulatory (inhibitor) subunit 15A

F02 Hs.719926 NM_002715 PPP2CA Protein phosphatase 2, catalytic subunit, alpha isozyme

F03 Hs.280342 NM_002734 PRKAR1A Protein kinase, cAMP-dependent, regulatory, type I, alpha (tissue

specific extinguisher 1)

F04 Hs.1905 NM_000948 PRL Prolactin

F05 Hs.196384 NM_000963 PTGS2 Prostaglandin-endoperoxide synthase 2 (prostaglandin G/H

synthase and cyclooxygenase)

F06 Hs.408528 NM_000321 RB1 Retinoblastoma 1

F07 Hs.19413 NM_005621 S100A12 S100 calcium binding protein A12

F08 Hs.275243 NM_014624 S100A6 S100 calcium binding protein A6

F09 Hs.639 NM_004057 S100G S100 calcium binding protein G

F10 Hs.516726 NM_003469 SCG2 Secretogranin II

F11 Hs.510078 NM_005627 SGK1 Serum/glucocorticoid regulated kinase 1

F12 Hs.158322 NM_003053 SLC18A1 Solute carrier family 18 (vesicular monoamine), member 1

G01 Hs.487046 NM_000636 SOD2 Superoxide dismutase 2, mitochondrial

G02 Hs.520140 NM_003131 SRF Serum response factor (c-fos serum response element-binding

transcription factor)

G03 Hs.12409 NM_001048 SST Somatostatin

G04 Hs.514451 NM_001050 SSTR2 Somatostatin receptor 2

G05 Hs.463059 NM_003150 STAT3 Signal transducer and activator of transcription 3 (acute-phase

response factor)

G06 Hs.633301 NM_001058 TACR1 Tachykinin receptor 1

G07 Hs.713281 NM_003239 TGFB3 Transforming growth factor, beta 3

G08 Hs.435609 NM_000360 TH Tyrosine hydroxylase

G09 Hs.732539 NM_003246 THBS1 Thrombospondin 1

G10 Hs.241570 NM_000594 TNF Tumor necrosis factor

G11 Hs.643896 NM_003373 VCL Vinculin

G12 Hs.53973 NM_003381 VIP Vasoactive intestinal peptide

H01 Hs.520640 NM_001101 ACTB Actin, beta

H02 Hs.534255 NM_004048 B2M Beta-2-microglobulin

H03 Hs.592355 NM_002046 GAPDH Glyceraldehyde-3-phosphate dehydrogenase

H04 Hs.412707 NM_000194 HPRT1 Hypoxanthine phosphoribosyltransferase 1

H05 Hs.546285 NM_001002 RPLP0 Ribosomal protein, large, P0

H06 N/A SA_00105 HGDC Human Genomic DNA Contamination

H07 N/A SA_00104 RTC Reverse Transcription Control

H08 N/A SA_00104 RTC Reverse Transcription Control

H09 N/A SA_00104 RTC Reverse Transcription Control

Methods

44

H10 N/A SA_00103 PPC Positive PCR Control

H11 N/A SA_00103 PPC Positive PCR Control

H12 N/A SA_00103 PPC Positive PCR Control

2.4 Compounds

Substance Function Company

4α-PDD TRPV4 agonist Sigma Aldrich Chemie

GmbH

GSK1016790A TRPV4 agonist GlaxoSmithKline

GSK2193874 TRPV4 antagonist GlaxoSmithKline

Ruthenium red General TRP channel blocker

Sigma Aldrich Chemie

GmbH

Digitonin Necrosis inducer Sigma Aldrich Chemie

GmbH

Staurosporine Apoptosis inducer Sigma Aldrich Chemie

GmbH

IL-1β Interleukin-1β R&D Systems

TNF-α Tumor necrosis factor-α R&D Systems

2.5 Calculations & Statistics

For statistical analyses GraphPad Prism Software for Windows version 7 was used.

Different treatment groups were compared by unpaired two-tailed t test, or one-way

ANOVA. ANOVAs were corrected for multiple comparisons with Tuckey´s, Dunnett's or

Holm-Sidak's multiple comparisons test as appropriate. Significance levels are shown as

*p ≤ 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 or “ns” for not significant p > 0.05.

Methods

45

2.6 Ethics statement

All human blood samples were obtained from volunteers with prior written informed

consent. The blood donation was voluntary and in compliance with data protection

standards. Please note that the current process of the donation service at Boehringer

Ingelheim in Biberach is approved by the appropriate external review board of the federal

state of Baden-Württemberg, Germany named Ethik-Kommission der Landesärztekammer

Baden-Württemberg. Blood donation from healthy subjects is voluntary. Ensure data

protection for the participants and prior written informed consent is obtained from each

blood donor. The same principles was applied to the blood donation at the time of blood

sampling for the current manuscript. Please note that we have obtained blood from less

than 5 healthy volunteers for the purpose of our investigation and the blood draw was not

part of a clinical trial but for basic research purposes only. Therefore, no prospective ethics

approval was necessary for the blood donation. In any case, the principles of voluntary

donation, data protection and prior written informed consent were applied.

Human cells sourced from commercial vendors were verified to have associated signed

informed consents in place. This process was endorsed by a panel of senior company

scientists and physicians.

All animal experimental procedures were performed in accordance with European and

local animal welfare regulations and approved by the Regierungspräsidium Tübingen in

Germany with the approved animal experimental licenses TVV 13-014-02 (approval date:

10.03.2013) and TVV 15-001-02 (approval date: 22.05.2015).

Results

46

3 Results

3.1 Results: Role of TRPV4 in regulating endothelial membrane integrity

For better understanding TRPV4 biology and its role in the regulation of membrane barrier

integrity, its activation and inhibition was investigated in different in vitro and in vivo

models focusing on permeability using two reported selective activators of TRPV4,

GSK1016790A and 4α-PDD (Thorneloe et al. 2008, Willette et al. 2008) and the potent

and selective TRPV4 blocker GSK2193874 (Thorneloe et al. 2012).

3.1.1 TRPV4 mediated calcium influx

TRPV4 mediated calcium influx has been identified as key regulator of endothelial

permeability in previous study (Hamanaka et al. 2007, Yin et al. 2008). To investigate the

different mode of action of GSK1016790A and 4α-PDD, TRPV4 mediated calcium influx

was studied with these TRPV4 agonists (described in section 2.1.3). Pharmacological

activation and inhibition of TRPV4 on calcium influx was analysed in the FLIPRTETRA

using the FLIPR Calcium 6 Assay kit in human umbilical vein endothelial cells

(HUVECs). TRPV4 activation with GSK1016790A resulted in a direct, significant and

strong increase in intracellular calcium concentration in a dose-dependent manner (Figure

3A, 3C), that could be nearly completely block by preincubation for 15 min with 1 µM of

the TRPV4 antagonist GSK2193874 (Figure 3C). Activation of TRPV4 with 4α-PDD led

to a very small but significant increase of intracellular calcium concentration, that occurred

hours after agonist addition (Figure 3B, 3D) and that was blocked by inhibition with

GSK2193874 (Figure 3D).

Results

47

Figure 3: TRPV4 mediated Calcium influx with the FLIPR Calcium 6 Assay in HUVECs. (A) Calcium influx

measured in the FLIPRTETRA before and after addition of different concentrations of the TRPV4 agonist GSK1016790A

(Ag). (B) Calcium influx measurement after addition of different concentrations of the TRPV4 agonist 4α-PDD. (C)

Calcium influx maxima in cells preincubated for 15 min in presence or absence of a TRPV4 antagonist GSK2193874 [1

µM] (Ant) and treated afterwards with different concentrations of the TRPV4 agonist GSK1016790A (Ag). (D) Calcium

influx in cells preincubated for 15 min in presence or absence of a TRPV4 antagonist GSK2193874 [1 µM] (Ant) and

treated afterwards with different concentrations of the TRPV4 agonist 4α-PDD. Data are shown as mean ± SEM; (n=3;

*p < 0.05; ****p < 0.0001 vs control or as indicated above graphs; one-way ANOVA Tukey's multiple comparisons

test). Grid lines labelled with Ag showing time points of TRPV4 agonist addition in the graphs.

3.1.2 TER measurement in HUVECs

Transient receptor potential vanilloid 4 (TRPV4) has been suggested to be a critical

regulator of endothelial barrier integrity. Pharmacological activation of TRPV4 was

therefor studied in a cellZscope allowing continuous measurement of

transepithelial/transendothelial electrical resistance (TER) (described in section 2.1.1).

During method establishment a TER improvement was observed when endothelial cells

were cultured for 24 hours under the more physiological hypoxic conditions (1% O2, 5%

CO2 at 37°C) prior to TER measurement. Under these conditions it was possible to

establish a model with an assay window high enough to test compound effect on TER in

HUVECs. An initial TER improvement was observed when cells were cultured 24 h under

hypoxic conditions (Figure 4B) compared to cells cultured under normoxia (Figure 4A).

Results

48

With cells cultured under hypoxia the assay window was improved and compound effect

on TER could be investigated. A dose-dependent decrease in TER was observed after

addition of the TRPV4 agonist GSK1016790A. Interestingly, the cytokines TNF-α [100

ng/ml] and IL-1β [10 ng/ml] also induced a reduction in TER after compound addition.

Figure 4: Effect of hypoxia on TER in HUVECs. (A) TER measurement in the cellZscope before and after addition of

different concentrations of the TRPV4 agonist GSK1016790A (Ag), TNF-α or IL-1ß in cells preincubated at normoxia.

(B) TER measurement after addition of different concentrations of the TRPV4 agonist GSK1016790A (Ag), TNF-α or

IL-1β in cells preincubated for 24h at hypoxia (1% O2, 5% CO2 at 37°C). Grid lines labelled with cpd showing time

points of compound addition in the graphs.

3.1.3 Effect of TRPV4 agonism on TER

Addition of different concentrations (1 nM, 3 nM, 10 nM, 30 nM, 100 nM) of a selective

TRPV4 agonist GSK1016790A resulted in a significant concentration-dependent decrease

in TER and the effect occurred directly after agonist addition (Figure 5A). A significant

and dose dependent decrease in TER is shown in Figure 5C after 1.5 h with a calculated

EC50 of about 3 nM (Figure 5E). Alternatively an EC50 could be calculated from the area

under the curve from the normalized TER curves with an EC50 of about 4 nM (Figure 5F).

In contrast, when endothelial cells were stimulated with 4α-Phorbol 12,13-didecanoate

(4α-PDD), the TER drop began only about 5 h after agonist addition and significant TER

reduction was only observed with a high concentration of the agonist [10 µM] (Figure 5B),

shown also after 7 h of agonist exposure in Figure 5D.

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Figure 5: Effect of TRPV4 agonism on TER in HUVECs. (A) Representative TER measurement in the cellZscope

before and after addition of different concentrations of the TRPV4 agonist GSK1016790A (Ag). (B) TER measurement

after addition of different concentrations of the TRPV4 agonist 4α-PDD. (C) TER measurement in the cellZscope 1.5 h

after addition of different concentrations of the TRPV4 agonist GSK1016790A (Ag). (D) TER measurement 7 h after

addition of different concentrations of the TRPV4 agonist 4α-PDD. (E) Dose-response curve on TER 1.5 h after addition

of different concentrations of the TRPV4 agonist GSK1016790A (Ag) with an EC50 of 3.18 nM. (F) Dose-response

curve on TER calculated with the area under the curve (AUC) from normalized TER curves after addition of different

concentrations of the TRPV4 agonist GSK1016790A (Ag) with an EC50 of 4.34 nM. Grid lines labelled with Ag

showing time points of TRPV4 agonist addition in the graphs. Data are shown as mean only or mean ± SEM; (n=3-5;

performed statistical test: one-way ANOVA Tukey's multiple comparisons test; *p < 0.05; **p < 0.01; ****p < 0.0001 vs

control).

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3.1.4 TRPV4 agonism effect on vascular permeability assay with FITC-Dextran in

HUVECs

To verify that TRPV4 mediated reduction in TER was associated with a functional change

in permeability, the TRPV4 effect was investigated in parallel using another model that

measures transendothelial FITC-Dextran permeability (described in section 2.1.2).

HUVECs cultured till confluence on transwell filters were exposed to different

concentrations of the TRPV4 agonist GSK1016790A for 3 h or 4α-PDD for 21 h and

fluorescein isothiocyanate-dextran (FITC-Dextran) given on the apical side of each

transwells was measured in the medium from the basal side of the receiver plate to identify

permeability increase. A significant concentration dependent permeability increase was

observed after addition of the TRPV4 agonist GSK1016790A (Figure 6A) with a

calculated EC50 of about 5 nM (Figure 6B). 4α-PDD showed a permeability increase with

a concentration of 10 µM (Figure 6C). This data showed similar results on TRPV4

mediated permeability increase compared to TER measurement, but had the disadvantage

that permeability changes could not be recorded continuously over time and that the assay

window was more difficult to achieve in this model. We therefore used TER measurement

as a more appropriate model for further investigations on TRPV4 mediated permeability

increase.

Figure 6: Effect of TRPV4 agonism on vascular permeability assay in HUVECs. (A) Representative FITC-Dextran

measurement on the basal site of the transwell plate 3 h after addition of different concentrations of the TRPV4 agonist

GSK1016790A (Ag). (B) Dose-response curve on FITC-Dextran measurement 3 h after addition of different

concentrations of the TRPV4 agonist GSK1016790A (Ag) with an EC50 of 5.27 nM. (C) Representative FITC-Dextran

measurement on the basal site of the transwell plate 21 h after addition of different concentrations of the TRPV4 agonist

4α-PDD. Data are shown as mean ± SEM; (n=6-8, performed statistical test: one-way ANOVA Holm-Sidak's multiple

comparisons test or Dunnett's multiple comparisons test; **** p < 0.0001 vs control).

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3.1.5 Effect of TRPV4 agonism and antagonism on TER

Preincubation with different doses of the TRPV4 antagonist GSK2193874 resulted in a

significant concentration-dependent inhibition of the TRPV4 agonist GSK1016790A (30

nM) effect on TER 1.5 h after agonist addition (Figure 7A, 7C) with a calculated IC50 of

about 200 nM (Figure 7E). In contrast the TRPV4 antagonist GSK2193874 was not able to

inhibit the effect of the TRPV4 agonist 4α-PDD (Figure 7B, 7D) under this assay

procedure. Additionally TRPV4 agonism and antagonism effect with GSK1016790A and

GSK2193874 were also investigated in human small airway epithelial cells (SAECs) by

our group (cultured as described in section 2.1.10) in TER measurement (described in

section 2.1.1). SAECs showed a strong initial TER without the need of preincubation in

hypoxia. Similar to the observation in HUVECs, TRPV4 agonism with GSK1016790A

resulted in a direct and significant reduction in TER that could be inhibited by

preincubation with the TRPV4 antagonist GSK2193874 in SAECs.

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Figure 7: Effect of TRPV4 agonism and antagonism on TER in HUVECs. (A) TER measurement with cells

preincubated with different concentrations of the TRPV4 antagonist GSK2193874 (Ant) and afterwards with 30 nM of

the TRPV4 agonist GSK1016790A (Ag). (B) TER measurement with cells preincubated in presence or absence of the

TRPV4 antagonist GSK2193874 [1 µM] and treated afterwards with the TRPV4 agonist 4α-PDD [10 µM]. (C) TER

measurement in the cellZscope 1.5 h after addition of the TRPV4 agonist GSK1016790A (Ag) with cells preincubated

with different concentrations of the TRPV4 antagonist GSK2193874 (Ant). (D) TER measurement in the cellZscope 7 h

after addition of the TRPV4 agonist 4α-PDD with cells preincubated in presence or absence of the TRPV4 antagonist

GSK2193874 (Ant). (E) Dose-response curve on TER 1.5 h after addition of 30 nM TRPV4 agonist GSK1016790A (Ag)

with cells preincubated with different concentrations of the TRPV4 antagonist GSK2193874 (Ant) with an IC50 of 188.9

nM. Grid lines labelled with Ag showing time points of TRPV4 agonist addition in the graphs. Data are shown as mean

only or mean ± SEM; (n=3-5; performed statistical test: one-way ANOVA Tukey's multiple comparisons test; *p < 0.05;

** p < 0.01; ***p<0.001; **** p < 0.0001 vs control).

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3.1.6 Effect of TRPV4 activation on vascular permeability in vivo

After having demonstrated that TRPV4 activation with the agonist GSK1016790A led to a

permeability increase in vitro and that this effect can be inhibited by the TRPV4 antagonist

GSK2193874 in endothelial cells, further investigations on TRPV4 activation were

performed in a murine vascular permeability model (described in section 2.2.1). Evans

blue given intravenously (i.v.) was used to monitor vascular leakage in different organs

after 15 min exposure with the TRPV4 agonist GSK1016790A [300 µg/kg] given

intravenously. Vascular leakage could not be observed in the different tissues (lung

parenchyma, bronchus, kidney, liver and colon) of C57BL/6J mice (Figure 8A) and

suggest that Balb/c mice may be the more appropriate animal model in this permeability

assay, an observation also made by others showing that mannitol permeability is greater in

Balb/c gut compared to C57BL/6J by assessing lipopolysaccharide (LPS) in portal vein

plasma (Volynets et al. 2016). In the tissues of kidney, colon and liver of Balb/c mice

permeability increase could not be observed. In contrast in lung tissues of Balb/c mice a

significant vascular leakage was observed in the lung parenchyma and an elevated but not

significant Evans blue signal was observed in the bronchus (Figure 8B) and may be

explained by an higher TRPV4 protein expression in the mice lungs compared to the colon,

liver and kidney like the situation seen in humans

(https://www.proteinatlas.org/ENSG00000111199-TRPV4/tissue, in the Human Protein

Atlas, available from www.proteinatlas.org (Uhlén et al. 2015)).

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Figure 8: Effect of TRPV4 activation on vascular permeability in different tissues of C57BL/6J and Balb/c mice.

(A) Measurement of Evans blue in formamide extracts of mice tissues (lung parenchyma, bronchus, kidney, liver and

colon) after protein leakage induction for 15 min with 300 µg/kg of the TRPV4 agonist GSK1016790A (Ag) given

intravenously in C57BL/6J mice. (B) Measurement of Evans blue in formamide extracts of mice tissues after protein

leakage induction for 15 min with 300 µg/kg of the TRPV4 agonist GSK1016790A (Ag) given intravenously in Balb/c

mice. Data are shown as mean ± SEM; (n=4; performed statistical test: Unpaired t-test; *p < 0.05; **p < 0.01; ***p <

0.001; ****p < 0.0001 vs negative control).

3.1.7 Effect of TRPV4 activation on lung vascular permeability in Balb/c mice

Following the previous observations, subsequent investigations on the TRPV4 effect on

vascular permeability were performed in lung tissues of Balb/c mice. Figure 9A showing

graphs of mice exposed for different periods of time with the TRPV4 agonist

GSK1016790A (10 µg) given via intratracheal instillation. No significant permeability

increase was observed in the lung parenchyma after 5, 20 or 45 min (Figure 9A). In

contrast significant vascular leakage in the bronchus was induced by the TRPV4 agonist

GSK1016790A after 20 and 40 min of exposure (Figure 9A). A 30 min preincubation time

was selected for subsequent experiments with different concentrations of the TRPV4

agonist GSK1016790A given i.v. or i.t for comparison. After 30 min of GSK1016790A

exposure given intravenously, a significant permeability increase was observed in the lung

parenchyma and bronchus with the highest concentration of 600 µg/kg, but was not

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significantly different from control with a concentration of 200 and 400 µg/kg (Figure 9B).

After 30 min of GSK1016790A exposure given intratracheally, no significant permeability

increase was observed after agonist addition with different doses (10 µg, 20 µg, 30 µg) in

the lung parenchyma of Balb/c mice, but a significant and dose-dependent permeability

increase was observed in the lung bronchus compared to the negative control group (Figure

9B).

Figure 9: Effect of TRPV4 activation on vascular permeability in Balb/c mice lung tissues. (A) Measurement of

Evans blue in formamide extracts of mice lung tissue (lung parenchyma and bronchus) after protein leakage induction for

different periods of time (5, 20 and 45 min) with 10 µg of the TRPV4 agonist GSK1016790A (Ag) given intratracheal

(i.t.) in Balb/c mice. (B) Measurement of Evans blue in formamide extracts of mice lung tissues (lung parenchyma and

bronchus) after protein leakage induction for 30 min with different concentrations of the TRPV4 agonist GSK1016790A

(Ag) given intravenous (i.v.) or intratracheal (i.t) in Balb/c mice. Data are shown as mean ± SEM; (n=4; performed

statistical test: one-way ANOVA Dunnett's multiple comparisons test; *p < 0.05; **p < 0.01; ***p<0.001; ****p <

0.0001 vs negative control).

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3.1.8 Effect of TRPV4 activation and inhibition on lung vascular permeability in

vivo

Having demonstrated that vascular permeability increase is induced in a dose-dependent

manner by the TRPV4 agonist GSK1016790A, the question was asked if the TRPV4

agonist effect can be inhibited by the orally active TRPV4 antagonist GSK2193874 in the

same model (described in section 2.2.1). Again, permeability increase in the lung

parenchyma was absent after 30 min of TRPV4 agonist GSK1016790A exposure (30 µg)

given intratracheally (Figure 10A). In contrast the permeability increase induced by

GSK1016790A in the bronchus was dose-dependently inhibited by preincubation for 2

hours with different concentrations (3, 30 and 300 mg/kg) of the TRPV4 antagonist

GSK2193874 given orally (Figure 10B).

Figure 10: Effect of TRPV4 activation and inhibition on vascular permeability in Balb/c mice lung tissues. (A)

Measurement of Evans blue in formamide extracts of lung parenchyma of mice pretreated with different concentrations

of the TRPV4 antagonist GSK2193874 (Ant) given orally (p.o.) and after protein leakage induction for 30 min with 30

µg of the TRPV4 agonist GSK1016790A (Ag) given intratracheal (i.t.). (B) Measurement of Evans blue in formamide

extracts of lung bronchus of mice pretreated with different concentrations of the TRPV4 antagonist GSK2193874 (Ant)

given orally (p.o.) and after protein leakage induction for 30 min with 30 µg of the TRPV4 agonist GSK1016790A (Ag)

given intratracheal (i.t.). Data are shown as mean ± SEM; (n=4; performed statistical test: one-way ANOVA Dunnett's

multiple comparisons test; *p < 0.05; **p < 0.01; ***p<0.001; ****p < 0.0001 vs positive control).

3.1.9 TRPV4 antagonist reverses the effect of TRPV4 agonism

Additionally the question was asked whether the effect of TRPV4 activation can not only

be prevented but also reversed by TRPV4 antagonists that would make a TRPV4 inhibitor

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even more attractive as a drug candidate. HUVECs were treated firstly with the TRPV4

agonist GSK1016790A and afterward with the TRPV4 antagonist GSK2193874 in TER

and intracellular calcium concentration measurement (described in section 2.1.1 and

section 2.1.3). In TER measurement HUVECs that were first stimulated with 15 nM of the

TRPV4 agonist GSK1016790A and that were subsequently left untreated for 30 min after

agonist exposure, showed a drop in TER that remained until the end of the experiment. In

contrast HUVECs treated within the first 30 min of agonism with 1 µM of the TRPV4

antagonist GSK2193874 showed a recovery in TER too that of the control group, treated

only with 0.1% DMSO (Figure 11C). The same picture was observed in the intracellular

calcium influx measurement with the FLIPRTETRA

. HUVECs first treated with the TRPV4

agonist GSK1016790A [300 nM] and 15 min later with the TRPV4 antagonist

GSK2193874 [1 µM], showed a large and significant increase in intracellular calcium

concentration directly after agonist addition, that could be significantly reduced from the

time point of the antagonist addition compared to the group, that remained treated with the

agonist only (Figure 11A, 11B). In the calcium measurement a too low concentration of the

TRPV4 agonist GSK1016790A resulted in a assay window not high enough to be reversed

and the effect of a too high concentration of GSK1016790A could not be reversed by

TRPV4 antagonism with GSK2193874 (Figure 11B).

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Figure 11: TRPV4 antagonist can reverse the effect of TRPV4 agonism on TER and calcium influx in HUVECs.

(A) Intracellular calcium concentration measurement with the FLIPR Calcium 6 Assay in the FLIPRTETRA on HUVECs

treated first with 300 nM of the TRPV4 agonist GSK1016790A (Ag) for 15 min and afterward treated without or with 1

µM of the TRPV4 antagonist GSK2193874 (Ant). (B) Area under the curve (AUC) from the intracellular calcium

concentration measurement in relative fluorescence units (RFU) over time in the FLIPRTETRA with HUVECs treated first

for 15 min with different concentration of GSK1016790A and afterward treated with or without 1 µM of the TRPV4

antagonist GSK2193874. (C) TER measurement on HUVECs in the cellZscope treated first with 15 nM of the TRPV4

agonist GSK1016790A (Ag) and 30 min afterward treated either with or without 1 µM of the TRPV4 antagonist

GSK2193874 (Ant). Data are shown as mean only or mean ± SEM (for C n=2-3; for B and C n=3; ****p < 0.0001; one-

way ANOVA Tukey's multiple comparisons test). Grid lines labelled with Ag showing time points of TRPV4 agonist

addition in the graphs and grid lines labelled with Ant showing time points of TRPV4 antagonist addition in the graphs.

3.1.10 TRPV4 mediated cytotoxicity

Because of the strong and direct effect of TRPV4 activation during TER measurement the

link between pharmacological activation of TRPV4 and the corresponding functional

observations on barrier integrity was questioned. Despite the fact that at the time our

investigations were made no literature reported directly a cytotoxic effect of the two

widely published TRPV4 agonists GSK1016790A and 4α-PDD, we hypothesized that the

effect of TRPV4 activation during TER measurement may also be caused by a TRPV4-

mediated cytotoxicity. To investigate whether cell viability is impacted by TRPV4

activation, HUVECs were exposed to different concentrations of TRPV4 agonists with or

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without preincubation with 1 µM of the TRPV4 antagonist GSK2193874 for 1 h and

lactate dehydrogenase (LDH) release was recorded to monitor cytotoxic effects (described

in section 2.1.4 and 2.3.5). Cells exposed up to 3 µM 4α-PDD showed no significant LDH

release compared to the control groups after 12 h (Figure 12B), whereas cells exposed to

10 µM 4α-PDD showed no increase in LDH release after 3 h, but LDH release was

observed to begin after 8 h reaching a maximum after 12 h (Figure 12A, 12B). This effect

could not be blocked when cells were preincubated with the TRPV4 antagonist

GSK2193874 (Figure 12A, 12B). In contrast activation of TRPV4 with the agonist

GSK1016790A led to a rapid concentration-dependent increase in cytotoxicity as measured

by LDH release (Figure 12C), even at low concentrations of the agonist (Figure 12D). In

contrast to 4α-PDD, the effect of TRPV4 activation with GSK1016790A could completely

be blocked by preincubation with the TRPV4 antagonist GSK2193874 (Figure 12E) and

was also inhibited in a dose-dependent manner by the TRPV4 antagonist GSK2193874,

when challenged against 30 nM of the TRPV4 agonist GSK1016790A (Figure 12F).

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Figure 12: TRPV4 mediated lactate dehydrogenase (LDH) release in HUVECs. (A) Kinetic of HUVECs

preincubated for 1 h in the presence or absence of the TRPV4 antagonist GSK2193874 [1 µM] (Ant) and afterwards

exposed to different concentrations of the TRPV4 agonist 4α-PDD (time points 3, 8, and 12 h). (B) Dose-response of

cells preincubated in the presence or absence of the TRPV4 antagonist GSK2193874 [1 µM] and afterwards incubated

with different concentrations of the TRPV4 agonist 4α-PDD for 12 h. (C) Kinetic of cells exposed to the TRPV4 agonist

GSK1016790A (15 or 100 nM) for up to 90 min. (D) Dose-response of cells incubated with different concentrations of

the TRPV4 agonist GSK1016790A for 4 h. (E) Cells preincubated in the presence or absence of the TRPV4 antagonist

GSK2193874 [1 µM] and afterwards incubated with different concentrations of the TRPV4 agonist GSK1016790A for

3.5 h. (F) Dose response curve of the LDH release with cells preincubated with different concentrations of the TRPV4

antagonist GSK2193874 and afterwards treated with 30 nM of the TRPV4 agonist GSK1016790A for 3 h. Data are

shown as mean only or mean ± SEM; (n=6; *p < 0.05; ****p < 0.0001 vs control; one-way ANOVA Tukey's multiple

comparisons test).

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3.1.11 Time point of TRPV4 induced cytotoxicity and calcium dependent TRPV4

induced LDH release

Additionally the time points, when a significant cytotoxic effect occurred was investigated

when HUVECs were treated with either 15 nM or 100 nM of the TRPV4 agonist

GSK1016790A monitored by LDH release (described in section 2.1.4 and 2.3.5). Cells

exposed to 15 nM of the TRPV4 agonist GSK1016790A showed no significant LDH

release for up to 45 min and a significant cytotoxic effect was observed after 1 h. When

cells were treated with 100 nM GSK1016790A, a cytotoxic effect was not observed for up

to 20 min and LDH release was significantly increased after 25 min of agonist exposure

compared to control group (Figure 13A, 13B).

After having shown that the TRPV4 agonist GSK1016790A induces a large increase in

intracellular calcium concentration in HUVECs and also induces cytotoxicity, further

investigations were made on the question whether the cytotoxic effect induced by the

TRPV4 activator GSK1016790A is dependent on extracellular calcium influx. Therefor

HUVECs were incubated for 1 h in absence or presence of calcium with 100 nM

GSK1016790A in HBSS and LDH release was recorded (described in section 2.1.4 and

2.3.5). The TRPV4 agonist GSK1016790A showed, in cells incubated in HBSS with

calcium, the same cytotoxic effect as in cells treated with the agonist in medium. 100 nM

of the agonist induced a strong and significant increase in LDH release, that could be

blocked by preincubation with the TRPV4 antagonist. In contrast the TRPV4 agonist

GSK1016790A [100 nM] caused no cytotoxic effect compared to control, when incubated

in HBSS without calcium (Figure 13C).

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Figure 13: Kinetic and calcium dependent TRPV4 induced LDH release in HUVECs. (A) Kinetic of cells exposed to

the TRPV4 agonist GSK1016790A [15 nM] for up to 90 min showing time points of significant LDH release. (B) Kinetic

of cells exposed to the TRPV4 agonist GSK1016790A [100 nM] for up to 90 min showing time points of significant

LDH release. (C) Fold change cytotoxicity in HUVECs preincubated in presence or absence of the TRPV4 antagonist

GSK2193874 [1 µM] (Ant) and afterwards exposed to 100 nM of the TRPV4 agonist GSK1016790A in HBSS with or

HBSS without calcium for 1 h. Data are shown as mean ± SEM; (n=6; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0 vs

control; one-way ANOVA Tukey's multiple comparisons test).

3.1.12 Life cell imaging of HUVECs exposed to the TRPV4 agonist GSK1016790A

HUVECs, preincubated with or without the TRPV4 antagonist GSK2193874 [1µM] were

exposed to different concentrations of the TRPV4 agonist (15, 30 and 100 nM) in a Cell-

IQ® for live cell imaging at controlled temperature and gaseous environment (37°C in 5%

CO2, humidified air) allowing biological cellular responses to be monitored in real time

(described in section 2.1.6). HUVECs exposed to the TRPV4 agonist GSK1016790A [30

nM] were observed to swell and bubble after 15 min (same picture with 15 nM, data not

shown). After a cell swelling phase, induced by the TRPV4 agonist GSK1016790A, cells

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were observed to burst (Figure 14, Figure 15), although mitosis was also observed in some

cells after 3 h of agonist exposure. In contrast the effect of TRPV4 agonism with

GSK1016790A [100 nM] inducing cellular swelling followed by bursting of the cells was

absent, when cells were preincubated with the TRPV4 antagonist GSK2193874 (Figure

14).

Figure 14: Life cell imaging of HUVECs incubated in presence or absence of the TRPV4 agonist GSK1016790A.

Left row showing untreated control group at different time points (0, 15, 60 and 180 min). Middle row showing cells

exposed to 30 nM of the TRPV4 agonist at different time points. Row on the right showing cells preincubated for 1 h

with 1 µM of the TRPV4 antagonist GSK2193874 and afterwards exposed to 100 nM of the TRPV4 agonist

GSK1016790A.

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Figure 15: Life cell imaging of HUVECs incubated in presence of the TRPV4 agonist GSK1016790A. Cells exposed

to 30 nM of the TRPV4 agonist GSK1016790A for 10, 23, 24 and 25 min. Arrows pointing to cell that swell and then

burst.

3.1.13 TRPV4 activation in the RealTime-Glo™ Annexin V Apoptosis and Necrosis

Assay

In TER measurement and LDH release assays different modes of action were observed

with the two different TRPV4 agonists GSK1016790A and 4α-PDD. Therefore the

question was made whether the observed effects could be explained by different cytotoxic

processes. To investigate whether the TRPV4 induced cytotoxicity is an apoptotic or

necrotic process, HUVECs were exposed to different concentrations of the TRPV4

agonists GSK1016790A or 4α-PDD after being preincubated in presence or absence of the

TRPV4 antagonist GSK2193874 [1 µM] and necrotic or apoptotic process was recorded in

real time using the RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay (described

in section 2.1.5). The TRPV4 agonist GSK1016790A showed a direct, strong and

concentration-dependent increase in the fluorescence signal from the DNA-intercolating

dye, similar to the necrosis inducer digitonin, that could be significantly blocked, when

pretreated with the TRPV4 antagonist GSK2193874 (Figure 16A, 16B). In contrast the

agonist 4α-PDD [10 µM] led to an significant increase in the fluorescence signal, that

began only after 8 h reaching the maximum signal after more than 15 hours and that could

not be blocked by the TRPV4 antagonist GSK2193874 [1 µM] (Figure 16A, 16B).

Luminescence detection of phosphatidylserine (PS) on the outer leaflet of the cell was not

increased by the TRPV4 agonist GSK1016790A compared with the control group. The

agonist 4α-PDD [10 µM] in contrast led to an increase in the luminescence signal

beginning after 5 h and reaching its maximum signal at a time point of 8 h. The effect of

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4α-PDD could not be blocked by pretreatment with the TRPV4 antagonist GSK2193874 [1

µM] (Figure 16 C, 16D).

Figure 16: TRPV4 activation in the RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay. (A) Fluorescence

measurement (in relative fluorescence units RFU) over time (indicating necrosis) beginning 30 min after agonist addition

in HUVECs preincubated in presence or absence of the TRPV4 antagonist GSK2193874 [1 µM] (Ant) and afterwards

treated with different concentrations of the TRPV4 agonists GSK1016790A (Ag), 4α-PDD or staurosporine (apoptosis

inducer) or digitonin (nekrose inducer). (B) Fluorescence measurement in HUVECs preincubated in presence or absence

of the TRPV4 antagonist GSK2193874 [1 µM] (Ant) and afterwards treated with different concentrations of the TRPV4

agonist GSK1016790A (Ag) or 4α-PDD after 3 h. (C) Luminescence measurement (in relative light units RLU) over time

(indicating apoptosis) beginning after agonist addition in HUVECs preincubated in presence or absence of the TRPV4

antagonist GSK2193874 [1 µM] (Ant) and afterwards treated with different concentrations of the TRPV4 agonists

GSK1016790A (Ag), 4α-PDD or staurosporine as an apoptosis inducer. (D) Luminescence measurement in HUVECs

preincubated in presence or absence of the TRPV4 antagonist GSK2193874 [1 µM] (Ant) and afterwards treated with

different concentrations of the TRPV4 agonists GSK1016790A, 4α-PDD or staurosporine after 8 h. Data are shown as

mean only or mean ± SEM; (n=4; ****p < 0.0001 vs control; one-way ANOVA Tukey's multiple comparisons test).

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3.2 Results: Role of TRPV4 in stretch induced pathological cellular response

Major content in this part of the thesis are published in PLOS ONE with the title “TRPV4

inhibition attenuates stretch-induced inflammatory cellular responses and lung barrier

dysfunction during mechanical ventilation” (Pairet et al. 2018).

After having shown that pharmacological activation of TRPV4 leads to a disruption of

membrane integrity and permeability increase in vitro and in vivo and that this effect could

be inhibited with the orally active TRPV4 antagonist GSK2193874, the second part of this

thesis focussed on the effect of a more physiologically relevant mean to activate TRPV4,

namely lung cell stretch, due to over-distention of lung region during mechanical

ventilation and the role of TRPV4 in modulating a cellular response to this physical

stimuli.

3.2.1 Effect of TRPV4 agonism on cells Ca2+

influx

To investigate the effect of lung cell stretch, epithelial cells were chosen as a more

appropriate model to investigate cell stretch induced cellular response rather than

endothelial cells, who would be more suitable to study the effect of shear stress. Because

of this consideration and the observed permeability increase in the bronchus of Balb/c mice

after TRPV4 activation in our murine vascular permeability model, human bronchial

epithelial cells NCI-H292, shown to express TRPV4 by our group (RT-PCR, CT value ≈

26), were selected to study the effect of cell stretch. In a first step, to confirm the presence

of functional TRPV4 activity, human lung epithelial cells (NCI-H292) were incubated with

the TRPV4 agonist GSK1016790A and intracellular calcium influx was measured. A

significant increase in intracellular calcium concentration was observed after TRPV4

agonist addition with EC50s ranging from 1-2 nM, and the significant increase in calcium

after agonist addition [2 nM] was concentration-dependently inhibited by the TRPV4

antagonist GSK2193874 with an IC50 of approximately 50 nM and an IC95 of

approximately 1 µM (Figure 17A, 17B). The TRPV4 agonist 4α-PDD led also to a

intracellular calcium concentration increase in a dose-dependent manner in NCI-H292 with

an EC50 of about 5 µM and the effect of 4α-PDD [4 µM] could be also inhibited by the

TRPV4 antagonist GSK2193874 concentration-dependently with an IC50 of about 45 nM

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(Figure 17C, 17D). In subsequent studies, 1µM GSK2193874 was used as a maximal

efficacious, but not a supra-physiological concentration.

Figure 17: Concentration-dependent inhibition of TRPV4 effect on Ca2+ response. (A) Representative Ca2+ influx

measurement in NCI-H292 cells stimulated with different concentrations of the TRPV4 agonist GSK1016790A with an

EC50 of 1.48 nM. (B) Ca2+ influx measurement in NCI-H292 cells stimulated with the TRPV4 agonist GSK1016790A [2

nM] and challenged against different concentrations of the TRPV4 antagonist GSK2193874 (0.1 nM, 0.3 nM, 1 nM, 3

nM, 10 nM, 30 nM, 100 nM and 300 nM) preincubated for 15 min before agonist addition in the FLIPRTETRA.

Concentration-dependent inhibition of the agonist effect through TRPV4 antagonism with an IC50 of 48,67 nM. (C) Ca2+

influx measurement in NCI-H292 cells stimulated with different concentrations of the TRPV4 agonist 4α-PDD with an

EC50 of 4.58 µM. (D) Ca2+ influx measurement in NCI-H292 cells stimulated with the TRPV4 agonist 4α-PDD [4 µM]

and challenged against different concentrations of the TRPV4 antagonist GSK2193874 (0.1 nM, 0.3 nM, 1 nM, 3 nM, 10

nM, 30 nM, 100 nM and 300 nM) preincubated for 15 min before agonist addition in the FLIPRTETRA. Concentration-

dependent inhibition of the agonist effect through TRPV4 antagonism with an IC50 of 44.58 nM (data are mean ± SEM;

n=3).

3.2.2 Effect of stretch on cells Ca2+

influx

To investigate whether TRPV4 is involved in the mechanical strain induced stress response

in NCI-H292, we studied the Ca2+

response after uniaxial cell stretch with various

combinations of stretch speeds and distances on the Stretch/compression device (described

in section 2.1.8). After a single stretch to 80% length increase and back to relaxation within

800 ms an increase in the intracellular Ca2+

concentration was observed, that began directly

Results

68

in the first seconds and reached is maximum signal at about 25 sec after stretch (Figure

18). Interestingly some cells showed a direct increase of intracellular calcium

concentration within the first 5 sec after stretch and in other cells calcium influx began

only after 10 sec after stretch. This observation led to the question, if the cells that

responded directly to stretch, may release a second messenger activating other cells with

some time delay (indirect responders).

Figure 18: Life cell imaging of stretch induced calcium influx in NCI-H292. Ca2+ response in NCI-H292 cells 5 sec

before and 5, 10 and 25 sec after a single uniaxial cell stretch to 80% length increase and back to relaxation within 800

ms with cells loaded with the Ca2+ dye fluo-4 [2 µM] and 0.2% Pluronic F127 under the fluorescence microscope (20

fold). Arrows pointing at cells reacting at different time points.

After having observed that cell stretch led to a calcium influx in NCI-H292, further

investigations were performed on these cells with the same stretch protocol and the role of

TRPV4 in mediating this stretch induced cellular response was studied (described in

section 2.1.8). After a single stretch to 80% length increase and back to relaxation within

800 ms an about 2.5 fold increase in the intracellular Ca2+

concentration was observed

compared to the baseline signal before stretch (Figure 19A) and this effect was

significantly decreased by 43% with the TRPV4 antagonist GSK2193874 [1 µM],

comparing the area under the curves (AUC) of the percent baseline signal after stretch

from the control and the GSK2193874 treated groups (Figure 19A, 19B).

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Figure 19: TRPV4 mediated calcium influx after stretch. Ca2+ response in NCI-H292 cells 10 s after a single uniaxial

cell stretch to 80% length increase and back to relaxation within 800 ms. Cells were loaded with the Ca2+ dye fluo-4 [2

µM] and 0.2% Pluronic F127 and the average fluorescence values of each cell before and 10 s after the strain were

determined. The strain-induced change after stimulation was expressed as the % change intensity compared to baseline

signal before stretch. (A) Ca2+ response 10 s after stretch for 60 seconds from the control and the GSK2193874 [1 µM]

treated groups. (B) Area under the curve (AUC) of the Ca2+ response after stretch (40-100 sec), significantly decreased by

43% with the TRPV4 antagonist GSK2193874 [1 µM], comparing AUC of the percent baseline signal after stretch from

the control and the GSK2193874 treated groups. For (A) and (B) data are mean ± SEM; (control n=121; GSK2193874

n=94, summary of 13 experiments; ****p ˂ 0.0001 vs control; Unpaired two-tailed t-test).

3.2.3 Effect of TRPV4-agonist on cell cytokine release

To investigate the functional consequences of an increase in intracellular calcium

concentration, the effect of the TRPV4 agonist GSK1016790A on human lung epithelial

cells was examined for cytokine release (described in section 2.1.7). NCI-H292 cells were

incubated for 24 h in the presence or absence of a non-cytotoxic dose of GSK1016790A [3

nM] (Figure 20E) with or without treatment with the TRPV4 antagonist GSK2193874 [1

µM]. GSK1016790A [3 nM] increased the release of IL-6 by 2.8 fold and IL-8 by 12.4

fold and this effect could be completely blocked by the TRPV4 antagonist (Figure 20A,

20B). The TRPV4 agonist also induced a 2 fold increase in IL-1α and MDC that was

abolished by TRPV4 antagonism (Figure 20C, 20D). Other cytokines were measured

without significant effect (IL-12p70, IL-17A, IL-18, IL-1β, MCP-1, TNF-α).

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Figure 20: TRPV4 mediated cytokine release. Representative experiment of NCI-H292 cells incubated for 24 h in the

presence or absence of the TRPV4 agonist GSK1016790A [3 nM] (ag) with or without pre-treatment with the TRPV4

antagonist GSK2193874 [1 µM] (ant). (A, B) Release of IL-6 and IL-8 through TRPV4 activation compared to medium

(ctrl) and 0.1% DMSO control (veh) that could be blocked by the TRPV4 antagonist. (C, D) TRPV4 mediated release of

IL-1α and MDC. (E) LDH signal in NCI-H292 cells incubated for 24 h in the presence or absence of the TRPV4 agonist

GSK1016790A [3 nM] (ag) with or without pre-treatment with the TRPV4 antagonist GSK2193874 [1 µM] (ant). Data

are mean ± SEM; (n =6; ****p ˂ 0.0001 vs agonist control; one-way ANOVA Tukey's multiple comparisons test).

3.2.4 Effect of TRPV4 antagonism on stretch induced cytokine release

We further investigated the effect of mechanical stretch on cytokine release and the role of

TRPV4 in the airway epithelial response. NCI-H292 cells seeded on a collagen I coated

silico-elastic membrane were exposed to a cyclic stretch with an amplitude of 20% and a

frequency up to 0.5 Hz for 24h. Both IL-6 and IL-8 release were marginally increased

compared with non-stretched cells (data not shown). Increasing the stress stimulus with a

cyclic strain of 30% (from a minimum strain of 8% to a maximum of 30%) and a

frequency of 1.25 Hz for 24 h (described in section 2.1.9), resulted in an 3.4 fold increase

of IL-8 release and an 6.8 fold increase of IL-6 release compared to non-stretched cells

(Figure 21A, 21B). The stretch induced IL-8 increase could be reduced by 34% with the

TRPV4 antagonist GSK2193874 [2 μM] and was decreased by 86% by the general TRP-

channel blocker Ruthenium red [10 μM] (Figure 21A). A similar observation was seen

with IL-6 (Figure 21B). These data showed that a high magnitude mechanical stretch

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71

results in a cytokines IL-6 and IL-8 release in NCI-H292 epithelial cells and that this effect

is significantly reduced by about 30% by TRPV4 antagonism, but also suggest that other

mechano-sensing channels could play a role in the secretion of cytokines induced by

mechanical strain. Other cytokines were measured without significant effect (IL-18, IL-

17A, IL-1α, IL-1β, MCP-1, TNF-α).

Because stretch-induced activation of TRPV4 has been reported to be mediated via p38

and ERK pathways in fetal mouse distal lung epithelial cells (Nayak et al. 2015) and has

been shown to mediate stretch-evoked Ca2+

influx and ATP release in primary urothelial

cell cultures when comparing WT and TRPV4-KO cells (Mochizuki et al. 2009), further

investigations were also made in NCI-H292 on stretch with ATP (described in section

2.3.4) and phosphorylated/total ERK1/2 (described in section 2.3.3) has readout. Different

stretch protocols with stretch amplitudes of 20% and 30% and different time points

(ranging from 1 min to 24 h) were performed with or without preincubation with the

TRPV4 antagonist GSK2193874, but no significant increase in pERK and ATP release

was observed after stretch and further investigations on cellular stretch were performed

with cytokines as readouts.

Figure 21: TRPV4 mediated stretch-induced cytokine release. Representative experiment of NCI-H292 cells seeded

on silico-elastic membranes and exposed to cyclic equibiaxial stretch (cyclic 30% strain with 1.25 Hz) for 24 h in the

presence or absence of the TRPV4 antagonist GSK2193874 [2 µM] (ant). (A) Stretch induced release of IL-8 compared

to unstretched control (ctrl) reduced by 34 % with the TRPV4 antagonist (ant) and reduced by 86% with Ruthenium red

[10 µM] (RR). (B) IL-6 release via stretch that was reduced through TRPV4 antagonism by 33% (ant) and reduced by

80% with Ruthenium red [10 µM] (RR) addition. Data are mean ± SEM; (n =3; *p ˂ 0.05; ****p ˂ 0.0001 vs stretch

control; one-way ANOVA Tukey's multiple comparisons test).

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72

3.2.5 TRPV4 mediated regulation of genes in the Human cAMP / Calcium Signaling

PathwayFinder

Pathway analysis following a TRPV4-mediated increase in intracellular calcium

concentration were performed. The effect of the TRPV4 agonist 4α-PDD on NCI-H292

human lung epithelial cells was examined in the Human cAMP / Calcium Signaling

PathwayFinder (described in section 2.3.6). This data represent a data set of 3 samples per

group pooled to one sample each group, after exposure of the cells to the TRPV4 agonist

and after two washing steps. The purified RNA concentration from the control group was

142.3 ng/ml, 116 ng/ml for the TRPV4 antagonist GSK2193874 treated group, 90.35 ng/ml

for the TRPV4 agonist 4α-PDD treated group and 104.1 ng/ml for the group pretreated

with the TRPV4 antagonist GSK2193874 and afterwards treated with 4α-PDD. The 4α-

PDD treated group indicated a loss of cells during agonist exposure and we already

identified it as a cytotoxic effect (monitored by LDH-release) induced by 4α-PDD [10 µM]

after 24 h. Nevertheless the same amount of purified RNA from each group had to further

be used for cDNA preparation, that normalised somehow the loss in cells observed during

experimental procedure. Interestingly the gene regulation of Protein phosphatase 1,

regulatory (inhibitor) subunit 15A (PPP1R15A) showed an about 45 fold upregulation

compared to the control group after TRPV4 agonist exposure with 4α-PDD, that was

nearly completely blocked by preincubation with the TRPV4 antagonist GSK2193874 [1

µM] (Table 2).

Table 2: Regulation of genes in the Human cAMP / Calcium Signaling PathwayFinder compared to control in

NCI-H292. Cells preincubated for 2 h in presence or absence of the TRPV4 antagonist GSK2193874 [1 µM] (Ant) and

afterward incubated in presence or absence of the TRPV4 agonist 4α-PDD [10 µM] for 24 h.

Up-Down Regulation

(Fold regulation in expression comparing to control group)

Gene Symbol Control GSK Ant

4α-PDD

GSK Ant / 4α-PDD

ADRB1 1 -1.071 1.5746 2.8442

AHR 1 -1.1942 -1.1227 -1.0217

AMD1 1 -1.2466 -3.4248 -2.535

Continuation of table 2 on page 73

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73

Continuation of table 2

Up-Down Regulation

(Fold regulation in expression comparing to control group)

Gene Symbol Control GSK Ant

4α-PDD

GSK Ant / 4α-PDD

AREG 1 -1.9711 -1.031 -1.4917

ATF3 1 -1.0585 3.3058 1.948

BCL2 1 -1.6841 -1.3379 -1.2483

BDNF 1 -1.4825 -1.5294 -1.7789

BRCA1 1 -1.1096 -1.6853 -1.7171

CALB1 1 3.0251 4.4475 -1.0563

CALB2 1 -1.9889 -1.305 -1.3268

CALM1 1 -1.1495 -1.2075 -1.1297

CALR 1 -1.077 1.5115 1.3746

CCNA1 1 -1.0845 -1.0512 -1.0563

CCND1 1 -3.9862 -8.3861 -4.4878

CDK5 1 -1.2008 -1.0497 -1.0526

CDKN2B 1 -1.0845 -1.0512 -1.0563

CGA 1 -1.1607 -1.1027 -2.6244

CHGA 1 -2.0042 -12.826 -2.4402

CNN1 1 -1.6099 -2.7057 1.136

CREB1 1 -1.1503 -1.5519 -1.3976

CREM 1 -1.1567 1.0281 -1.1157

CTF1 1 -1.014 1.0338 1.1376

CYR61 1 -1.7219 -5.6061 -3.8557

DDIT3 1 -1.0454 28.5022 18.8175

DUSP1 1 -1.967 -1.2959 -3.0335

EGR1 1 -3.6859 1.077 -1.629

EGR2 1 -1.1647 2.8324 4.1612

ENO2 1 1.0432 1.3315 1.4241

FGF6 1 -1.0845 -1.0512 -1.0563

FOS 1 -1.2754 1.0989 1.455

FOSB 1 -1.3086 6.3335 3.2445

GCG 1 -1.0845 -1.0512 -1.0563

GEM 1 1.452 3.7218 5.8038

GIPR 1 -1.5497 1.9185 1.9252

HK2 1 -1.3698 -1.3168 -1.0822

HSPA4 1 -1.1065 -1.2825 -1.3594

HSPA5 1 -1.8468 9.1198 6.4755

IL2 1 -1.0845 -1.0512 -1.0563

IL6 1 -1.0762 1.5856 1.6449

INHBA 1 -1.0432 -1.3775 -2.6684

Continuation of table 2 on page 74

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74

Continuation of table 2

Up-Down Regulation

(Fold regulation in expression comparing to control group)

Gene Symbol Control GSK Ant

4α-PDD

GSK Ant / 4α-PDD

JUNB 1 -1.6189 -2.2815 -1.9265

JUND 1 -1.1282 1.2781 1.1785

KCNA5 1 -1.0845 -1.0512 -1.0563

LDHA 1 -1.2702 -2.5883 -2.0994

MAF 1 8.334 -1.0512 5.9587

MIF 1 1.4702 1.0056 1.0154

NCAM1 1 -1.0845 -1.0512 4.8906

NF1 1 -1.0021 -1.0303 -1.0147

NOS2 1 -1.6178 -1.9903 -1.87

NPY 1 -1.0845 -1.0512 -1.0563

NR4A2 1 1.0622 2.4623 1.9834

PCK2 1 -1.1785 2.9959 2.1886

PCNA 1 -1.4103 -3.4967 -2.4589

PENK 1 -1.0845 -1.0512 -1.0563

PER1 1 -1.8378 1.3426 1.3444

PLAT 1 -1.7691 -1.8545 -2.0849

PLN 1 1.0028 -1.7065 1.4631

PMAIP1 1 -1.1096 1.6099 1.9305

POU1F1 1 -1.0845 -1.0512 -1.0563

POU2AF1 1 -1.0845 -1.0512 -1.0563

PPP1R15A 1 -1.5922 45.0983 2.4453

PPP2CA 1 -2.7151 -2.4419 -2.3343

PRKAR1A 1 1.2176 1.0504 1.1073

PRL 1 -1.0845 -1.0512 -1.0563

PTGS2 1 -3.0759 -1.5966 -1.8251

RB1 1 -1.0425 -1.0622 -1.3232

S100A12 1 -1.8417 -5.8767 -1.0785

S100A6 1 -1.0725 1.1235 1.0673

S100G 1 -1.6245 -2.0111 -5.9835

SCG2 1 9.3179 3.387 4.5726

SGK1 1 -1.3986 -3.7659 -4.1439

SLC18A1 1 -1.0845 -1.0512 -1.0563

SOD2 1 -374.5459 -17.5938 -20.6204

SRF 1 -1.4856 -2.3006 -1.9212

SST 1 -1.0845 -1.0512 -1.0563

SSTR2 1 -1.9106 -35.1146 -1.287

Continuation of table 2 on page 75

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75

Continuation of table 2

Up-Down Regulation

(Fold regulation in expression comparing to control group)

Gene Symbol Control GSK Ant

4α-PDD

GSK Ant / 4α-PDD

STAT3 1 -1.2193 -1.456 -1.3557

TACR1 1 -1.9903 1.1753 1.6958

TGFB3 1 -1.5199 -2.0014 -1.6737

TH 1 1.0119 2.8959 3.4919

THBS1 1 -2.117 -11.2902 -7.19

TNF 1 -5.7837 -1.6178 -2.2863

VCL 1 -1.4044 -1.6947 -1.6178

VIP 1 -1.0845 10.9208 4.7601

ACTB 1 -12.0587 -12.6758 -8.6638

B2M 1 -1.3482 -1.3444 1.0807

GAPDH 1 -1.1527 -1.6586 -1.3528

HPRT1 1 -1.2025 -2.6009 -1.8687

RPLP0 1 1 1 1

HGDC 1 -1.0845 -1.0512 -1.0563

RTC 1 -1.3736 -1.2605 -1.1258

RTC 1 -1.0725 -1.092 -1.0875

RTC 1 -1.1583 -1.215 -1.1966

PPC 1 -1.3059 -1.0622 1.057

PPC 1 -1.4123 -1.0461 -1.0098

PPC 1 -1.105 1.007 1.0777

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3.2.6 Effect of stretch on macrophages cytokine release

The results on stretch induced cytokine release suggested, that the effect is only partially

modulated by TRPV4 in NCI-H292 and other stretch activated channels and/or integrins

may play a role, but also led to the question, if stronger effector cells exist regarding

TRPV4 mediated stretch-induced cytokine release. We further asked the question, if

macrophages as a major source of cytokine secretion and adhering to lung cells (Tao and

Kobzik 2002) could be the stronger effector cells regarding TRPV4 mediated cytokine

release.

To answer this question, human monocytes were seeded on silico-elastic membranes and

differentiated with GM-CSF (M1 subtype) or M-CSF (M2 subtype) for seven days

(described in section 2.1.10). Macrophages were exposed to the same equibiaxial stretch

protocol (cyclic 30% strain with 1.25 hz) as for the lung epithelial cells, but significant

cytokine release was observed after 36 h and 48 h (described in section 2.1.9). The

mechanical stretch induced stress on the M1 macrophages resulted in a significant increases

in IL-1α (4.3-fold; 48h; Figure 22A), IL-1β (3.2-fold; 48h; Figure 22B), IL-8 (1.5-fold;

48h; Figure 22C), IL-6 (1.5-fold; 48h; Figure 22D), and MCP-1 (2.2-fold; 36h, Figure

22E) compared to unstretched cells. All cytokine release was abolished, by the TRPV4

antagonist GSK2193874 [2 μM]. M2 macrophages showed an about 3 fold increase in

MCP-1 after 36 h and an about 2 fold increase of TNF-α after 48h stretch, that were both

blocked by TRPV4 antagonism with GSK2193874 [2 µM] (Figure 22F, 22G) but no

significant increases in IL-1α, IL-1β, IL-6 or IL-8. Other cytokines were measured without

significant effect (IL-10, IL-12p70, IL-2 and MDC).

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77

Figure 22: TRPV4 mediated stretch-induced cytokine release in macrophages M1 and M2. Representative

experiment of macrophages seeded on silicoelastic membranes and exposed to cyclic equibiaxial stretch (cyclic 30%

strain with 1.25 Hz) for up to 48 h in the presence or absence of the TRPV4 antagonist GSK2193874 [2 µM] (ant). (A-E)

Stretch induced cytokine release in M1 macrophages compared to unstretched control (ctrl) that could be blocked with the

TRPV4 antagonist GSK2193874 [2 μM] (ant). (F,G) Stretch induced release of MCP-1 and TNF-α in M2 macrophages

that could be blocked by TRPV4 inhibition. Data are mean ± SEM; (n = 3; *p ˂ 0.05; **p ˂ 0.01; ***p ˂ 0.001; ****p ˂

0.0001 vs stretch control; one-way ANOVA Tukey's multiple comparisons test).

3.2.7 TRPV4 antagonist effect on mechanical ventilation induced cytokine release

and permeability increase in vivo

Having demonstrated that the orally active TRPV4 antagonist GSK2193874 can inhibit

vascular leakage induced by TRPV4 activation with GSK1016790A in the bronchus of

Balb/c mice in vivo, and having shown that TRPV4 plays a role in mediating the stretch

induced stress on human lung epithelial cells and macrophages, we further wanted to

examine the effect of the TRPV4 antagonist GSK2193874 in a murine mechanical

ventilation model on Balb/c mice with high tidal volumes (described in section 2.2.2). No

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78

increase in cytokine release or protein concentration in BALF was observed, when mice

were subjected to a tidal volume (TV) of 20 ml/kg. However, we observed a 23.3 fold

increase of KC/GRO level (Figure 23A) and an IL-6 release that was 15.3 fold higher

(Figure 23B) after mechanical ventilation with 30 ml/kg TV compared to control group

(6.5 ml/kg TV). Furthermore a tidal volume of 30 ml/kg ventilation resulted in a 2.6 fold

increased protein concentration in BALF compared to the normal ventilated control group

(Figure 23C). All were significantly blocked with the TRPV4 inhibitor GSK2193874.

Additionally LDH release was measured in the BALF of mice lungs after mechanical

ventilation showing an increase in LDH signal after a 30 ml/kg TV ventilation compared to

the non-ventilated control group, but that could not be inhibited by TRPV4 antagonism

with GSK2193874 (Figure 23D). Lung elastance and resistance was also measured during

assay procedure showing an elevated but not significant increase in resistance and

elastance in the 30 ml/kg TV ventilated group compared to the 6.5 ml/kg TV control, that

was absent in the 30 ml/kg TV group pretreated with the TRPV4 antagonist GSK2193874

(Figure 23E, 23F). Other cytokines were measured without significant increase (IFN-γ, IL-

1β, IL-2, IL-4, IL-5, IL-10, IL-12p70 and TNF-α.)

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79

Figure 23: TRPV4 antagonist effect on ventilation induced cytokine release and protein concentration in BALF.

Balb/c mice were anesthetized and mechanically ventilated (VT) in presence or absence of the TRPV4 antagonist

GSK2193874 [90 mg/kg] (ant) with different ventilation protocols; with tidal volumes of 20 ml/kg and 30 ml/kg with a

frequency of 75/min and a control group ventilated with a normal tidal volume of 6.5 ml/kg and a frequency of 150/min

for 3 h and a non-ventilated control group (no VT). (A,B) Increase release of the cytokine KC/GRO and IL-6 after a 30

ml/kg ventilation that could be blocked by TRPV4 antagonism (ant). (C) Increased protein concentration in BALF was

observed with a 30 ml/kg ventilation that could be blocked by TRPV4 inhibition (ant). (D) Fold change in LDH signal in

BALF after ventilation with the different tidal volumes for 3 hours with or without TRPV4 inhibition (ant). (E,F)

Elastance and resistance measurements during mechanical ventilation with the different tidal volumes (6.5 ml/kg and 30

ml/kg) in presence or absence of the TRPV4 antagonist GSK2193874 [90 mg/kg] (ant) for 3 h. Data are mean ± SEM;

(n=8; *p ˂ 0.05; **p ˂ 0.01; ***p ˂ 0.001 vs 30 ml/kg VT control; one-way ANOVA Tukey's multiple comparisons

test).

Discussion

80

4 Discussion

This work investigated on the role of the cation channel TRPV4 in endothelial/epithelial

barrier integrity and represents a step by step approach to assess TRPV4 antagonists as

potential drug for the treatment of patients with the need of mechanical ventilation. We

first studied pharmacological activation and inhibition on endothelial cells, using two

widely used selective activators of TRPV4 and a selective potent and orally active TRPV4

blocker GSK2193874 on in vitro permeability assays. Afterwards TRPV4 activation and

inhibition on vascular permeability was investigated in vivo with the TRPV4 activator

GSK1016790A and the TRPV4 channel blocker GSK2193874 given orally.

After compound characterisation in in vitro and in vivo models of permeability, the next

part of the thesis investigated on the pathophysiological role of TRPV4 activation via a

physical stimulus. Therefore TRPV4 activation via cell stretch and the resulting

pathological cellular response was investigated in human bronchial lung epithelial cells

and macrophages in vitro with the TRPV4 antagonist GSK2193874 and afterwards the

effect of the orally given TRPV4 blocker GSK2193874 was investigated on a murine

disease related model of VILI using mechanical ventilation with high tidal volumes.

4.1 Role of TRPV4 in regulating endothelial membrane integrity

To investigate TRPV4 biology and its role in regulating the endothelial membrane

integrity, we used two reported selective activators of TRPV4, GSK1016790A and 4α-

Phorbol 12,13-didecanoate (4α-PDD) (Thorneloe et al. 2008, Willette, Bao et al. 2008)

and a potent and selective TRPV4 blocker GSK2193874 (Thorneloe et al. 2012).

During method establishment initial TER improvement was observed, when HUVECs

were cultured 24 hours under more physiological condition (1% O2, 5% CO2 at 37°C), here

namely hypoxia, in prior to TER measurement. Through this effect it was possible to

establish a model with an assay window high enough to test compound effect on TER in

HUVECs in the cellZscope. Under this assay procedure a dose-dependent decrease in TER

was observed after addition of the TRPV4 agonist GSK1016790A. We also observed that

the cytokines TNF-α [100 ng/ml] and IL-1β [10 ng/ml] induced a reduction in TER after

compound addition. It is reported that hypoxia can induce a large variety of biological

active agents in endothelial cells, e.g. vascular endothelial growth factor (VEGF) (Namiki

Discussion

81

et al. 1995), that has been further implicated to mediate loss of trans-epithelial resistance

(TER) (Ghassemifar et al. 2006). Furthermore Hypoxia-induced hyperpermeability of rat

glomerular endothelial cells has been reported, involving hypoxia-inducible factor-2α

(HIF-2α), to mediate changes in the expression of occludin and ZO-1 inducing

permeability (Luo et al. 2018).

However, in our model hypoxia improved initial TER in HUVECs and we concluded that

this assay procedure 1% O2, 5% CO2 at 37°C represented more physiological conditions

for human umbilical vein endothelial cells rather than hypoxic conditions, leading to an

improvement of initial TER, that remained constant during electrical resistance

measurement in the cellZscope. This observation led to the question, if in general cells

should be cultured under more physiological O2 conditions, e.g. culturing lung epithelial

cells and intestine derived cells under separate conditions. A thought that is shared in the

literature and nicely reviewed (Carreau et al. 2011), evaluating the consequences of

physioxia on cells and importantly emphasizing the discrepancy between in vivo and in

vitro tissues and cells oxygen status, which can have significant impact on experimental

outcome. Pointing out, that the values corresponding to the physioxia of different tissues

are ranging between 11% and 1% O2, whereas current in vitro experimentations are usually

performed in 19.95% O2, an artificial context as far as oxygen balance is concerned,

concluding that most of the experiments performed in so-called normoxia might be

misleading (Carreau et al. 2011). In this review the pO2 in umbilical vein blood, that is in

direct contact to human umbilical vein endothelial cells, is also mentioned to be normally

between 20 and 30 mmHg (≈ 2.6-3.9% O2) (Gluckman et al. 1989, Carreau et al. 2011),

indicating that 1% O2 should represent more physiological levels of oxygen (physioxia) in

HUVECs than the so-called normoxia (19.5% O2) in the incubator.

TRPV4 agonism with GSK1016790A on the endothelial cell layer resulted in a strong

decrease in TER and increase in permeability, which could be dose-dependently blocked

by the TRPV4 antagonist. In contrast, 4α-Phorbol 12,13-didecanoate (4α-PDD) reduced

TER only approximately 5 h after agonism and only with a high concentration of 4α-PDD

[10 µM] and could not be blocked by the TRPV4 antagonist.

Further investigations in vitro showed that the effects on TER correlated with the

intracellular calcium influx measurement over time in HUVECs. Whilst GSK1016790A

resulted in a direct, significant and strong increase in intracellular calcium concentration in

Discussion

82

a dose-dependent manner, high concentrations of 4α-PDD induced a small, late-onset

increase of intracellular calcium concentration. However, the agonist effect could be

blocked by TRPV4 inhibition with GSK2193874. These data suggest that GSK1016790A

induced TER reduction is mediated via TRPV4 in an intracellular calcium-influx

dependent manner in HUVECs, whereas 4α-PDD-mediated TER reduction may be

independent of TRPV4 activation, although we cannot exclude the possibility that

GSK2193874 and 4α-PDD do not share a competitive binding site. Interestingly, the effect

of the TRPV4 agonist GSK1016790A on TER and intracellular calcium influx could not

only be blocked by preincubation with the TRPV4 antagonist, but it was also possible to

reverse the TRPV4 activation on both calcium influx and TER reduction by administration

of the antagonist after agonist addition within a certain time range. These data suggest that

not only GSK2193874 is capable of displacing GSK1016790A from its binding site, but

that reduction in endothelial barrier permeability can be reversed if the intracellular

calcium concentrations fall within a certain time frame (approximately 30 minutes in these

experiments). Efficacy of TRPV4 inhibition, when administered after TRPV4 activation,

was also reported in vivo in a murine model of chemically induced acute lung injury, where

protection from the acute lung injury response to intra-tracheal instillation of hydrochloric

acid was attenuated by administration of TRPV4 inhibitors (GSK2220691 and

GSK2337429A) 30 min after IT HCL (Balakrishna et al. 2014). Beyond this limited time

window, the loss of barrier permeability appears more permanent and that additional, non-

calcium-dependent processes are responsible.

In these studies, we used HUVECs as a cellular test system. However, we also replicated

selected findings in primary epithelial cells.

Furthermore, these data are consistent with the ex vivo findings in which TRPV4 in murine

isolated lungs regulates vascular permeability and its activation, whether via physical

stimuli such as mechanical stress or with pharmacological tools leads to an increase

endothelial and epithelial permeability in an intracellular calcium-influx dependent manner

(Alvarez et al. 2006, Hamanaka et al. 2007, Jian et al. 2008, Yin et al. 2008). It has also

been reported, that 4α-PDD activity on Ca2+

influx and whole-cell currents in human

embryonic kidney (HEK) cells is approximately 300 fold less potent than GSK1016790A

and had only a weak ability to contract bladder strips compared to GSK1016790A.

Furthermore 4α-PDD has been reported to be less selective compared to GSK1016790A

(Thorneloe et al. 2008, Thorneloe et al. 2017), that is consistent with our experimental

Discussion

83

observations. Additionally the exclusivity of 4α-PDD for TRPV4 has been put in question,

by the fact that it can activates mouse DRG neurons independently of TRPV4, by the fact

that it stimulated a dose-dependent increase in [Ca2+]i in neurons from WT and TRPV4-

KO mice, with the proportion of responding neurons and magnitude of increase unaffected

by the genotype (Alexander et al. 2013).

After having demonstrated that TRPV4 activation with the agonist GSK1016790A led to a

permeability increase in vitro and that this effect can be inhibited by the selective and

orally active TRPV4 antagonist GSK2193874 in endothelial cells, further investigations on

TRPV4 activation were performed in murine vascular permeability in vivo models. The

dye Evans blue given intravenously (i.v.) was used to monitor vascular leakage in different

organs after protein leakage induction with the TRPV4 agonist GSK1016790A. Results of

our in vivo investigations on TRPV4 activation in murine vascular permeability models are

consistent with prior findings, demonstrating that activation of TRPV4 with

GSK1016790A produced acute circulatory collapse and failure of the pulmonary

microvascular permeability barrier in rats and mice (Willette et al. 2008). The TRPV4

activators 4α-PDD has also been reported to increased lung endothelial permeability in a

dose-dependent manner in isolated rat lung, that was absent in TRPV4-/- rats (Alvarez et

al. 2006). More importantly our results extent prior finding by demonstrating for the first

time, that lung permeability increase in the bronchus of Balb/c mice induced by the

selective TRPV4 activator GSK1016790A can be inhibited by the selective TRPV4

activator GSK2193874 given orally.

We also questioned the link between pharmacological activation of TRPV4 and the

corresponding functional observations on barrier integrity when there is no affirmed signal

transduction pathway that can be followed to substantiate such a link. We hypothesized

that such effects may also be caused by cytotoxicity. Interestingly, we observed differential

cytotoxic effects in endothelial cells induced by the two TRPV4 agonists at concentrations

within the pharmacological range. 4α-PDD showed a time-dependent release of lactate

dehydrogenase, a cytotoxicity marker released by damaged cells, beginning after 8 h and

reaching a maximum after 12 h, which could not be blocked with the TRPV4 antagonist

GSK2193874. Necrosis was confirmed with a DNA-intercalating dye, but was preceded by

an increase in phosphatidylserine on the outer leaflet of the cell membranes, indicating an

apoptotic process followed by secondary necrosis, which was apparently independent of

TRPV4, again suggesting a possible off-target mechanism in HUVECs. In contrast

Discussion

84

activation of TRPV4 with the agonist GSK1016790A in HUVECs lead to a rapid

concentration-dependent increase in both LDH release and DNA dye intercalation within

the first hour, even with a concentration of the agonist in the low nanomolar range, that

could completely be blocked with the TRPV4 antagonist. Furthermore this effect was

dependent upon extracellular calcium. No cytotoxic effect occurred, when cells were

incubated with GSK1016790A [100 nM] in HBSS in the absence of calcium. Live cell

imaging showed that within the first few minutes after TRPV4 activation with

GSK1016790A cellular swelling and blebbing occurred, followed by apparent bursting of

the plasma membrane. Cellular swelling and blebbing has also been reported in the

literature (Alvarez et al. 2006) following lung exposure to TRPV4 agonists resulting in a

loss of barrier function. Because of the chronological relationship between

GSK1016790A-mediated increases in intracellular calcium concentrations, the cellular

swelling and cytotoxicity, we speculate that this effect maybe a consequence of rapid water

entry into the cell following the rapid increase and high concentrations of intracellular

calcium.

The physiological Ca2+

concentration in extracellular biologic fluids (and media mimicking

these conditions) ranges from 1.6 to 2 mM, in contrast the cytosolic free Ca2+

concentration is kept by cells around 100 nM producing an extremely large

electrochemical gradient between extracellular and intracellular Ca2+

concentrations,

meaning that for a cell at rest the [Ca2+

] is ~ 20.000 times lower in the cytoplasm than

outside the cell (Berridge et al. 2003, Clapham 2003, Bootman 2012). TRP channels

modulate the cations flux through plasma membranes down their electrochemical

gradients, thereby playing an important role in raising the free intracellular Ca2+

concentration (Pedersen et al. 2005, Bootman 2012). TRPV4 has been implicated in the

control of regulatory volume decrease (RVD), a regulatory response to cell swell of cells

exposed to hypotonic solutions, that is normally associated with changes in intracellular

calcium concentrations (Arniges et al. 2004). TRPV4 has been shown to provide the Ca2+

signal, required to activate further Ca2+

potassium channel and the subsequent RVD in

epithelial cells and also interacts with aquaporins to control RVD in astrocytes (Arniges et

al. 2004, Benfenati et al. 2011, Jo et al. 2015). This is an important observation suggesting,

that a disruption of cell volume regulation may have crucial consequences for cell

signalling, barrier integrity and cell viability (Benfenati et al. 2011). Finally TRPV4–AQP4

interactions have been promoted to constitute a molecular system that fine-tunes astroglial

Discussion

85

volume regulation by integrating osmosensing, calcium signaling, and water transport and,

when over-activated, triggers pathological cell swelling (Jo et al. 2015). These prior

finding support our speculation that pharmacological activation of TRPV4, leads to a

permanent opening of TRPV4 channels in HUVECs, that we do not expect to mimic the

situation in real life when activated by a physiological trigger, and led to an extreme

calcium-influx followed by water entry, disturbing cell volume regulation and leading to

the observed excessive cell swelling and the followed disruption of the cell membrane.

Alternatively, it has been suggested that intracellular calcium mediates expression of

ligands that bind to and activate death receptors such as CD95 (Kass and Orrenius 1999),

although within the time frame of these experiments, it seems unlikely that transcriptional

changes could occur. Another possibility is, that mitochondria may respond to an apoptotic

Ca2+

signal by the selective release of cytochrome c or through enhanced production of

reactive oxygen species and opening of an inner mitochondrial membrane pore (Kass and

Orrenius 1999). Our findings are supported by the observations of Olivan-Viguera et al.

(2018), who showed in parallel similar TRPV4 mediated cytotoxic effects on melanoma

cells and keratinocytes.

Taken together we have shown that the effects of pharmacological TRPV4 activation on

TER and vascular permeability assays in vitro resulted in permeability increase. We further

demonstrated that TRPV4 activation with GSK1016790A led to lung permeability increase

in vivo in the bronchus of Balb/c mice and that this effect could be inhibited by

preincubation with the orally active TRPV4 antagonist GSK2193874. Additionally we

explained the functional effects of TRPV4 activation on TER with different cytotoxic

effects induced by two widely-published TRPV4 agonists in endothelial cells. We

conclude that in this test system in HUVECs, 4α-PDD may not be a selective activator of

TRPV4 and mediates TER reduction via apoptosis. In contrast GSK1016790A selectively

activates TRPV4, but that TER reduction is also a consequence of cellular necrosis, during

which the cells swell led to membrane disruption and collapse of the cells. Cell death plays

an important role in regulating barrier integrity and TRPV4 mediated cytotoxicity in

endothelial cells, but also in epithelial cells, is poorly described in the literature and we

believe that these findings add significant context to many reported and further studies

concerning the role of TRPV4 in endothelial and epithelial barrier-function.

Discussion

86

4.2 Role of TRPV4 in stretch induced pathological cellular response

TRPV4 is a force-sensitive Ca2+

-permeable cation channel expressed in many pulmonary

tissues and cells including bronchiolar and alveolar epithelia, alveolar macrophages and the

endothelium (Alvarez et al. 2006, Hamanaka et al. 2010, Yin and Kuebler 2010) and has

been shown to be a particularly promising candidate for the initiation of the acute calcium-

dependent permeability increase during ventilation in isolated mouse lungs (Hamanaka et

al. 2007). Therefore in the second part of this study we investigated the potential of a

TRPV4 inhibitor for the improvement of mechanical ventilation induced pathological

response of lung cells, using the TRPV4 antagonist GSK2193874 in both in vitro and in

vivo models of pathophysiological cell stretch.

Prior to the investigations on cell stretch, compound characterisation was performed on

human lung epithelial cells (NCI-H292), which confirmed a TRPV4 agonism evoked Ca2+

response with GSK1016790A and 4α-PDD, that could be concentration-dependently

reduced and blocked by TRPV4 antagonism with GSK2193874. NCI-H292 also showed an

extension-evoked Ca2+

response after uniaxial cell stretch, which was significantly reduced

by 43% with TRPV4 inhibition. This is consistent with previously reported data in which

TRPV4 mediated stretch-evoked Ca2+

influx contributes to the increase in membrane

permeability due to lung over-distention following high PIP ventilation (Hamanaka et al.

2007) and in mice primary urothelial cells, where the Ca2+

increase was partially reduced

in TRPV4-KO compared to WT cells during stretch stimuli (Mochizuki et al. 2009). In this

study we firstly showed, that stretch evoked Ca2+

influx in human lung epithelial cells and

the involvement of TRPV4. Also the increase in Ca2+

concentration could not be

completely blocked by TRPV4 antagonism leading to the conclusion, that the TRPV4

independent calcium influx could be regulated by other mechanosensory channels and

systems, perhaps with different thresholds, that might play a role on the initial calcium

response of the lung epithelium to extension, e.g. TRPV2 has also been demonstrated to

participates in strain-induced Ca2+

entry in rat primary alveolar type II (ATII) cells (Fois et

al. 2012).

In our experiments, cells had to be stretched with a magnitude of 80% within 400 ms in

order to induce TRPV4 activation. One possible reason for the need of such large stretch

amplitudes is, that in these experiments the mechanical strain system stretches cells in a

uniaxial direction, compared to the multidirectional extension in the mechanically

Discussion

87

ventilated lung in vivo. Another explanation may be, that these cellular experiments were

performed at room temperature (due to technical reasons), whereas in the lung, the

epithelial temperature, particularly in the lower airways, is likely to be higher, which may

impact the heat-sensitive TRPV4 channel. The need for large stretch amplitudes for the

TRPV4 mediated strain-evoked calcium entry was also observed on mice primary

urothelial cells under similar conditions (Mochizuki et al. 2009).

VILI has been described as a cellular response to mechanical stress, that includes a rapid

increase in vascular permeability followed by cytokine release (Dreyfuss and Saumon

1998, Dos Santos and Slutsky 2000). Deformation per se can trigger inflammatory

signalling and it is possible, that alveolar epithelial cells may play an active role in

ventilator-induced lung injury (Vlahakis et al. 1999). Mechanical ventilation has been

reported to be able to induce cytokine upregulation in both injured and healthy lungs and

that the underlying mechanisms include cellular responses to stretch with the frequently

involved cytokines IL-8 and probably IL-6, IL-1β and TNF-α, making cytokines good

surrogate endpoints in exploring the pathogenesis and pathophysiology of VILI in

experimental studies (Halbertsma et al. 2005).

We further tested the hypothesis that the initial calcium response to stretch results also in

an inflammatory response in human epithelial cells. TRPV4 activation of epithelial cells

with the synthetic TRPV4 agonist GSK1016790A resulted in a release of the pro-

inflammatory cytokines IL-6 and IL-8 in vitro, that could also be completely blocked by

addition of the TRPV4 antagonist GSK2193874. This is consistent with a more recent

study in fetal mouse distal lung epithelial cells, which demonstrated that TRPV4 may play

an important role in the transduction of mechanical signals in the lung epithelium by

modulating the release of the cytokine IL-6 via p38 and ERK pathways (Nayak et al.

2015).

Cyclical stretching of lung epithelial cells (NCI-H292) in equibiaxial direction also

increased release of the cytokines IL-6 and IL-8 after stretch, that could also be reduced by

about 30% with GSK2193874 and was decreased by about 80% by addition of the general

TRP-channel blocker Ruthenium red, suggesting that this effect is only partially modulated

by TRPV4 and other stretch activated channels and integrins may play a role. It is not

unreasonable, that the applied stretch of 30% in our studies mimic the deformation by the

lung epithelium in situ when mechanically ventilated with recommended ventilator settings

Discussion

88

(Slutsky 1993) during which the lung volume more than doubles. Interestingly the

reduction of the stretch-evoked increase in intracellular calcium on NCI-H292, that was

reduced by 43% with TRPV4 antagonist addition, is similar to the about 30% reduction of

the stretch induced cytokine release via TRPV4 antagonism, suggesting that the size of

changes in Ca2+

influx extrapolate directly to cytokine release.

Furthermore, during simultaneous live cell imaging and uniaxial mechanical strain, we

observed that some cells (NCI-H292) showed a direct increase of intracellular calcium

concentration and in other cells calcium influx began only 10 sec after stretch, leading to

the hypothesis that the directly responding cells to stretch may release a second messenger

activating other cells with some time delay (indirect responders). TRPV4 has been shown

to mediate stretch-evoked Ca2+

influx and also ATP release in primary urothelial cell

cultures when comparing WT and TRPV4-KO cells (Mochizuki et al. 2009) and ATP has

been demonstrated to interact with TRPV4 has a positive modulator of channel activity

(Lorenzo et al. 2008). We therefore questioned, if ATP could be the second messenger

activating the indirect responding cells. Therefor we studied, if stretch induces a TRPV4

mediated ATP release in NCI-H292 in the biaxial cell stretch system with different

protocols and time points with or without preincubation with the TRPV4 antagonist

GSK2193874, but we could not observe significant increases in ATP after stretch under

our assay procedure.

Macrophages are a major source of cytokine secretion and are known to adhere directly to

lung epithelial cells (Tao and Kobzik 2002) and because of this property may also be

exposed to stretch during mechanical ventilation, and have indeed been shown to be

activated by strain in vitro resulting in an increase in IL-8 (Pugin et al. 1998). Therefore we

examined the effect of mechanical stretch on isolated human macrophages in vitro. The

mechanical stretch induced stress on the pro-inflammatory M1, and to a lesser extent in the

tissue remodelling M2, macrophages resulted in an significant increase of the cytokines

IL-1α, IL-1β and the chemokine MCP-1 and also a small but significant increases in IL-6

and -8. Interestingly, and in contrast to the findings in epithelial cells, the stretch induced

increase of the cyto- and chemokine levels could be nearly completely abolished by the

TRPV4 antagonist GSK2193874. Particularly interesting is that IL-1α has been shown to

directly increase vascular endothelial cell permeability in vitro (Royall et al. 1989), and

therefore may play an important role in the stretch induced pulmonary vascular

permeability increase reported during ventilation leading to VILI. Additionally we also

Discussion

89

observed TER reduction induced by IL-1β and TNF-α in HUVECs. More importantly we

have recently shown that macrophage-derived IL-1α and IL-1β promotes permeability

increases in primary human epithelial cells (SAECs) differentiated in air-liquid interface

and additionally shown that IL-1α, IL-1β and TNF-α but not IL-4, IL-13, GM-CSF, M-

CSF or IL-10 are able to promote permeability increase in SAECs also in the absence of

macrophages (Mang et al. 2018). These findings indicate that the TRPV4 mediated stretch

induced release of the cytokines IL-1α and IL-1β from macrophages could not only affect

the vascular endothelium, but also directly act on the lung epithelium permeability increase

leading to edema formation and alveolar flooding during ventilation, that may induce or

aggravate VILI. M1 macrophages may mimic an alveolar phenotype, because they express

PPARγ, that has been shown to be an alveolar macrophage marker (Schneider et al. 2014).

Furthermore, in the hyperinflammatory environment associated with ARDS, macrophages

are more likely to be driven initially towards a pro-inflammatory, rather than a tissue

remodelling phenotype and TRPV4 has also been reported to mediate polarization of

macrophages toward an M1-like phenotype (Misharin et al. 2013, Scheraga et al. 2016,

Scheraga et al. 2017).

However, the signal transduction pathway from a mechanical stimulus resulting in an

inflammatory response remains elusive. One possibility is that TRPV4 inhibitors block

other Ca2+

dependent processes, such as the release of cytokines. TRPV4 has also been

shown to play a role in the transduction of mechanical signals in the distal epithelium by

modulating inflammation (IL-6 secretion) via p38 and ERK pathways (Nayak et al. 2015).

We also investigated in NCI-H292 the effect of stretch on phosphorylated/total ERK1/2 in

the biaxial cell-stretch system with different protocols and time points with or without

preincubation with the TRPV4 antagonist GSK2193874, but no significant increase in

pERK was observed under our assay procedure. Release of inflammatory cytokines IL-6

and -8 after TRPV1 activation has been shown to be mediated through MAPK signalling in

corneal epithelium (Zhang et al. 2007). These two pathways may also be important

downstream activators of TRPV4 in mediating an inflammatory response after stretch.

Having demonstrated that both human lung epithelial cells and macrophages play an active

role in the TRPV4-mediated stretch induced cytokine release, we wanted to evaluate the

effect of the TRPV4 antagonist GSK2193874 in a murine model of mechanical ventilation

similar to other models reported to induce lung injury (Hegeman et al. 2013, Michalick et

al. 2017). LDH release measured in the BALF of mice lungs after mechanical ventilation

Discussion

90

showed an increase in LDH signal after 30 ml/kg tidal volume (TV) ventilation compared

to the non-ventilated control group that could not be significantly reduced by TRPV4

antagonism with GSK2193874. Lung elastance and resistance showed an elevated but not

significant increase in resistance and elastance in the 30 ml/kg TV ventilated group

compared to the 6.5 ml/kg TV control, that was absent in the 30 ml/kg TV group pretreated

with the TRPV4 antagonist GSK2193874. Studies already shown, that both sterile (e.g.,

ventilator-induced stretch) and infectious triggers of ARDS result in stiffening of the lung

tissue (reduced compliance) (Perlman et al. 2011, Meng et al. 2015, Scheraga et al. 2017).

Our data also indicate that a lung stiffening effect may be induced by mechanical

ventilation with high tidal volumes and that this effect might be attenuated by TRPV4

inhibition, when comparing the 30 ml/kg TV ventilated control group and the 30 ml/kg TV

ventilated with GSK2193874 treated group, but the effect was not significant because of a

too high variability. LDH release during mechanical ventilation with high tidal volume

could not be significantly reduced by TRPV4 inhibition in this model system. Although

TRPV4 deficiency or inhibition by HC-067047 has been demonstrated to attenuated

histological features of mice lung injury in a model of VILI (Michalick et al. 2013).

More importantly we investigated the effect of high tidal volume ventilation on lung

cytokine release and permeability increase. Protein infiltration into the lung as a

measurement of barrier dysfunction was chosen for a number of reasons. We had

previously observed that protein infiltration is both quantitative and more sensitive than the

semi-quantitative histological assessments. Alveolar water and alveolar collapse would

have been lost during the fixation and dehydration steps and therefore only interstitial

oedema-related tissue damage would have been observed, which itself is susceptible to

fixation artefacts. We also considered that lung strain during mechanical ventilation is

poorly defined (Protti et al. 2014, Carrasco Loza et al. 2015) and difficult to estimate

because of the heterogeneous local lung susceptibility during MV (Carrasco Loza et al.

2015). Because of the heterogeneous susceptibility between regions of the lung we opted to

use a total pulmonary barrier function index rather than a local one.

The high tidal volume ventilation (30 ml/kg TV) protocol significantly enhanced protein,

IL-6 and KC/GRO concentrations in BALF compared to the normal ventilated group (6.5

ml/kg TV), all of which could be nearly completely blocked with the TRPV4 antagonist.

These data are consistent with the in vitro findings in macrophages, which were almost

entirely TRPV4-dependent, but less so with the in vitro findings in epithelial cells, which

Discussion

91

were only partially dependent on TRPV4. This may suggest, that macrophages may be

stronger effector cells during ventilation contributing to a pathological response compared

to epithelial cells, a hypothesis which is consistent with the findings of others (Frank et al.

2006, Eyal et al. 2007, Hamanaka et al. 2010) and that these effects are TRPV4-dependent.

An important role for alveolar macrophages in mechanical ventilation models has also

been demonstrated by depletion of macrophages in rat lungs using clodronate-filled

liposomes resulting in an attenuation of ventilator-induced lung injury, where high volume

ventilation resulted not only in a activation-associated adhesion of alveolar macrophages,

but also in an increased alveolar protein leak and lung edema formation, that was

attenuated by depletion of macrophages (Frank et al. 2006, Eyal et al. 2007). A more

important investigation linked TRPV4 channels and macrophages in the role of modulating

VILI. In this study the ventilator induced lung injury was markedly attenuated in TRPV4-

KO mice, whereas reintroduction of TRPV4-WT macrophages in TRPV4-KO mice

reconstituted the lung injury response to mechanical ventilation, showing that macrophages

TRPV4 activation plays a crucial role in initiating this injury (Hamanaka et al. 2010). One

study demonstrated that the inflammatory response to particles was amplified by contact-

dependent interactions between alveolar macrophages and epithelial cells (Tao and Kobzik

2002) and it is also possible that a similar interaction occurs during ventilation. The

importance of TRPV4 in preventing VILI was indirectly addressed in another study, in

which inhalation of nanoparticles containing Ruthenium red prevented ventilator damage

and vascular permeability for several days (Jurek et al. 2014). However Ruthenium red is

known to impact calcium handling in cells via effects on other TRP channels (Vincent et

al. 2009, Yamashiro et al. 2010, Takahashi et al. 2011) and non-TRP proteins (Deinum et

al. 1985, Sasaki et al. 1992, Cardoso and De Meis 1993, Yamada et al. 2000) and it

therefore remains a possibility, that the effect of Ruthenium red on ventilator-induced

oedema in this study may be modulated via other TRP channels or via modulation of

downstream intracellular calcium handling (Jurek et al. 2014).

In summary we have shown, that mechanical stretch evoked intracellular Ca2+

influx and

induced the release of pro-inflammatory cytokines that was partially dependent upon

TRPV4 in human lung epithelial cells, but also induced the release of pro-inflammatory

cytokines from M1 macrophages, that was entirely dependent upon TRPV4. In a murine

ventilation model with high tidal volumes, TRPV4 inhibition attenuated pulmonary barrier

permeability increase and pro-inflammatory cytokines secretion. Taken together, these data

Discussion

92

suggest TRPV4 inhibitors may have utility as a prophylactic pharmacological treatment to

improve pathological responses of lung cells exposed to stretch during ventilation and

potentially may have utility in the support of patients receiving mechanical ventilation.

4.3 Summary and clinical relevance

At present time no effective pharmacological treatments exist for ARDS (Matthay et al.

2012) and only patient management strategies exist to ensure gas exchange while

minimizing the risk of VILI such as a protective mechanical ventilation (Matthay et al.

2012). ARDS can arise as a result of infection and can be the consequence of a non-

infectious cause like mechanical ventilation (lung ventilator stretch) (Rezoagli et al. 2017).

Despite the fact that mechanical ventilation (MV) is an important tool for life support of

ARDS patients, it also has the potential to exert pathological mechanical forces on

different lung cells leading to VILI (Slutsky and Imai 2003). There is an urgent need to

identify the molecular mechanism underlying mechanotransduction leading to the

pathological response of the lung during MV and TRPV4 has been shown to be a

particularly promising candidate for the initiation of the acute calcium-dependent

permeability increase during ventilation (Hamanaka et al. 2007).

Therefore this study investigated the potential of a TRPV4 antagonist to improve MV

induced pathological response in lung cells, using the TRPV4 antagonist GSK2193874 in

both self-established in vitro and in vivo models of permeability and afterward in models of

pathophysiological cell stretch. We have shown that pharmacological TRPV4 activation

leads to TER drop in endothelial cells and that the effect can be inhibited by the TRPV4

antagonist GSK2193874. We further demonstrated that TRPV4 activation with

GSK1016790A led to lung permeability increase in vivo in the bronchus of Balb/c mice

and firstly shown that this effect can be inhibited by preincubation with the TRPV4

antagonist GSK2193874 given orally. Additionally we explained the functional effects of

TRPV4 activation on TER with differential cytotoxic effects induced by two widely-

published TRPV4 agonists in endothelial cells.

We further investigated the effect of TRPV4 inhibition on the cellular response to cell-

stretch and firstly shown in human lung epithelial cells, that mechanical stretch evoked

intracellular Ca2+

influx and induced the release of pro-inflammatory cytokines that was

Discussion

93

partially dependent upon TRPV4 and suggest that other stretch activated channels could

play a role. More importantly we firstly shown the stretch-induced release of pro-

inflammatory cytokines from human macrophages, that was entirely dependent upon

TRPV4 and suggest that macrophages may be the stronger effector cells regarding

mechanical stretch induced TRPV4 activation and the pathological cellular response

occurring during MV.

It is not unreasonable that the applied stretch of 30% in our studies mimic the deformation

of the lung epithelium in situ when mechanically ventilated with recommended ventilator

settings (Slutsky 1993), during which the lung volume more than doubles and could be

even higher when considered that lung strain is poorly defined during MV (Protti et al.

2014, Carrasco Loza et al. 2015) and difficult to estimate because of the heterogeneous

local lung susceptibility during ventilation (Carrasco Loza et al. 2015), especially in regard

of lungs from patients with ARDS. Measured inspiratory capacity in lungs of ARDS

patients is reduced to almost one third of the predicted inspiratory capacity (Beitler et al.

2016) pointing to the fact that almost two third of the total lung volume are not

participating in inspiration during MV. Injured regions of the lung will receive smaller

fractions of the total tidal volume from the inspired tidal volumes, e.g. due to alveolar

collapse and fluid extravasation, therefore other lung areas will receive the majority of the

tidal volume leading to massive over-distension of this areas and local damage perhaps

even with protective ventilation strategies (Carrasco Loza et al. 2015, Bellani et al. 2016).

Studies recommended inspired volume of more than 20 ml/kg to induce lung injury in

healthy young mice during ventilation, but also showed that 40 ml/kg ventilation is

accompanying a high mortality of the laboratory animals (Wilson et al. 2012). In regard of

these facts the 30 ml/kg ventilation we used mimic the clinical features and mechanical

forces occurring during VILI without leading to animal mortality during experimental

procedures and is similar to other models reported to induce lung injury (Hegeman et al.

2013, Michalick et al. 2017).

Furthermore MV has been reported to be able to induce cytokine upregulation in both

injured and healthy lungs and that the underlying mechanisms include cellular responses to

stretch with the frequently involved cytokines IL-8 and probably IL-6, IL-1β and TNF-α

making cytokines good surrogate endpoints in exploring the pathogenesis and

pathophysiology of VILI in experimental studies (Halbertsma et al. 2005).

Discussion

94

Interestingly we observed an significant cell stretch induced and TRPV4 mediated increase

in the cytokines mentioned to be linked to ARDS and VILI, with IL-6 and IL-8 in human

lung epithelial cells and IL-1α, IL-1β, IL-6, IL-8 in M1 macrophages and TNF-α in M2

macrophages. In regard of the clinical relevance of our results on lung epithelial cells

exposed to cellular stretch, our data point to the fact that the effect on cytokine release is

partly modulated by TRPV4 in epithelial cells and suggest that other stretch activated

channel, like other TRP channels may play a role, indicated by a near complete blocked

effect on cytokine release with the general TRP channel blocker Ruthenium red. The

suggestion that other stretch activated channels might play a role in lung cell stretch during

mechanical ventilation is supported by others, who point out the increasing evidence for

the importance of stretch-activated ion channels (SACs) in the activation of lung-resident

and inflammatory cells and indicating that the time has come to seriously consider SACs as

new therapeutic targets against VILI and ARDS (Schwingshackl 2016). Also for a clinical

drug proof of concept a general SACs blocker might be inappropriate and negative side

effects difficult to estimate. In contrast regarding the clinical relevance of our data on

stretch induced and TRPV4 mediated cytokine release in macrophages, that was entirely

dependent upon TRPV4, suggest that macrophages are the stronger effector cells.

Importantly is that alveolar macrophages in MV models have been shown to have the

potential to induce VILI by themselves, demonstrated by depletion of macrophages in rat

lungs resulting in an attenuation of VILI monitored by alveolar protein leak and lung

edema formation (Frank et al. 2006, Eyal et al. 2007). Furthermore TRPV4 channels of

macrophages have been linked to modulate VILI by a study demonstrating, that VILI was

attenuated in TRPV4-KO mice, whereas reintroduction of TRPV4-WT macrophages in

TRPV4-KO mice reconstituted the lung injury response to MV (Hamanaka et al. 2010).

Furthermore our data showing the stretch induced and TRPV4 mediated cytokine release

of IL-1α and IL-1β in macrophages can be linked to another study of our group showing

that macrophage-derived IL-1α and IL-1β promotes permeability increases in primary

human epithelial cells differentiated in air-liquid interface (Mang et al. 2018) and could be

a possible explanation for the permeability increase observed during MV in the literature,

but also in our murine model of ventilation, pointing out that macrophage TRPV4

activation plays a crucial role in initiating the lung injury response to MV by promoting a

permeability increase of the lung epithelium through local release of IL-1α, Il-1β but also

TNF-α (M2), making macrophages TRPV4 a very specific and promising new drug target.

We also demonstrated the efficacy of the TRPV4 antagonist GSK2193874 in a murine

Discussion

95

ventilation model with high tidal volumes in vivo, where the orally given TRPV4

antagonist attenuated both pro-inflammatory cytokines secretion and also pulmonary

barrier permeability increase. The oral bioavailability of the TRPV4 antagonist

GSK2193874 (Thorneloe et al. 2012), demonstrated in our murine ventilation model, gives

this compound a key advantage, by the mean that it can potentially be dose repeatedly for

chronic use and to the time points it is required.

Taken together, these data suggest TRPV4 inhibitors, with special attention on the orally

active TRPV4 antagonist GSK2193874, may have utility as a prophylactic

pharmacological treatment to improve pathological responses of lung cells exposed to

stretch during ventilation and potentially may have utility in the support of patients

receiving mechanical ventilation by attenuating permeability increase and cytokine release.

One possibility is that TRPV4 inhibitors could be given prophylactically to patients who

require mechanical ventilation and exhibit certain risk factors for the development of

ARDS such as sepsis, low blood pH, elevated lactate, low albumin, low respiratory

compliance and patient weight (Xiaoming et al. 2008).

4.4 Next steps

In regard of the outcome of this thesis, the following major points would be part of further

investigation on TRPV4:

During experimental procedure the observation was made, that cells reacted differently to

cellular stretch. While some cells responded to stretch in a direct increase of intracellular

calcium concentration, in other cells calcium influx began only after some time delay. This

observation led to the question, if the directly responding cells to stretch may release a

second messenger activating afterwards other cells and would be very interesting to be

further investigated.

We further observed that TRPV4 in lung epithelial cells partly modulated calcium influx

and stretch induced cytokine release and that stretch induced cytokine release was in

contrast nearly completely abolished by the general stretch activated channel (SAC)

blocker Ruthenium red. For a clinical drug proof of concept a general SACs blocker might

be inappropriate and in this concept it would be very important to identify the other

channels, perhaps TRP channels, leading to the observed stretch induced cellular response,

Discussion

96

by co-administration of different selective TRP channel blocker in this system or by

genetic deletion of suspected channels that might play a role. Furthermore TRPV channels

in general have been demonstrated to be activated by specific, largely non-overlapping

temperature ranges, e.g. TRPV4 is activated by temperature ranging between 24 and 38°C,

TRPV1 by temperatures greater than 43°C and TRPV2 by temperature greater than 52°C

(Watanabe, Vriens et al. 2002, Clapham 2003). Except of only two TRP channels

(TRPM4, TRPM5) that are impermeant for calcium, all other TRP channels are Ca2+

permeable (Pedersen et al. 2005, Owsianik et al. 2006) and several members have been

described to be implicated in mechanotransduction such as TRPA1, TRPC1, TRPC3,

TRPC6 , TRPM7, TRPP2 , TRPV1, TRPV2 and TRPV4 (reviewed in Yin and Kuebler

(2010). In this concept it would be very interesting to investigate which TRP channels

react to similar stretch amplitudes in calcium measurement and furthermore if there are

groups, that react to non-overlapping stretch amplitudes for mechanosensation like the

situation reported in temperature sensing.

We further suggested, that macrophages TRPV4 could be the driving force in regard of

stretch induced and TRPV4 mediated cytokine and permeability increase, by the fact that

stretch induced cytokine release in macrophages was entirely dependent upon TRPV4 and

that the involved cytokines IL-1α and Il-1β derived from macrophages have also been

shown by our group to be able to induce lung permeability increase in primary human lung

epithelial cells (Mang et al. 2018). Further investigations in co-culture models with

macrophages and lung epithelial cells on a lung-on-a-chip model (Huh 2015), allowing cell

stretch experiment in regard of permeability increase, could answer the question if

macrophages TRPV4 activated by stretch is the major driving force of epithelial barrier

permeability increase. Therefore comparing the stretch induced permeability increase of

lung epithelial cells alone to the stretch induced permeability increase of co-culture models

of macrophages and epithelial cells with or without TRPV4 inhibition. A major concern of

this study will be, if this method allows amplitudes of cell stretch high enough to induce

permeability increase strong enough to further compare the effect of TRPV4 inhibition.

Another important point regarding stretch induced cytokine release mediated by TRPV4,

would be to identify the possible pathway leading to this cellular response. We already

began to investigate the possible pathway enhanced by TRPV4 mediated calcium influx,

when pharmacologically activated by TRPV4 agonism with 4α-PDD in NCI-H292 cells

using the Human cAMP / Calcium Signaling PathwayFinder. Although 10 µM 4α-PDD

Discussion

97

also induced a cytotoxic effect, cell loss was somehow normalised by the mean, that the

same amount of purified RNA from each group had to be used for cDNA preparation. An

interesting result of this assay was the gene regulation of Protein phosphatase 1, regulatory

(inhibitor) subunit 15A (PPP1R15A) also named DNA damage-inducible protein 34

(GADD34), showing an about 45 fold upregulation compared to the control group after

TRPV4 agonist exposure with 4α-PDD [10µM], that was nearly completely blocked by

preincubation with the TRPV4 antagonist GSK2193874 [1µM]. Interestingly, a study

showed that dextran sodium sulfate (DSS) induced inflammatory responses were

downregulated by GADD34 deficiency, where the expression of pro-inflammatory

mediators such as TNF-α, IL-6, and iNOS/NOS2 was higher in the colons of WT mice

than in GADD34-KO mice (Tanaka et al. 2015). In this concept it would be now very

interesting to further investigate, in a similar way, the pathway leading to a stretch induced

and TRPV4 mediated release of cytokines with the help of RT² Profiler™ PCR Arrays,

especially in macrophages, where we have shown that the stretch induced cytokine release

was entirely dependent upon TRPV4.

Finally suggesting that TRPV4 inhibitors may have utility as a prophylactic

pharmacological treatment for patient with the need for mechanical ventilation, especially

for patient with risk factors for the development of ARDS such as patient with a “hyper-

inflammatory” subphenotype (Thompson et al. 2017), it will be an interesting point to

make further investigations, that may further allow to enclose and identify such patient

populations, that could benefit from a pharmacological treatment with TRPV4 inhibitors.

TRP channels implication in diseases has been shown by their correlation between the

level of channel expression and the disease symptoms, e.g. TRPV1 expression is

considerably increased in the airway nerves of patients exhibiting chronic cough

(Groneberg et al. 2004). In this regard it would be an important point to investigate, if there

is a significant difference in TRPV4 expression between patients with risk factors for

ARDS developing ARDS compared to patients with risk factors for ARDS not developing

ARDS.

Abstract

98

5 Abstract

Acute respiratory distress syndrome (ARDS) is a rapidly progressive form of acute

respiratory failure characterized by severe hypoxemia and noncardiogenic pulmonary

edema and remains a syndrome with a high incidence frequently resulting in death. At

present time no effective pharmacological treatments exist for ARDS and only patient

management strategies exist to ensure gas exchange while minimizing the risk of

ventilation induced lung injury (VILI) such as a protective mechanical ventilation. ARDS

can arise as a result of infection and can be the consequence of a non-infectious cause like

mechanical ventilation. Despite the fact that mechanical ventilation is an important tool for

life support of ARDS patients, it also has the potential to exert pathological mechanical

forces on different lung cells leading to VILI. There is an urgent need to identify the

molecular mechanism underlying mechanotransduction leading to the pathological

response of the lung during mechanical ventilation and the force sensitive calcium

permeable ion channel transient receptor potential vanilloid 4 (TRPV4) has received

specific attention to be a particularly promising candidate for the initiation of the acute

calcium-dependent permeability increase during ventilation.

In this study we investigated the potential for TRPV4 inhibition in a step by step approach

as a treatment of ARDS with particular attention to ventilation induced pathological

response in lung cells, using the selective TRPV4 antagonist GSK2193874 in both self-

established in vitro and in vivo models of permeability and afterwards in models of

pathophysiological cell-stretch. We first investigated on the role of TRPV4 in modulating

membrane barrier integrity and shown that pharmacological TRPV4 activation leads to

transepithelial/transendothelial electrical resistance (TER) drop in endothelial cells and that

the effect can be inhibited by the TRPV4 antagonist GSK2193874. We further

demonstrated that TRPV4 activation with GSK1016790A led to lung permeability increase

in vivo in the bronchus of Balb/c mice and have shown that this effect can be inhibited by

preincubation with the TRPV4 Antagonist GSK2193874 given orally. Additionally we

explained the functional effects of TRPV4 activation on TER with differential cytotoxic

effects induced by two widely-published TRPV4 agonists in endothelial cells and gave

time points for their occurrence and believe that these findings add significant context to

Abstract

99

many reported and further studies concerning the role of TRPV4 in endothelial and

epithelial barrier-function.

We further investigated the effect of TRPV4 inhibition on the cellular response to cell-

stretch and shown in human lung epithelial cells, that mechanical stretch evoked

intracellular Ca2+

influx and induced the release of pro-inflammatory cytokines that was

partially dependent upon TRPV4 and suggest that other stretch activated channels could

play a role. More importantly we firstly shown the stretch-induced release of pro-

inflammatory cytokines from human macrophages that was entirely dependent upon

TRPV4 and suggest that macrophages may be the stronger effector cells regarding

mechanical stretch induced TRPV4 activation and the pathological cellular response

occurring during MV. Interestingly we observed a significant cell-stretch induced and

TRPV4 mediated increase in the cytokines that have been linked to ARDS and VILI. In a

murine ventilation model with high tidal volumes we demonstrated that the orally given

TRPV4 Antagonist GSK2193874 attenuated both pulmonary barrier permeability increase

and pro-inflammatory cytokines secretion.

Taken together, these data suggest TRPV4 inhibitors, with special attention on the orally

active TRPV4 antagonist GSK2193874, may have utility as a pharmacological treatment to

improve pathological responses of lung cells, especially targeting macrophages TRPV4,

exposed to cell-stretch during ventilation and potentially may have utility in the support of

patients receiving mechanical ventilation by attenuating both permeability increase and

cytokine release. This opens the possibility for the use of TRPV4 inhibitors

prophylactically in patients with the need for mechanical ventilation with risk factors for

the development of ARDS such as sepsis, low blood pH, low respiratory compliance,

obesity and with an hyper-inflammatory subphenotype, that may further allow to enclose

and identify patient populations, that could benefit from a pharmacological treatment with

TRPV4 inhibitors.

References

100

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Acknowledgement

119

Acknowledgement

“We” in this thesis indicates several people made this work possible and contributed to its

outcome.

Firstly, I would like to express my sincere thanks to my supervisor Dr. David Lamb for the

continuous support and guidance during my doctorate and for giving me the opportunity to

conduct my doctoral thesis work at Boehringer Ingelheim Pharma GmbH & Co. KG in

collaboration with the University of Ulm. Thank you for supporting my ideas and

encouraging my research with your positive way of thinking, helping me to focus on the

essential goals and also for reading and making suggestions to this written work.

I extent my thanks to my collaborators and further supervisors Prof. Dr. Paul Dietl and

Prof. Dr. Manfred Frick from the Institute of General Physiology at the University of Ulm

for the welcome during my research visit, for offering me lab space for experiments and

for your guidance and mentorship during my thesis. In this team I would also like to give

special thanks to Dr. Giorgio Fois for his technical advice and sharing knowledge during

my research visit.

Many thanks also goes to whole the members of the department of Immunology and

Respiratory Diseases Research of Boehringer Ingelheim, giving me the opportunity to join

their team, giving me access to the laboratory and research facilities, helping me to deepen

and expand my understanding of biology in health and disease by sharing their knowledge

and making it possible to achieve my PhD in such a great team. In this team I would like to

give special thanks to my fellow lab mates Samuel Mang, Ingrid Christ, Martina Keck,

Tobias Kiechle, Nadine Laufhäger, Melanie Kühnbach and Julia Gindele for technical

advice and help and for being such good companions contributing to an excellent

atmosphere not only at work.

Although they already know, I would like to thank my parents and my brothers for always

supporting me throughout my thesis and my life in general.

Last but not the least, I would like to thank my friends, my rugby and fitness mates for

changing my mind, when I needed to. Finally I want to thank my love Debora, for

everything in the last years.