The regulation of pulmonary vascular permeability by PAF

Role of caveolae and acid sphingomyelinase

Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH

Aachen University zur Erlangung des akademischen Grades eines Doktors der

Naturwissenschaften genehmigte Dissertation

vorgelegt von

M. Eng. Yang Yang

aus QingHai, China

Berichter: Universitätsprofessor: Univ.-Prof. Dr. rer. nat. Stefan Uhlig

Universitätsprofessor: Univ.-Prof. Dipl. Ing. Dr. Werner Baumgartner

Tag der mündlichen Prüfung: 12. 01. 2011

Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar

I

Dedication

This dissertation is dedicated to the very precious people that I have in my life. These

people have instilled in me an unquenchable thirst for knowledge, undying zest for

success, and an unparalleled determination. To my loving parents, Guohao Hao and

Zhaoan Yang, thanks for allowing your only son many opportunities to explore the

world as I matured in your care. I am thankful for the strength, courage and resilience

that I have inherited from you. And certainly Ivy Yu, without you beside me I am

certain all these would not have been possible. Thank you all greatly from the

deepest crevices of my being for inspiring, motivating, and supporting me.

II

Acknowledgements

Throughout my Ph.D. program, I have received a tremendous amount of assistance

and encouragement from my advisor and mentor. My deepest gratitude goes first and

foremost to Professor Stefan Uhlig, my supervisor, for his constant guidance and

encouragement. He has walked me through all the stages of my dissertation

research here at institute of pharmacology and toxicology, RWTH Aachen. Without

his consistent and illuminating instruction, this thesis could not have reached its

present form. I also would like to thank Professor Werner Baumgartner and Dr.

Christian Martin for being supervisors of my dissertation.

I would like to thank our partner group of Professor Wolfgang Kübler, Charité

Universitätsmedizin, Berlin, for the Real-time fluorescence imaging performances and

all the other excellent experiments and assistances. It is a pleasure and privilege to

work with you for the last three years. I want to thank our cooperating group of

Professor Werner Baumgartner for the size and number of caveolae measurement.

My gratitude is also given to our cooperating group of Professor Veit Flockerzi,

Universität des Saarlandes for the TRPC antibodies developments and group of

Professor Arenz Christoph, Humboldt Universität zu Berlin for the ASM inhibitors

developments. I also would like to thank group of Thomas Gudermann for providing

TRPC6 knockout mice and group of Dieter Adam for providing ASM knockout mice.

I have spent my last three years at institute of pharmacology and toxicology, RWTH

Aachen. To all my colleagues and friends within the institute, sprinkled in various

research groups, a special thanks to you for your attention and many help not only

my work, but also my life. Thank Dr. Ulrike Uhlig and Dr. Christian Martin for the

theoretical and experimental support. Thank Anke Kowallik and Nadine Ruske for

being excellent technicians. Thank Dr. Daniela Dreymüller, Lucy Kathleen Reiss,

Stephanie Siegl, Marco Schlepütz, Franz Martin Heß, Dennis Lex, Oliver Pack,

Chrisovalantis Papadopoulos and Ulf Soppa for being not just colleagues but friends

also. Last, but certainly not least, our kind and warm secretaries Mrs. Marion Peters

and Mrs. Vera Lothmann.

To our departed colleague Jie Gao, may she rest in peace.

III

Publications

1. Yang Y, Yin J, Baumgartner W, Samapati R, Solymosi EA, Reppien E, Kuebler WM, and Uhlig S. Platelet-activating factor reduces endothelial nitric oxide production: role of acid sphingomyelinase. Eur Respir J 36: 417-427, 2010.

2. Kuebler WM, Yang Y, Samapati R, and Uhlig S. Vascular barrier regulation by

PAF, ceramide, caveolae, and NO - an intricate signaling network with discrepant effects in the pulmonary and systemic vasculature. Cell Physiol Biochem 26: 29-40, 2010.

3. Roth AG, Drescher D, Yang Y, Redmer S, Uhlig S, and Arenz C. Potent and

selective inhibition of acid sphingomyelinase by bisphosphonates. Angew Chem Int Ed Engl 48: 7560-7563, 2009.

4. Rudi Samapati, Yang Yang, Jun Yin, Christoph Arenz, Alexander Dietrich,

Thomas Gudermann, Dieter Adam, Xyy Wu, Chrostoph Stoerger, Veit Flockerzi, Stefan Uhlig and Wolfgang M. Kuebler. Regulation of lung vascular permeability by ASM dependent activation of TRPC6 In lung endothelial cells. Manuscript in preparation.

5. Yang Y and Uhlig S. The role of sphingolipids in respiratory disease. The journal of

Therapeutic Advances in Respiratory Disease. Manuscript in preparation.

Conferences presentions

1. Y. Yang, J. Jin, Rudi Samapati, C. Stoerger, V. Flockerzi, W.M. Kuebler and S Uhlig. PAF-induced recruitment of caveolin-1 reduces endothelial NO levels-Role of acid sphingomyelinase. 8th European respiratory society – lung science conference 2010, Estorul, Portugal.

2. R. Samapati, Y. Yang, S. Uhlig and W.M. Kuebler. PAF causes vascular barrier

failure by acid sphingomyelinase- and ceramide-dependent activation of endothelial TRPC channels. Symposium of microcirculation and vascular biology, 2009, Bern, Swiss land.

3. Y. Yang, J. Jin, Rudi Samapati, W.M. Kuebler and S Uhlig. PAF-induced recruitment of caveolin-1 reduces endothelial NO levels. 5th International symposium on respiratory diseases,ISRD 2008, Shanghai, China.

IV

Table of contents

Dedication------------------------------------------------------------------------------------------------- I

Acknowlegements--------------------------------------------------------------------------------------- I

Publications--------------------------------------------------------------------------------------------- III

1 Introduction-------------------------------------------------------------------------------------------- 1

1.1 Concise lung morphology----------------------------------------------------------------------------------------------1

1.2 Acute respiratory distress syndrome (ARDS) ---------------------------------------------------------------------4

1.3 The regulation of vascular permeability ----------------------------------------------------------------------------8

1.4 Platelet activating factor ---------------------------------------------------------------------------------------------- 18

1.5 Sphingolipids------------------------------------------------------------------------------------------------------------ 22

1.6 Lipid rafts and caveolae ---------------------------------------------------------------------------------------------- 31

1.7 The isolated perfused lung model --------------------------------------------------------------------------------- 39

2 The aim of the study -------------------------------------------------------------------------------41

3 Material and Methods------------------------------------------------------------------------------42

3.1 Materials ----------------------------------------------------------------------------------------------------------------- 42

3.2 Methods ------------------------------------------------------------------------------------------------------------------ 45

4 Results ------------------------------------------------------------------------------------------------52

4.1 PAF induced edema formation ------------------------------------------------------------------------------------- 52

4.2 PAF induced caveolin recruitment and endothelial nitric oxide synthase (eNOS) distribution ------ 53

4.2.1 Number and size of caveolae ------------------------------------------------------------------------------------ 53

4.3 PAF activates ASM to decrease endothelial NO production ------------------------------------------------ 61

4.4 PAF activates ASM to increase endothelial Ca2+ levels ------------------------------------------------------ 66

4.5 Effects of steroids------------------------------------------------------------------------------------------------------ 73

5 Discussion --------------------------------------------------------------------------------------------77

5.1 Regulation of caveolin-1 by PAF ----------------------------------------------------------------------------------- 78

5.2 Endothelial permeability regulation by caveolin and NO in lungs ------------------------------------------ 80

5.3 Caveolae, calcium influx and permeability regulation--------------------------------------------------------- 82

5.4 Endothelial permeability regulation in the lungs and in extra-pulmonary organs----------------------- 88

5.5 Concluding remarks --------------------------------------------------------------------------------------------------- 93

6 Summary----------------------------------------------------------------------------------------------95

7 References -------------------------------------------------------------------------------------------96

V

Abbreviation ACE: angiotensin converting enzyme AGEPC: acetyl-glyceryl-ether-phosphorylcholine ASM: acid sphingomyelinase ALI: acute lung injury ARDS: Acute respiratory distress syndrome AJs: adherens junctions ATP: Adenosine-5'-triphosphate BAL: bronchoalveolar lavage Cav-1: caveolin-1 CSD: caveolin scaffolding domain CBM: caveolin binding motif CFTR: cystic fibrosis transmembrane conductance regulator COX: cyclooxygenase COPD: Chronic obstructive pulmonary disease CTxB: cholera toxin binding subunit Ca2+: calcium ion DAD: diffuse alveolar damage DAG: diacylglycerol eNOS: endothelial nitric oxide synthase ER: endoplasmic reticulum FAK: focal adhesion kinase FRET: fluorescence resonance energy transfer FGF: fibroblast growth factor GST: glutathione S-transferase HMEC: human microvascular endothelial cell GAP: GTPase-activating protein GTP: guanosine triphosphate GEF: guanine nucleotide exchange factors HDL: high density lipoproteins HUVECs: human umbilical vein endothelial cells HUVEC: human umbilical endothelial cell IP3: inositol trisphosphate IPL: isolated perfused rat lung IRDS: infant respiratory distress syndrome IQGAP: IQRas GTPase activating protein LDL: low-density lipoprotein

LTC: leukotrienes C LTD: leukotrienes D LPS: lipopolysaccharide MAEC: mouse aortic endothelial cell MLCK: myosin light chain kinase MLCP: myosin light chain phosphatise MAPK: mitogen activated protein kinase NGF: nerve growth factor NOS: nitric oxide synthase NO: nitric oxide NPD: Niemann-pick disease LPS: lipopolysaccharides PAEC: pulmonary artery endothelial cell PAF: platelet activating factor PAK: p21-activated kinase PAR: Proteaseactivated receptor PGE2: Prostaglandin E2 PKC: Protein kinase C PLA2: phospholipase A2 PLC: phospholipase C PMCA: plasma membrane Ca2+- ATPase PIP2: phosphotidyl inositol bisphosphate PTEN: phosphoinositide phosphatase ROC: receptor operated channel ROCK: Rho-associated protein kinase S1P: Sphingosine-1-phosphate SERCA: sarco/endoplasmic reticulum Ca2+-ATPase SNAP: S-nitroso-N-acetyl-penicillamine SHP: Src-homology-2-domain-containing protein tyrosine phosphatise SOC: store operated channel STIM: Stromal-Interacting Molecule TGF: transforming growth factor TJs: tight junctions TNF-α: tumour necrosis factor-α TRPC: canonical transient receptor potential channel TXA2: thromboxane A2 VEGF: vascular endothelial growth factor VSMC: vascular smooth muscle cell

Introduction

1

1 Introduction The Lungs, the most essential and important respiration organ in all air-breathing

animals. In mammals and the more complex life forms, the two lungs are located in

the chest on either side of the heart. Their principal function is to transport oxygen

from the atmosphere into the bloodstream, and to release carbon dioxide from the

bloodstream into the atmosphere. Air progresses through the mouth or nose, and

travels through the oropharynx, nasopharynx, the larynx, the trachea, and a

progressively subdividing system of bronchi and bronchioles until it finally reaches

the alveoli where the gas exchange of carbon dioxide and oxygen takes place [1].

1.1 Concise lung morphology Human lungs weight 900 to 1000 g, of which nearly 40% to 50% is blood. At end

expiration, the gas volume is about 2.5 L, whereas at maximal inspiration it may be 6

L [2-3]. The human lungs are located in two cavities on each side of the heart.

Although similar in appearance, the two are not identical. And both are separated into

lobes by fissures, with three lobes on the right and two on the left [3].

1.1.2 Airways The airways, connecting the atmosphere and the terminal respiratory units, are

critically important to the lung functions. As seen in figure. 1, the lower airways are

divided into three major groups: bronchi, membranous bronchioles and respiratory

bronchioles/gas exchange ducts. The bronchi enter the lungs and branch into a tree-

like appearance. The bronchi branches into bronchioles, respiratory bronchioles and

eventually to the terminal respiratory units whereas the most of the gas exchange

progresses [2-4].

1.1.3 Terminal respiratory units The terminal respiratory units comprise all alveolar ducts, together with their

accompanying alveoli. In the human lungs, this unit contains approximately 100

alveolar ducts and 2000 alveoli and in total there are about 150,000 such units in the

entire lung. The terminal alveoli are wrapped by the capillaries with a total surface

area of 70 m2 in humans [4]. The gas exchange can be accomplished in this so-

called alveolar-capillary unit which is built up by several thin layers: the endothelium

Introduction

2

facing the blood, the interstitium and the alveolar epithelium facing the air. In human

lungs, the total alveolar surface is about 50-100 m2 [5].

In the terminal respiratory units, gas-phase diffusion is so rapid that the partial

pressures of oxygen and carbon dioxide are even throughout the unit. Oxygen will

diffuse from the gas adjacent to the alveolar wall through the air-blood barrier into the

red blood cells flowing in the capillaries, where it combines with haemoglobin;

meanwhile the carbon dioxide diffuses in the opposite direction [3].

1.1.4 Pulmonary circulation The pulmonary circulation starts at the right ventricle though the pulmonary artery

and ends in the left ventricle with oxygen enriched arterial blood from the lung. The

normal pulmonary circulation is a low-resistance and low-pressure circuit. In humans,

the pulmonary artery enters each lung at the hilum in a loose connective tissue

adjacent to the main bronchus. The pulmonary artery travels adjacent to and

branches with each airway generation down to the respiratory bronchiole. The

arrangements of the pulmonary arteries and the airways are a continual reminder of

the relationship between ventilation and perfusion that determines the efficiency of

normal lung functions [4].

The capillary network is long and crosses one or several alveoli of the terminal

respiratory unit. Theoretically, gas exchange may occur through the thin wall of

almost any pulmonary vessel at normal alveolar oxygen tensions, however, only little

oxygen and carbon dioxide exchange occurs before the true capillaries. This could

be explained by the rapid linear velocity of blood flow in pulmonary arterioles [6]. As

blood enters the vast alveolar wall capillary network, its velocity slows, and the

majority of gas exchange occurs in the sufficient time and huge exchange surface.

1.1.5 Type I and type ll cells The alveolar wall is mainly formed of flat alveolar type l pneumocytes and cuboidal

type II pneumocytes. Type II cells attach via tight junctions to neighbouring type I

cells to form a relatively impermeable seal between alveolar air and connective tissue

spaces. The function of type I cells is to facilitate gas exchange, but the type I cells

also express water channels and have remarkable water permeability [7].

Furthermore these cells also express epithelial Na+ channels and membrane Na+/K+-

ATPase. These observations collectively imply that type I cells may also play a major

Introduction

3

role in pulmonary water fluxes [8-9]. The type II cell is the major synthesizing and

secreting factory of the alveolar epithelium and implements epithelial repair via its

ability to proliferate. The proteins produced by the type II cell include surfactant-

associated proteins that affect surfactant recycling, adsorption of surfactant lipids to

an air-liquid interface, and immuno-modulating functions, receptors for growth

factors, growth factors; enzymes, matrix proteins, epithelial mucins, and others. The

presence in type II cells of various ion channels and transporters supports earlier

evidence that type II cells are actively involved in fluid resorption and trans-epithelial

water fluxes [10-11].

Type II cells can proliferate and generate both new type II and new type I cells, but

type I cells are believed to be incapable of cell division. During post-injury repair, the

postmitotic progeny of type II cells can differentiate into type I cells with normal

morphology, a process that is also presumed to occur during the normal turnover of

type I cells [1, 4].

1.1.6 Lung endothelium Pulmonary capillaries are lined by continuous (non-fenestrated) endothelial cells.

These attenuated cells have an individual area of 1000 to 3000 µm2 and an average

volume of 600 µm3 [4]. These endothelial cells form a semi-permeable barrier which

is very important in regulating and controlling the macromolecules and fluid passing.

Pulmonary capillary endothelial cells have organelles involved in endocytosis through

caveolae, multivesicular bodies, and lysosomes. These endocytic apparatus appears

to participate in receptor-mediated uptake and transport of albumin, low-density

lipoproteins, and thyroxine [12-13]. Another route for passage of solutes and water is

between adjacent endothelial cells and this passage route is restricted by specialized

junctional complexes called adhering junctions (AJs) [14-15].

Introduction

4

Fig. 1: Complete respiratory system, Adapted from Wikipedia, http://en.wikipedia.org/ wiki/Respiratory_system, last reviewed 23.09.2010.

1.2 Acute respiratory distress syndrome (ARDS) 1.2.1 Definition and background Acute respiratory distress syndrome (ARDS), also known as respiratory distress

syndrome (RDS) as its name suggests, is a syndrome, reflecting a series of clinical

and physiologic observations caused by a variety of direct and indirect issues [1, 4].

ARDS is characterized by inflammation of the lung parenchyma leading to impaired

gas exchange with concomitant systemic release of inflammatory mediators causing

inflammation and hypoxemia and is frequently part of a multiple organ failure

syndrome. This condition is often fatal, usually requiring mechanical ventilation and

Introduction

5

admission to an intensive care unit. A less severe form is called acute lung injury

(ALI) [4].

The first description of ARDS appeared in a remarkable case study in 1967 [16].

Ashbaugh and colleagues described 12 patients in age 11 to 48, who presented

respiratory distress, hypoxemic respiratory failure, and patchy bilateral infiltrates on

chest radiographs. The syndrome was characterized by rapid onset, as most of the

patients developed respiratory distress within 48 to 72 hours. Most of the cases were

preceded by severe trauma or viral infection. Many patients required positive-

pressure ventilation and exhibited low respiratory system compliance. This syndrome

was initially termed the adult respiratory distress syndrome, to distinguish it from the

respiratory distress syndrome seen in infant (IRDS). Subsequently, by recognizing

that this type of pulmonary edema can also developed in children, it was renamed the

acute respiratory distress syndrome [4, 16].

In 1994, a consensus conference of American and European investigators published

their definition of ARDS, which has since been widely adopted. Aiming for simplicity,

ARDS was defined as a syndrome of acute onset, with bilateral infiltrates on chest

radiography consistent with pulmonary edema, pulmonary artery occlusion pressure

less than or equal to 18 mm Hg (or absence of clinical evidence of left atrial

hypertension), and hypoxemia as measured by the ratio of the arterial partial

pressure of oxygen (PaO2) to the fraction of oxygen inspired (FiO2). Recognizing the

spectrum of severity of the disease, the consensus panel recommended that a PaO2/

FiO2 ratio of less than or equal to 300 define an entity termed acute lung injury (ALI).

ARDS, the most severe form of ALI was defined as occurring if the PaO2/FiO2 ratio is

less than or equal to 200 [17].

To summarize and simplify, ARDS is an acute (rapid onset) syndrome (collection of

symptoms) that affects the lungs widely and results in a severe oxygenation defect,

but without heart failure.

1.2.2 Pathophysiology of ARDS In the past, ARDS has also been called non-cardiogenic pulmonary edema, which is

not entirely correct but nonetheless is descriptive. Unlike cardiogenic pulmonary

edema, the edema in ADRS is caused by increased permeability though the alveolar-

capillary barrier. Following endothelial and epithelial damage, a protein-rich fluid

floods into the alveolar spaces. Type II cells are damaged, causes surfactant

Introduction

6

production decrease and alveoli collapse. This leads to decreased respiratory system

compliance and profound hypoxemia.

The pathologic features of ARDS are described in three stages: The first one has

been termed diffuse alveolar damage (DAD). There are hyaline membranes and

protein-rich fluid in the alveolar spaces, as well as epithelial disruption and infiltration

of the interstitium and air spaces with neutrophils. Areas of hemorrhage and

macrophages can also be found in the alveoli. The second stage is so-called

proliferative stage. In this one hyaline membrane are reorganized and fibrosis begins

to form, together with the obliteration of pulmonary capillaries and interstitial

deposition. In the last fibrotic stage, precipitating pulmonary fibrosis appears in a

subset of patients [4, 18].

One of the hallmarks of ALI/ARDS is the accumulation of neutrophils in the

microvasculature of the lung [19]. Neutrophils and some T-lymphocytes can quickly

migrate into the inflamed lung parenchyma and contribute to the amplification of the

inflammation. Neutrophils can generate an array of cytotoxic compounds, which

include reactive oxygen species, cationic peptides, eicosanoids, and proteolytic

enzymes. In addition, once activated, neutrophils can release growth factors and

cytokines (such as tumour necrosis factor-α (TNF-α) and interleukin-1β) that can also

be secreted from epithelial and endothelial cells. All these factors act to sustain and

perpetrate the inflammatory response [20].

The mechanisms of the ARDS/ALI have been and will be intensively studied. So far

we know that the pathogenesis is mediated by plethora of inflammatory mediators,

and by recruitment and activation of inflammatory cells through their intricate

networking and cross talking. In this study, we will raise a new hypothesis that

platelet activating factor (PAF) regulates edema formation and vascular permeability

in ARDS/ALI via acid sphingomyelinase (ASM).

1.2.3 Pulmonary edema and acute lung injury Pulmonary edema occurs when more fluid is filtered into the lungs more than is

absorbed. Accumulation of fluid can have serious consequences on lung function,

because efficient gas exchange cannot occur in fluid-filled alveoli and interstitium.

Pulmonary edema is mainly caused by either failure of the heart to remove fluid from

the lung circulation and increase capillary pressure ("cardiogenic edema") or by a

Introduction

7

direct injury to the lung parenchyma ("non-cardiogenic edema") [21]. Major causes of

cardiogenic and non-cardiogenic pulmonary edema are listed in table 1.

Table 1: Causes of cardiogenic and non-cardiogenic pulmonary edema [1, 4]

Cardiogenic edema Non-cardiogenic edema

Congestive heart failure Inhalation of toxic gases

Heart attack with left ventricular failure Aspiration, e.g., gastric fluid or in case of drowning

Arrhythmias (tachycardia/fast heartbeat or bradycardia/slow heartbeat

Pulmonary contusion, i.e., high-energy trauma

Hypertensive crisis Multiple blood transfusions

Pericardial effusion with tamponade infections

Fluid overload, e.g., from kidney failure or intravenous therapy

Multi-trauma, neurogenic causes

Upper airway obstruction

Other/unknown

The essential form that regulate fluid exchange in the lungs can be expressed in the

Starling equation [22] for the microvascular endothelial barrier:

Jv = Kf [(Pc - Pi) - σ (πc - πi)]

Where [Pc − Pi] − σ[πc − πi] is the net driving force; P and π are the hydrostatic and

oncotic pressure in the capillary (c) and interstitial (i) compartments; Kf is the filtration

coefficient, a proportionality constant; Jv is the net fluid movement between the

endothelial barrier. The Starling equation classifies pulmonary edema into

“hydrostatic” type and “permeability” type: hydrostatic type caused by hydrostatic

or/and oncotic pressure gradient and permeability type caused by vascular

permeability and endothelial barrier dysfunction (see Fig. 2).

Introduction

8

Fig. 2: Schematic representation of the pulmonary capillary tissue-lymphatic system and starling equation [23]. The lymph flow removes the overflow in the system.

The edema caused by altered permeability of pulmonary endothelial barrier for water

and proteins has been called permeability type edema. This is a characteristic

hallmark of inflammatory responses and contributes to the morbidity and mortality in

anaphylaxis, sepsis and acute lung injury (ALI) respectively it’s most severe form, the

acute respiratory distress syndrome (ARDS) [19, 24].

Several mechanisms normally protect the lungs against edema such as lymphatic

pumps, resorption and endothelial as well as epithelial barriers. However, if the

barriers function is impaired and if protective osmotic pressure differences are lost,

even normal hydrostatic pressure results in significant fluid and protein influx into

interstitial and alveolar spaces despite increased the lymph flow. Increased

permeability edema is often rapid in onset and progression because once injured, the

barriers offer much less resistance to fluid flow. Subsequently, interstitial edema and

alveolar filling compromise gas exchange. In acute lung injury, functional residual

capacity is decreased as a consequence and this loss of respiratory units also

accounts for virtually all the observed decrease in lung compliance. In addition,

airflow resistance was increased as a result of decreased lung volume [25-26].

1.3 The regulation of vascular permeability The pulmonary endothelium, lining the blood vessels forms a size-selective and

semi-permeable barrier between the blood plasma and interstitium and controlling the

passage of macromolecules and fluid between blood and interstitial space [27]. The

Introduction

9

regulation of vascular permeability by transport of plasma proteins and solutes across

the endothelium involves two different routes: a) Paracellularly, via inter-endothelial

junctions. By losing the integrity of endothelial cells and cell junctions, the original

osmotic gradient can be demolished by protein transport across vascular wall through

gap formation [28-30]; b) Transcellularly, via transcytosis-mediated vesicular

transport. Small vesicles can be formed out of membrane invaginations such as

caveolae and selectively transport macromolecules like albumin across the

endothelium [30-31].

1.3.1 Regulation of paracellular permeability

The paracellular permeability of the endothelial barrier is maintained by the inter-

endothelial junctions. The junction structures connect adjacent endothelial cells and

regulate the transport of plasma proteins from the vessel lumen to interstitium [4].

Three types of inter-endothelial junctions are documented in the endothelium, tight

junctions (TJs), adherens junctions (AJs) and gap junctions. AJs and TJs function

promote adhesion of opposing cells in the monolayer and maintain the tightness of

the endothelial barrier, whereas gap junctions form channels between neighbouring

cells that pass water, ions, and other small molecules and transmit signals within the

contiguous monolayer of cells [27]. AJs, composed of the vascular endothelial (VE)-

cadherin complexes with catenins, are dominant and critically contribute to vascular

paracellular permeability regulation [27]. The general concept is that VE-cadherin-

mediated AJs established prior to TJs in endothelial cell cultures [32] and maintain

the architectural integrity of endothelium, whereas TJs are secondary and are only in

the brain microvasculature composing the blood-brain barrier [33-34]. Here, we

discuss the structure, dynamics, and regulation of the integrity of AJs in relation to

endothelial barrier function.

1.3.1.1 Endothelial barrier regulation by cadherin and catenin

In the intracellular junction domain, VE-cadherin interacts with proteins of the

Armadillo-repeat gene family, β-catenin, plakoglobin (γ-catenin), and p120-catenin.

The classical junction contains a proximal binding site for p120-catenin, and a distal

binding site for β-catenin and plakoglobin (see Fig. 3). Both β-catenin and

plakoglobin are linked to α-catenin, which may further interact with α-actinin and

vinculin [28]. The binding of β-catenin induces ordering of the last 100 residues of the

cadherin tail, which is intrinsically highly unstructured [35-36]. Plakoglobin binds the

Introduction

10

same region of VE-cadherin as β-catenin; however, it displays different dynamics at

AJs and recruits desmoplakin and vimentin to AJs [37-38]. Recent findings that over

expression of plakoglobin in human microvascular endothelial cells in culture (HMEC)

increased endothelial barrier function suggest that plakoglobin contributes to

strengthening of the endothelial barrier. Inhibition of plakoglobin expression leaded to

AJs disassembly and endothelial barrier failure under fluid shear stress [37],

indicating that plakoglobin has a role in stabilizing AJs and endothelial barrier

function.

P120-catenin, with β-catenin, also regulates the endothelial barrier at the level of VE-

cadherin expression. Both β-catenin and p120-catenin spatially regulate organization

of the actin cytoskeleton at the AJs. P120-catenin recruits the p190Rho GTPase-

activating protein (GAP), which stimulates GTP hydrolysis of RhoA [39]. β-Catenin

associates with IQRas GTPase activating protein 1 (IQGAP1), which is both an

effector and a regulator of the RhoGTPases Cdc42 and Rac1 [40]. The RhoGTPase

family members Cdc42, Rac, and RhoA are known to play a critical role in regulating

permeability of the endothelial barrier under basal conditions and in response to

external stimuli and will be discussed later in this section.

The role of α-catenin in the regulation of endothelial AJs has not yet been

determined. One theory suggests that α-catenin inhibits VE-cadherin recycling at the

cell surface and supports the functional and structural integrity of the endothelial

barrier [41-42].

Fig. 3: General schematic representation of the composition of adherens junctions (AJs) [30].

Introduction

11

1.3.1.2 Endothelial barrier regulation by kinases and phosphatises Phosphorylation of VE-cadherin and catenins provides the mechanism for remodeling

of AJs [43-45]. Kinases and phosphatases modulate the affinities of the proteins

within the cadherin-catenins complex and thereby determine AJ stability and

paracellular permeability. The high-affinity interaction between VE-cadherin and β-

catenin can be increased by differential phosphorylation of VE-cadherin or can be

diminished by phosphorylation of β-catenin [46].

The cytoplasmic tyrosine kinase c-Src plays a pivotal role in regulating vascular

endothelial permeability by transducing signals that mediate AJ destabilization and

acto-myosin contractility (Fig. 4). c-Src activation leads to tyrosine phosphorylation of

VE-cadherin and β-catenin, resulting in reduction of VE-cadherin binding to p120-

catenin or β-catenin and AJs destabilization [47-48]. c-Src phosphorylation provokes

the p21-activated kinase (PAK) that mediates phosphorylation of VE-cadherin and

stimulates a β-arrestin–mediated endocytosis [46]. The activation of protease

activated receptor-1 (PAR-1) by thrombin mediates the activation of c-Src and leads

to dissociating Src-homology-2-domain-containing protein tyrosine phosphatase

(SHP2) from AJs [49-50]. This dissociation provokes tyrosine phosphorylation of β-,

γ-, and p120-catenins, and AJ destabilization [50]. In contrast, the cytosolic C-

terminal Src kinase Csk binds to the phosphorylated Tyr685 of VE-cadherin and

protects it from Src-mediated phosphorylation [51].

Fig. 4: c-Src-mediated signalling in paracellular permeability regulation [30]. MLCK: myosin light chain kinase; PAK: p21-activated kinase; SHP2: Src-homology-2-domain-containing protein tyrosine phosphatises. Further details please see in text 1.3.1.2.

Introduction

12

c-Src can also regulate paracellular permeability by inducing tyrosine phosphorylation

of endothelial-specific myosin light chain kinase (MLCK) and resulting in a 2-3 fold

increase in kinase activity [52]. MLCK dependent phosphorylation of the regulatory

myosin light chain facilitates acto-myosin contraction [53]. Thus, c-Src mediated

MLCK activation provides an additional mechanism for regulating the reorganization

of the actin cytoskeleton by developing an endothelial cell shape change and

formation of inter-endothelial gaps with level of Ca2+ not increased.

1.3.1.3 Endothelial barrier regulation by calcium and ion channels Ca2+ (calcium ion) is a ubiquitous messenger regulating multiple processes including

paracellular endothelial permeability and vascular endothelial homeostasis [54]. The

basal cytosolic Ca2+ concentration in endothelial cells is about 100 nM. However

extracellular stimuli like thrombin, histamine or bradykinin can significantly increase

the cytosolic Ca2+ concentration and activate phospholipase C (PLC). As seen in Fig.

5, panel a, activated PLC further hydrolyzes phosphotidyl inositol bisphosphate

(PIP2) into inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 stimulates Ca2+

release from the IP3 sensitive endoplasmic reticulum (ER) Ca2+ store, which in turn

activates signalling molecules that mediate Ca2+ entry through store operated

channel (SOC) which might contain canonical transient receptor potential channels

(TRPC1, -4, -5). Alternatively, DAG can stimulate receptor operated channel (ROC)

mediated Ca2+ entry independently of Ca2+ store depletion. TRPC3 and -6 are

believed to participate in the ROC mediated Ca2+ influx [55]. Spontaneously, changes

in ER Ca2+ content are “sensed” by the stromal-interacting molecule (STIM1, STIM2)

which relay this signal to the Ca2+ channel (Orai1) [56-58]. These increases in

cytosolic Ca2+ concentration are short-lived, as excess Ca2+ is rapidly removed via

the plasma membrane Ca2+-ATPase (PMCA) [59-60] and sarco/endoplasmic

reticulum Ca2+-ATPase (SERCA) [60-61].

Protein kinase Cα (PKCα) is a member of a serine and threonine-specific protein

kinase family that is activated by Ca2+ and the second messenger DAG [62].

Although PKCα does not control basal permeability, it is a central regulator of the

endothelial permeability response to multiple mediators [63]. As seen Fig. 1.3.3,

Panel b PKCα selectively promotes phosphorylation of p120-catenin at Ser879,

leading to p120-catenin dissociation from VE-cadherin and AJ destabilization. PKCα

also modulates RhoA GTPase activation by phosphorylation of the upstream RhoA

regulators [64-65]. RhoA activation is accompanied by Ca2+/calmodulin-dependent

Introduction

13

activation of MLCK and MLC phosphorylation leading to actino-myosin contraction

and loss of endothelial barrier protection [66-67].

In this regard, PKCα cooperates with MLCK to elicit a coordinated spatial activation

of RhoA and reorganization of the actin cytoskeleton, resulting in endothelial

increased permeability. In addition, besides the direct effect of PKCα on the

cytoskeleton and AJs, PKCα also regulates endothelial barrier functions indirectly by

mediating the activation of other signalling pathways. For instance, PKCα induced

the phosphorylation of eNOS and increased NO production [68], suggesting a

coordinate role of PKCα and eNOS in the mechanism of endothelial barrier

hyperpermeability.

Fig. 5: Calcium signalling and paracellular permeability. Panel a shows the SOC and ROC operating Ca2+ influx. Panel b shows the Ca2+ regulated paracellular permeability. GPCR: G protein receptors; PLC: phospholipase C; DAG: diacylglycerol; SOC: store operated channels; ROC: receptor operated channel; PMCA: plasma membrane Ca2+-ATPase; STIM: Stromal-Interacting Molecule; SERCA: sarco/endoplasmic reticulum Ca2+-ATPase. TRPC: canonical transient receptor potential channels. Further details please see in text 1.3.1.3.

Introduction

14

1.3.1.4 Endothelial barrier regulation by RhoGTPase signalling The RhoGTPase family members Cdc42, Rac, and RhoA play a critical role in

regulating permeability of the endothelial barrier function especially in response to

external stimuli. The stabilization of AJs is associated with activities of Rac1 and

Cdc42, although some RhoA activity is required to maintain a subminimal amount of

MLC phosphorylation, which is critical for the formation of cortical actin bundles at the

sites of cell-cell adhesions [69]. Generally, Rac1 and Cdc42 operate the spatial

control of filopodial and lamellipodial activity of endothelial protrusions, whereas

RhoA facilitates the organization of actin stress fibers [70]. As seen in Fig. 6,

RhoGTPase controls paracellular permeability by multiple mechanisms that enable

activation of Cdc42 and Rac1 and inhibition of RhoA.

Fig. 6: RhoGTPase signalling in paracellular permeability regulation. IQGAP: IQRas GTPase activating protein; ROCK: Rho-associated protein kinase; MLCP: myosin light chain phosphatises. Further details please see in text 1.3.1.4.

p190RhoGAP plays a central role in inhibiting RhoA at the sites of cell-cell

adhesions, and its function depends on the interaction with p120-catenin, which

provides a platform for the physical interaction between p190RhoGAP and RhoA,

enabling the spatial inhibition of RhoA in AJs [39]. p190RhoGAP activity is positively

regulated by c-Src-mediated phosphorylation [71-72]. In contrast, Rho kinase

(ROCK)-mediated phosphorylation negatively regulates p190RhoGAP activity [73].

p190RhoGAP inhibits RhoA, and RhoA, in turn, is able to facilitate phosphorylation-

Introduction

15

induced inhibition of myosin light chain phosphatise (MLCP) by activating Rho kinase

(ROCK) [74-75]. The facilitation of MLCP leads to phosphorylation of MLC with

subsequence acto-myosin contraction and endothelial leakage [66-67].

p190RhoGAP mediated inhibition of RhoA required Rac1 activity [39], which is

regulated by Tiam-1 [76]. Tiam-1 regulates Rac1 and cooperates with p190RhoGAP

to inhibit RhoA and stabilize the AJs [77]. Vice versa, recruitment of Tiam-1 to the AJ

complex might also induce p190RhoGAP translocation and thereby sustained

inhibition of RhoA. In addition, the activities of IQGAP1 and Cdc42 are also important

in regulating AJ integrity [76]. IQGAP1 stabilizes Cdc42 and Rac1 in their active

GTP-bound forms [40], binds to β-catenin and can promote Rac1-mediated activation

of p190RhoGAP [78]. Further proof is that depletion of IQGAP1 destabilizes AJs,

whereas overexpression of IQGAP1 restores them in Rac1-deficient cells [79].

1.3.2 Regulation of transcellular permeability

The transcellular pathway has been defined as the receptor-mediated transport of

albumin from the vessel lumen to the interstitial space [80]. Transport occurs by

means of transcytosis, an energy-dependent trafficking of vesicles though the

endothelial cells [81]. The paracellular pathway allows free permeation of fluid and

solutes from blood plasma to the interstitial space but restricts the passage of

albumin and other large plasma proteins [82-83]. The difference in protein

concentration between plasma and interstitial space maintains the trans-endothelial

oncotic pressure, a key driving force that regulates tissue fluid homeostasis [84-85].

However, the abnormal transport of albumin from plasma to the interstitium via trans-

endothelial manner can increase the water content in the interstitial space and

thereby disturb the fluid balance across the endothelium leading to protein rich

edema [85].

Transcytosis requires a complex series of events involved caveolae and its structural

protein caveolin-1. Caveolae and caveolin will be discussed later in detail in the

section 1.6 “lipidraft and caveolae”. In the initial step of transcytosis, caveolar fission

is regulated by activation of dynamin at the caveolar neck. The following scission and

release of caveolar vesicle is associated with GTPase RhoG and GTP hydrolysis of

dynamin [80, 86-88]. Cav-1 is a structural protein of caveolae. Caveolin-1 deficient

mice demonstrated increased permeability due to albumin leakage in endothelia of

Introduction

16

small veins and capillaries [89-90], suggesting a transcellular pathways in regulating

tissue fluid balance.

As seen in Fig. 7, gp60 interacting with caveolin-1 leads to vesicle fission and

albumin transport [91-93]. Src-kinase family member c-Src and heterotrimeric G

proteins Gαi and Gαq are also recruited into caveolae [94]. Interaction of gp60 with

Cav-1 triggers activation of c-Src and Gαi dissociation from Gβγ [91, 93]. Activation

of c-Src and Gαi sequentially leads to tyrosine phosphorylation of Cav-1 and dynamin

[91, 93, 95-96], following recruitment of dynamin into caveolae and eventually

formation of the dynamin ring on caveolar necks [93, 97-98].

Fig. 7: Signalling mechanisms of caveolae-mediated albumin transport [30, 99].

Normally, albumin transcytosis is a constitutive process at part of the normal

maintenance of fluid balance across the endothelium. The accumulation of protein-

rich interstitial fluid associated with up-regulated transport of albumin only happens

under tissue injury and leads to protein rich edema [27, 100].

For instance, activation of neutrophils and their sequestration in pulmonary

microvessels lead to activation of albumin transcytosis and increased water content

in the alveolar space [101], and this process is Cav-1-dependent, as neutrophil

sequestration in lungs of caveolin-1 deficient mice did not induce hyperpermeability

to albumin [101].

Caveolae not only participate in albumin trafficking and transcellular permeability,

they also compartmentalize signalling molecules within caveolae microdomains and

facilitate dynamic interactions among signalling cascades. Cav-1 serves as a scaffold

that interacts with and modulates activity of binding partners [27, 80]. Here, in this

thesis we will describe that caveolin-1 negatively regulate eNOS activity in response

to PAF.

Introduction

17

1.3.3 Mediators regulating endothelial permeability Sphingosine-1-phosphate (S1P) is a bioactive lipid mediator produced by the

breakdown of the membrane phospholipid sphingomyelin [102-103]. S1P mediates

endothelial barrier function through activation of the five G protein–coupled receptors

(S1P1-S1P5), and different S1P receptor subtypes may regulate vascular permeability

antagonistically: endothelial barrier is enhanced by S1P1 but impaired by S1P2 and

S1P3 [104-105]. S1P1 coupling to G proteins induces signalling toward Rac1

activation and reorganization of the actin cytoskeleton into cortical bundles in concert

with cortactin, α-actinin 1 and 4, paxillin and the GPCR kinase–interacting proteins

[106-107]. S1P signalling also increases the abundance of VE-cadherin at the cell

surface and induces AJ enhancement [108] (for details, please see “sphingolipids

dection”).

Platelet-activating factor (PAF), a phospholipid produced by a variety of cell types

including endothelial cells, plays an important role in regulating vascular homeostasis

[109]. PAF activates a G protein–coupled receptor, PAF-R, localized at the cell

surface and in intracellular compartments [109]. The binding of PAF to the cell

surface receptor induces PLCs activation and leads to reorganization of actin

cytoskeleton associated vascular permeability [110-111]. PAF response on vascular

barrier homeostasis are also associated with PAF-R-mediated transcriptional

regulation of NO production and Ca2+ influx [109] (For the details please seen

following section of “platelet activating factor”).

Thrombin, a procoagulant serine protease, is a pro-inflammatory mediator, which

induces intravascular thrombosis and formation of fibrin clots [112]. Thrombin

mediates a transient increase in paracellular permeability. Signals through Gαq and

Gα12/13 proteins, lead to activation of PLCβ and sequentially to increased

intracellular Ca2+ concentration [74].

Vascular endothelial growth factor (VEGF) is a proangiogenic cytokine that is

essential for developmental and pathological angiogenesis [113-114]. Paracellular

permeability induced by the release of VEGF is mediated through activation of its

high-affinity receptor tyrosine kinase VEGFR2 [115-116]. The VEGF-VEGFR2

signalling induces an increased endothelial permeability via Src-mediated signalling

of VE-cadherin internalization in a relative long time course comparing with PAF

thrombin, and activation of PLC-γ that mediates an increase in intracellular Ca2+

concentration [117].

Introduction

18

Fibroblast growth factor (FGF) is an angiogenic cytokine that plays an important role

in the maintenance of endothelial barrier function through the regulation of AJ

integrity [118]. FGF binds to a high-affinity cell surface tyrosine kinase receptor

FGFR. Its interaction with p120-catenin leads to stabilization of VE-cadherin and

enhancement of endothelial barrier [118].

Bradykinin is an inflammatory mediator generated by cleavaging kinin peptides at the

sites of tissue injury [119]. The permeability response to bradykinin is mediated

through activation of the B2 receptor that is ubiquitously expressed in resting

endothelial cells [120]. The signal is coupled by Gαq, resulting in an increased

concentration of intracellular Ca2+ and subsequently in activation of endothelial nitric

oxide synthase (eNOS) [121].

In summary, Many signalling pathways of catenins, kinases [122-123], myosin light

chains (MLC) and Rho have been identified [63, 124-127] in endothelial permeability

regulation, but the exact mechanisms of endothelial barrier failure still requires further

study. Of note, there are only few mediators can alter pulmonary vascular

permeability in minutes or even seconds, and PAF is one of them. We will discuss in

the rest of thesis, new hypotheses and regulatory pathways of increased endothelial

permeability in the pulmonary vasculature induced by PAF, in particular with respect

to eNOS and TRPC channels.

1.4 Platelet activating factor Platelet-activating factor, also known as a PAF, PAF-acether or AGEPC (acetyl-

glyceryl-ether-phosphorylcholine) is a potent phospholipid activator and mediator of

many leukocyte functions, including platelet aggregation, inflammation, and

anaphylaxis. It is produced in response to specific stimuli by a variety of cell types,

including neutrophils, basophils, platelets, and endothelial cells.

This phospholipid was originally shown to induce aggregation of blood platelets

released from basophils stimulated with immunoglobulin E by J. Benveniste in 1972

[128-129], and its structure was elucidated as 1-alkyl-2-acetyl-sn-glycero-3-

phosphocholine by Constantinos A. Demopoulos in 1979 [130] (see Fig. 8).

Introduction

19

1.4.1 Chemistry and Biology of PAF PAF is an unusual lipid, as lipids with alkyl groups only in position sn-1 are not

common in animals. As seen in Fig. 8, the alkyl group is connected by an ether

linkage at the C1 carbon to a C16 or C18 chain; the acyl group at the C2 carbon is an

acetate unit whose short length increases the solubility of PAF, allowing it to act as a

soluble signal messenger; The C3 has a phosphocholine head group, just like

standard phosphatidylcholine.

Fig. 8: Structure of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF).

PAF was the first phospholipid known to have messenger functions, and to signal

though specific receptors rather than to act though its physico-chemical effects on the

plasma membrane or other cell membranes. There is a strict structural requirement

for binding to its unique single G-protein coupled receptor (PAF-R), which is

expressed by numerous cells including all those of the innate immune system. There

is a relatively high specificity for the ether bond in position sn-1 of PAF in comparison

to the 1-acyl analogue, together with considerable specificity for a short acyl chain in

position sn-2 and for the phosphorylcholine head group [128, 131-133].

PAF is synthesised by a variety of cells, but especially those involved in host

defence, such as platelets, endothelial cells, neutrophils, monocytes and

macrophages. PAF is synthesised continuously by cells, but at low level, controlled

by the activity of PAF acetyl hydrolases (see below). However, it is produced in much

greater quantities by inflammatory cells when receiving cell-specific stimuli. When

the cells are subjected to acute inflammatory stimulation, the activated enzymes

produce PAF in appreciable amounts, a distinct membrane-bound acetyltransferase

(LPCAT2) transfers an acetyl residue from acetyl-CoA to 1-alkyl-sn-glycero-3-

phosphocholine (lyso-PAF), generated by the action of phospholipase A2 on

phosphatidylcholine [134-136]. Alternatively, PAF also can be produced by

acetylation of 1-alkyl-sn-glycero-3-phosphate, which is converted to 1-alkyl-2-

Introduction

20

acetylglycerol and thence to PAF. But this ‘de novo’ pathway is believed to be non-

inflammatory [137-138].

Initially, PAF was shown to trigger aggregation of platelets at concentrations as low

as 10-11 M, and it induced a hypertensive response at very low levels as well. Recent

work has been concerned with the function of PAF as a mediator of inflammation,

and its role in the immune response. For example, it has a number of pro-

inflammatory properties, and it has been implicated in the pathogenesis of a number

of disease states, ranging from allergic reactions to stroke, myocardial infarction,

colitis and multiple sclerosis. For example, In asthma, platelet-activating factor is able

to act directly as a chemotactic factor and indirectly by stimulating the release of

other inflammatory agents. Administration of PAF can produce many of the

symptoms observed in asthma, partly via the formation of leukotrienes as secondary

mediators.

By binding to its specific receptor, PAF activates the cytoplasmic phospholipase A2

(PLA2) and phospholipase C (PLC), and tyrosine phosphorylation of various proteins.

The result of the latter is an increase in intracellular Ca2+; activation of protein kinase

C and mitogen activated protein kinase (MAPK) [139-140]. The G protein dependent

phosphatidylcholine specific phospholipase C mediates the following hydrolysis of

phosphatidylcholine and leads to generation of inositol 1,4,5-trisphosphate (IP3) and

diacylglycerol (DAG). IP3 increases intracellular Ca2+ via IP3 receptors (IP3R) as well

as DAG mediate protein kinase C (PKC) activation [138, 141].

1.4.2 PAF in vascular permeability and edema formation PAF plays a central role in many pulmonary disorders. In the lungs, PAF contracts

airway and vascular smooth muscle and causes vascular gaps in capillaries, venules

and veins [142-147]. All these actions take place in minutes and are all believed to be

associated with lipid mediators and lipid modifying enzymes (see Fig. 9).

Particularly, cyclooxygenase and lipoxygenase trigger bronchial and vascular smooth

muscle contractions via their products thromboxane and leukotrienes C4 and D4 [148-

149]. Rho kinase appears to regulate both thromboxane release and thromboxane

effects on smooth muscle contraction [150]. These evidences are mostly based on

pharmacological inhibitors of leukotrienes or thromboxane that prevented the

vascular and brocho-constriction in isolated perfused rat lung [151]. Cyclooxygenase

can also regulate the edema formation by prostaglandin E2 (PGE2) and activation of

Introduction

21

EP3 receptor [152]: In isolated perfused rat lung it was shown that PAF stimulates

PGE2 release and perfusion with neutralizing anti-PGE2 antibodies attenuated the

PAF-induced edema formation [152]; Sulprostone, an EP1/3-receptor agonist induced

the edema in isolated perfused rat lung but not the EP2-receptor agonist butaprost

[152].

Fig. 9: Overview on the mechanisms of PAF induced bronchocontriction, vasoconstriction and edema formation [148-149]. LTC4: leukotrienes C4; LTD4: leukotrienes D4; PGE2: Prostaglandin E2; TXA2: thromboxane A2; NO: nitric oxide. Further details please see in text 1.4.2.

There is another pathway which is not well studied so far is believed to be involved

with activation of acid sphingomyelinase (ASM) and ceramide production [111]. PAF

can rapidly increase intracellular ceramide level in several cell types [111, 153-154],

and this pro-inflammatory related ceramide is produced by ASM [111]. Ceramide can

induce edema formation both by lung perfusion and by airway instillation [111, 155];

and the ceramide specific antibodies can attenuate the PAF-induced edema

formation in perfused lung [111]. ASM Inhibition pharmacologically by D609 or

genetically by Smpd1 depletion did also prevent the PAF-induced edema formation

[111].

Introduction

22

It was the major aim of this study to reveal the role of the ASM-ceramide pathway in

regulation of pulmonary vascular permeability in response to PAF.

1.5 Sphingolipids The sphingolipids comprise a complex range of lipids in which fatty acids are linked

via amide bonds to a long-chain base or sphingoid. The term “sphingo-” was first

coined by J.L.W. Thudichum in 1884 because the enigmatic nature of these

molecules reminded him of the sphinx. Sphingolipids are extremely versatile

molecules regulating many physiological processes such as apoptosis, innate and

acquired immunity, vascular permeability, smooth muscle tone, and they contribute to

various pathological conditions [111, 156-157].

1.5.1 General comments of sphingolipids Sphingosine is the fundamental structural group to sphingolipids. Its phosphorylation

generates sphingosine-1-phosphate (S1P) and its acylation generates ceramide.

Sphingomyelins have a phosphorylcholine or phosphoroethanolamine molecule with

an ester linkage to the 1-hydroxy group of a ceramide. Figure. 10 shows the

metabolism of all these sphingolipids. Sphingolipids are synthesized in a pathway

that begins in the ER and is completed in the Golgi apparatus, but most of these

lipids are enriched in the plasma membrane and in endosomes where they perform

most of their functions [158]. Sphingolipids are virtually absent from mitochondria and

the ER, but constitute a 20-35 molar fraction of plasma membrane lipids [159].

Sphingolipid metabolites, such as ceramide and sphingosine-1-phosphate, have

been shown to play an important role in the signalling cascades involved in

apoptosis, proliferation, stress responses and vascular permeability [105, 111, 156,

160].

Introduction

23

Fig. 10: Overview of sphingolipid metabolism [156].

Ceramide consists of a long-chain sphingoid base linked to a fatty acid via an amide

bond. The level of ceramide in tissues in low, but they can still exert important and

various biological effects. Ceramide is formed as the key intermediate in the

biosynthesis of all the complex sphingolipids, in which the terminal primary hydroxyl

group is linked to carbohydrate, phosphate, etc. Unlike the sphingoid precursors,

ceramide is not soluble in water and is located in membranes where it participates in

raft formation [161-162].

Besides the de novo synthesis, ceramide is also produced during the catabolism of

other complex sphingolipids, for example by the activation of the sphingomyelinases

(acid-/neutral sphingomyelinase) on sphingomyelin or ceramide synthase on

sphingosine [163-164]. Many agonists including chemotherapeutic agents, tumor

necrosis factor (TNF), PAF, 1,25-dihydroxy-vitamin D3, endotoxin, interleukins, nerve

growth factor (NGF), irradiation and heat shock stimulate hydrolysis of sphingomyelin

to produce ceramide [111, 154, 165-167].

Ceramides are minor component of membranes in general, but their physical

properties ensure that they are concentrated preferentially into lateral liquid-ordered

Introduction

24

microdomains (a form of 'lipid raft' termed to ‘ceramide-rich platforms’) [168].

Ceramide is generated within these rafts by the action of acid sphingomyelinase that

causes small rafts to merge into larger units then modifying the membrane structure

and property [161-162, 168-169]. Specific receptor molecules and signalling proteins

cluster within such platform excluding potential inhibitory signals, while initiating and

greatly amplifying primary signals [156].

It is believed that ceramide-rich platforms amplify both receptor- and stress-mediated

signalling events and thence may influence various disease states. Assuming wide

range of biological functions in cellular signalling, ceramide is known as a pro-

apoptotic molecule and to respond to many forms of cellular stress. Ceramide has

also been implicated in the activation of various protein kinase cascades: Ceramide

was to shown to interact with ceramide-activated protein phosphatase and ceramide-

activated protein kinases to regulate glycogen synthesis, insulin resistance in

response to apoptotic stimuli [170-173]. In addition, ceramide can influence the

permeability of cell membranes via interaction with ion channels and by triggering

pore formation [174-175].

Sphingosine-1-phosphate (S1P), a zwitterionic lysophospholipid, is an important

cellular metabolite, derived from sphingosine. It is an inter-mediator in the irreversible

degradation of sphingolipids, and also has important signalling functions.

Like its precursors, sphingosine-1-phosphate is a potent messenger molecule which

is recognized as an ubiquitous regulator of cell proliferation and survival,

angiogenesis and endothelial barrier functions, cytoskeletal organization, cellular

Ca2+ homeostasis, cell-cell contacts, and adhesion [176-178]. Sphingosine-1-

phosphate exerts many of its extra-cellular effects through acting as a ligand for

specific G protein-coupled receptors on cell surfaces, designated S1P1 to S1P5. In

mammals, S1P1, S1P2, and S1P3 are found in all tissues, whereas S1P4 is restricted

to lymphoid tissues and lung and S1P5 to brain and skin [178]. Different S1P receptor

subtypes may act antagonistically: especially in the lung, endothelial barrier is

enhanced by S1P1 but impaired by S1P2 and S1P3 (see table 2) chemotaxis is

stimulated by S1P1 but inhibited by S1P2 [104-105].

There is accumulating evidence that sphingosine-1-phosphate and its receptors

regulate heart rate, blood flow in the coronary artery and the mechanisms have to be

identified [179-180]. It also has been suggested that S1P, together with other

lysolipids such as sphingosylphosphorylcholine and lysosulfatide, are responsible for

Introduction

25

the potent anti-atherogenic and anti-inflammatory signalling molecule nitric oxide

(NO) in the vascular endothelium [181]. In addition sphingosine-1-phosphate has a

pro-inflammatory function that responds to certain cytokines and bacterial

lipopolysaccharides (LPS) and induces up-regulation of the enzyme cyclooxygenase-

2 (COX-2) and thence produces the prostaglandin PGE2 [182-184].

Table 2: Pulmonary response of sphingolipids

Outcome Sphingolipids Receptors References ▲Endothelial barrier S1P S1P1 [105, 185]

▼Endothelial barrier S1P

Ceramide

S1P2, S1P3 [186-187]

[111]

▼Epithelial barrier S1P S1P3 [188-189]

▲Apoptosis Ceramide [190-192]

▲Survival S1P S1P1 [190, 193-194]

Airway contraction S1P S1P2, S1P3 [195]

Emphysema Ceramide [192]

Mast cell activation S1P S1P1 [196]

1.5.2 Regulation of pulmonary disease by sphingolipids 1.5.2.1 Sphingolipds and vascular permeability

Vascular permeability is regulated by sphingolipids in numerous ways. S1P enhances

endothelial barrier function, and this barrier stabilizing function appears to be mainly

mediated by S1P1 receptors and involves activation of Rac GTPase dependent

cytoskeletal reorganization and focal adhesion assembly [185]. S1P was also shown

to protect against lipopolysaccharides (LPS) induced pulmonary edema and ALI

[197-198]. On the other hand, S1P2 causes Rho-kinase–dependent inhibition of Rac

and impairing of adherence junctions [186], and S1P3 is known to increase epithelial

[188-189] and endothelial permeability [187].

S1P1 coupling to Gi induces signalling associated Rac1 activation and reorganization

of the actin cytoskeleton. Cortactin, α-actinin, paxillin, focal adhesion kinase (FAK),

and the GPCR kinase–interacting proteins GIT1 and GIT2 are also required [106-

107]. S1P-induced activation of Rac1 is mediated by Tiam-1 which is the Rac1

guanine nucleotide exchange factor. Recruitment of Tiam-1 into caveolae or cell-cell

space is the precondition for spatial modulation of the S1P-mediated Rac1 activation

and depends on phosphoinositide 3-kinase (PI3K) activity [199]. Recruitment of

Introduction

26

Tiam-1 to the caveolae facilitates its stabilization and abundance by inhibiting Tiam-1

degradation [200]. Tiam-1/Rac1 signalling cooperates and provokes p190RhoGAP

whose main function is inhibiting RhoA [77]. RhoA facilitates the inhibition of myosin

light chain phosphatise (MLCP) by activating Rho kinase (ROCK) [74-75]. The

inhibition of MLCP leads to phosphorylation of MLC and induces acto-myosin

contraction, which leads to endothelial barrier dysfunction [66-67, 201]. S1P

signalling also increases the abundance of VE-cadherin at the cell surface and

induces AJ assembly [108].

Interestingly, activation of S1P2 causes Rho-kinase dependent inhibition of Rac and

impairs adherence junctions [186], and signalling though S1P3 receptor is known to

increase epithelial [188-189] and endothelial permeability [187]. This bidirectional

phenomenon may be explained by coupling to different G proteins thus provoking

different signalling pathways (see Fig. 11). The enhancement of endothelial barrier

and activated Rac/Tiam is only regulated by S1P1 coupling to Gi. Activation of S1P2

on endothelial cell inhibits their migration and increases vascular permeability, most

likely through mechanisms involving Rho-dependent inhibition of Rac and stimulation

of the 3′-specific phosphoinositide phosphatase PTEN [202-203]. This S1P2 activated

Rac inhibition requires G12/13 and Gq. The G12/13 and Gq induces Rho activation by

direct physical and functional interaction between Gα12/13/ Gαq and Rho-guanine

nucleotide exchange factors (Rho-GEFs) [204]. S1P3 mediates vasoconstriction

through Gq coupled Ca2+ mobilization and G12/13 regulated Rho dependent myosin

light chain phosphatase inhibition in vascular smooth muscle cells (VSMCs) [205-

206]. In addition, S1P3 can also activate eNOS and provoke NO production in

vascular endothelial cells through Gi-and Akt-mediated phosphorylation of eNOS in

concert with a Gq-mediated, Ca2+/calmodulin-dependent activation [207].

Introduction

27

Fig. 11: signalling pathways of S1P receptors and G proteins

Ceramide is also involved in the regulation of vascular permeability. PAF can

stimulate the ceramide production rapidly by ASM activation [111, 154]. In the PAF-

induced edema, ASM inhibition or anti-ceramide antibody perfusion can mitigate the

edema formation in isolated perfused lung model [111, 142]. Ceramide can induce

edema formation by lung perfusion [111] or instillation in rat airways [155]. In cultured

human pulmonary microvascular endothelial cells, ceramide also increased vascular

permeability [208]. In addition, it seems that endothelial cell apoptosis does not

contribute to vascular permeability regulation [142, 208].

1.5.2.2 Ceramide and pulmonary infections

Ceramide-enriched micro domains seem to act as a platform that permits the spatial

and temporal organization of receptors and signalling molecules [161-162, 168-169].

Thus, it was shown that several receptors and signalling molecules including CD95

[209-211], CD40 [212], FcgRII [213], CD14 [214], CFTR [215], caspase 8 [216] and

platelet activating factor [111], were reported to trigger the formation of such

ceramide-enriched membrane platforms.

Ceramide-enriched membrane platforms are thought to be one important regulatory

factor in pulmonary infections. Pseudomonas. aeruginosa infection is one well

studied case. 40% of deaths in patients with ventilator-associated pneumonia are

caused by P. aeruginosa [217]. Infection triggers rapid forms of acid

sphingomyelinase transport to the extracellular leaflet of the cell membrane where

the ceramide-enriched membrane platforms [218]. It was shown that CD95 receptor

Introduction

28

and cystic fibrosis transmembrane conductance regulator (CFTR) molecules cluster

within this platform upon infection [218-219]. CD95 was previously shown to mediate

apoptosis of cells infected with P. aeruginosa and CFTR was known as a receptor for

P. aeruginosa, mediating internalization of the bacteria [220-221]. Additional

evidence is that ceramide-enriched platform destruction prevented NF-КB nuclear

translocation in respiratory epithelial cells and cellular apoptosis [215, 218].

1.5.2.3 Ceramide and cystic fibrosis

Mutations in CFTR cause cystic fibrosis which is the most common autosomal

recessive disorder [222]. Cystic fibrosis is characterized by chronic lung

inflammation, and frequent and chronic infections of the lung with P. aeruginosa, as

well as S. aureus, Burkholderia cepacia and Haemophilus influenzae. It was

unknown how CFTR deficiency results in increased pulmonary inflammation and the

high susceptibility to bacterial infections until a new explanation came up recently

implicating that ceramide and acid sphingomyelinase in that process. CFTR

deficiency results in an increase in pH that may affect enzymatic activity of acid

sphingomyelinase and acid ceramidase [223]. pH up to 6.0 almost inhibited acid

ceramidase whereas acid sphingomyelinase activity was only decreased by 30–40%

[223-224]. The accumulated ceramide can further trigger chronic pulmonary

inflammation, death of respiratory epithelial cells, and deposition of DNA in bronchi.

Of note, inhibition of ASM in CFTR deficient mice can prevent pulmonary infections of

P. aeruginosa.

1.5.2.4 Sphingosine-1-phosphate and allergic disorders

Asthma is characterized by bronchial epithelium abnormalities in function and

structure, accumulation of inflammatory cells in the bronchial mucosa and airway

tissue structure remodelling. Many mediators have been identified that play

significant roles in the initiation and progression of the disease, among them,

sphingosine-1-phosphate (S1P) appears to be an active metabolite that regulates

allergic responses in asthma and anaphylaxis. The first finding that S1P may play a

role in asthma was that S1P levels were elevated in the bronchoalveolar lavage

(BAL) fluid of asthmatics after antigen challenge, and this elevated S1P level was

associated with eosinophil numbers, a hallmark of asthma [225].

Introduction

29

Sphingosine-1-phosphate and mast cell function in the allergic response

Mast cells play central roles in immediate-type and inflammatory allergic reactions

that can result in asthma. Cross-linking of the high affinity immunoglobulin E receptor

FcεRI on master cells induces activation of sphingosine kinases, production of S1P

and subsequent activation of the S1P1 and S1P2 receptors [226]. Activation of S1P2

is required for mast cell degranulation while S1P1 activation is important for their

migration to sites of inflammation. S1P Binding to S1P1 induces cytoskeletal

rearrangements and triggers mast cell migration towards an antigen gradient [226],

by which low antigen concentrations could attract master cell to the action site via

S1P1. On the other hand, S1P2 mediates mast cells activation and promotes

degranulation [226]. Secretion of S1P by mast cells also provokes inflammation by

activating and recruiting other immune cells like eosinophils [227] and Th2

lymphocytes [228].

Sphingosine kinases phosphorylate sphingosine to form S1P. Two mammalian

isoforms of sphingosine kinases have been identified, named SphK1 and SphK2.

Furthermore, SphK1 and SphK2 display different catalytic properties, subcellular

locations and substrate specificity [176]. Crosslinking of the FcεRI activates SphK1

and SphK2, increases S1P production and decreases sphingosine [226, 229]. The

majority of studies of S1P in mast cells have been focused on SphK1; it was believed

that down-regulation of SphK1 but not SphK2 inhibited Ca2+ mobilization,

degranulation and migration of mast cells induced by antigen [226, 229-230].

However, recent studies have shown that both SphK1 and SphK2 were activated

upon FcεRI engagement in murine and immature human mast cells, and this required

Fyn kinase in bone marrow-derived mast cells [231]. Exogenous addition of S1P can

restore the migration and degranulation in Fyn deficient animals [231]. Furthermore, it

was recently shown that depletion of Sphk2, but not Sphk1, suppressed FcεRI-

mediated generation of S1P, as well as Ca2+ influx, PKC activation, degranulation

and cytokine secretion [232]. Sphingosine-1-phosphate and airway smooth muscle cells and airway remodeling Another defining feature of an asthmatic attack is the contraction of airway smooth

muscle. S1P stimulates contraction of human airway smooth muscles in a Rho-

dependent manner associated with activation of S1P2 and S1P3 [195]. S1P induced a

dose-dependent contraction in isolated bronchi from sensitized mice [233]. S1P also

caused airway hyperresponsiveness in the ovalbumin-sensitized mouse model of

asthma, and both S1P2 and S1P3 receptors expression. In addition, SphK1 and

Introduction

30

SphK2 activity were up-regulated in the ovalbumin-sensitized mouse asthma model

[233]. Inhibition of SphK impaired airway hyperresponsiveness in a murine asthma

model [234].

The regulation of S1P in airway remodelling has also been characterized. First, S1P

promoted the proliferation of both human airway smooth muscle cells [225] and

fibroblasts [235-236]. Furthermore, S1P produced by SphK1 has recently been

shown to be involved in fibroblast differentiation into myofibroblasts, which was

believed to be relevant to lamina reticularis thickening [237]. Additionally, it was

shown that transforming growth factor (TGF-β) stimulates SphK1, which in turn

released S1P that activates S1P2 and S1P3, leading to α-smooth muscle actin

expression in lung fibroblasts which is hallmark of their differentiation into

myofibroblasts [237].

1.5.2.5 Ceramide and pulmonary emphysema

Emphysema is a chronic, obstructive pulmonary disease primarily caused by

cigarette smoking. It is characterized by a destructive and permanent enlargement of

distal airspaces and alveolar walls, ultimately leading to impaired oxygenation.

Historically, the pathogenesis of emphysema has been linked to chronic lung

inflammation, and more recently it has been recognized that alveolar cell apoptosis is

a crucial step in the pathogenesis of this disease [238]. There is evidence showing

that ceramide is a crucial mediator of alveolar destruction in emphysema. In the

model of vascular endothelial growth factor blockade, formation of ceramide

predominantly increased in alveolar cells, which was mediated by an increased

activity of the ceramide synthase pathway and a feed forward activation of the ASM

[192]. Increased ceramide was critical to trigger alveolar cell death, oxidative stress

[239] and inhibition of ceramide, via anti-ceramide antibodies or inhibition of enzymes

controlling de novo ceramide synthesis prevented this phenomenon. It seems that

emphysema develops partially as the results of ceramide mediated oxidative stress

and apoptosis of alveolar endothelial and epithelial cells [192, 239], then endothelial

ceramide may be a novel target to prevent or treat emphysema and chronic

obstructive pulmonary disease (COPD).

1.5.2.6 Niemann-pick disease

Sphingomyelinases are characterized by their pH optimum, with acid, neutral and

alkaline sphingomyelinase identified [240-242]. Although all these three enzymes

Introduction

31

have general mechanisms for ceramide generation and cell signalling, the majority of

studies have focused specifically on the contributions of acid sphngomyelinase

(ASM). Niemann-pick disease (NPD, type A, B) is a rare genetic disorder that is

caused by ASM deficiency. Both forms of this disorder are inherited as recessive

traits, and both are due to mutations in the SMPD1 gene [243-244]. Type A NPD is

the infantile form, which is characterized by neurological deficits, enlarged livers and

spleens, pulmonary infections and death by age 2–3 [156]. In contrast, type B NPD is

the later onset form, in which patients exhibit little or no neurological symptoms but

may have severe and progressive visceral organ abnormalities, including

hepatosplenomegaly, pulmonary insufficiency, and cardiovascular disease [245].

Many of the clinical findings in NPD are likely related to lipid storage in lysosomes

and endosomes, and recent data have also revealed the important role of ASM in cell

membrane formation and function. Impaired pulmonary function is also a major

clinical finding in patients with type B NPD [246]. A similar outcome was observed in

ASM knockout mice [247].

1.6 Lipid rafts and caveolae 1.6.1 History and biology According to the fluid mosaic model of S. J. Singer and Garth Nicolson in 1972, the

biological membranes can be considered as a two-dimensional liquid where all lipid

and protein molecules can diffuse more or less easily [248]. Furthermore, the plasma

membrane contains different structures or domains that can be classified as a)

protein-protein complexes; b) lipid rafts, c) pickets and fences formed by the actin-

based cytoskeleton; and d) large stable structures, such as synapses or

desmosomes [248-249].

The original concept of lipid “rafts” was used as an explanation for the transport of

cholesterol from the Golgi network to the plasma membrane. The idea was more

formally developed in 1997 by Simons and Ikonen [250]. Eventually lipid rafts were

defined as "small (10-200nm), heterogeneous, highly dynamic, sterol- and

sphingolipid-enriched domains that compartmentalize cellular processes, and small

rafts can sometimes be stabilized to form larger platforms. Two types of lipid rafts

have been proposed: planar lipid rafts (also referred to as non-caveolar or glycolipid

rafts) and caveolae. Planar rafts are defined as being continuous with the plane of

the plasma membrane (not invaginated). Caveolae, on the other hand, are flask

Introduction

32

shaped invaginations of the plasma membrane that contain caveolin proteins and are

the most readily-observed structures in lipid rafts [251-252].

Caveolae (Latin for little caves, singular: caveola) were first identified by electron

microscopic examination by Yamada in 1950s [251], this “cave-like” domain is the

flask-shaped structures enriched in proteins as well as lipids such as cholesterol and

sphingolipids and have several functions in signal transduction [253]. Caveolae are

also believed to play a role in endocytosis, transcytosis, and the uptake of pathogenic

bacteria and certain viruses [254-255]. Since then, caveolae have been identified in a

wide variety of tissues and cell types. Particularly, caveolae have been well described

in adipocytes, fibroblasts where they are extremely abundant, endothelial cells, type I

pneumocytes, striated and smooth muscle cells. Additionally, ultra-structural analysis

of adipocytes has shown that as much as 20% of the total plasma membrane is

occupied by caveolae [253, 256].

Formation and maintenance of caveolae is primarily dependent on the protein

caveolin which has a cytoplasmic C-terminus and a cytoplasmic N-terminus, linked

together by a hydrophobic hairpin and inserted into the membrane. It has estimated

144 molecules of caveolin are present in a single caveolar structure [257], and the

relative amount of cholesterol in caveolae is much more. Some glycosphingolipids

and sphingomyelin are also enriched in caveolae relatively to the plasma membrane.

Interestingly, the density of lipids was found to be higher in the caveolae than in the

plasma membrane fraction from which the caveolae were isolated [258]. In summary,

caveolae represent a specialized, morphologically distinct sphingolipid-cholesterol

microdomain, which is stabilized by the unique caveolin protein.

Introduction

33

Fig. 12: Caveolae and caveolin. Panel a shows the electron micrograph in adipocytes; panel b indicates how caveolin is inserted to form caveolae membrane [99]. 1.6.2 Caveolins Caveolins are a family of integral membrane proteins which are the principal and

particular components of caveolae membranes. This 21~24 kDa protein together with

cholesterol and sphingolipids form the major structural components of caveolae

[249]. Caveolins also act as scaffolding proteins to compartmentalize and

concentrate signalling molecules within caveolar [99]. Various types of signalling

molecules, including G-protein subunits, receptor and non-receptor tyrosine kinases,

eNOS, and small GTPases can bind caveolin through its 'caveolin-scaffolding

domain' and this binding leads to inhibition of their activity [259].

The caveolin gene family has three members in vertebrates: CAV-1, CAV-2, and

CAV-3, respectively coding for the proteins caveolin-1, caveolin-2 and caveolin-3

[260]. All three members are membrane proteins with similar structure. CAV-1 the

first gene which was discovered, is derived by alternative translational initiation

and/or mRNA splicing of two different isoforms: caveolin-1α consisting of residues 1-

178 and the caveolin-1β containing residues 32-178, resulting in a protein of ~ 3 kDa

smaller size [261]. Caveolin-2 has three identified isoforms: caveolin-2α, -2β and -2γ,

and they are closely related to caveolin-1 as they colocalize and heterooligomerize

together with caveolin-1 [259, 262]. Caveolin-1 is most prominently expressed in

endothelial, fibrous and adipose tissue, and it seems to be co-expressed with

Introduction

34

caveolin-2. The expression of caveolin-3 is restricted to muscle cell type (cardiac,

skeletal, smooth muscle) [263-265]. Interestingly, ablation of Cav-1 and Cav-3

caused caveolae absence, but loss of Cav-2 has no apparent effect on caveolae

formation [266-268].

The so called “caveolin scaffolding domain” (CSD) is a region spanning 20 residues

in the caveolin sequence (mapping to residues 81-101 in the human caveolin-1

sequence) [94]. Using a glutathione S-transferase (GST)-CSD fusion protein as a bait

to select peptide ligands from a bacteriophage display library, Lisanti and colleagues

indentified a “caveolin binding motif” (CBM) that appeared to be presented in many

proteins located in caveolae (ΦΧΧΧΧΦΧΧ or ΦΧΦΧΧΧΧΦ, where Φ is an aromatic

amino acid) [269]. Different signalling proteins but containing CBM can all bind

caveolins and their activity are spontaneously inhibited [259].

1.6.3 Biological function role of caveolae With so many different types of molecules, proteins and receptors located in

caveolae (see table 3), it is evidential that they should play an important function role

in many cellular processes especially as macromolecules transportation [83, 270-

271], nitric oxide (NO) generation [272-273], ion-channel regulation [274-275],

angiogenesis regulation [276], shear stress and regulation of vascular permeability

[266, 277].

Table 3: Proteins and receptors located in caveolae

Proteins Functions References

Caveolin Signalling and major structure [249, 259, 278]

Flotillin Signalling and structure [279-280]

Actin Cell motility [278, 281]

SNAP Vesicular transportation [282]

G proteins G protein related receptor activation [278, 283]

IP3 receptor Calcium flux [284-285]

M6P receptor Receptor for mannose-6-phosphate [286]

Tyosine kinase Signalling and transcription [278, 287]

Ser/Thr kinase Signalling and phosphorylation [81, 278, 287-288]

eNOS NO production and signalling [259, 289]

nNOS Neuronal NO production and signalling [290]

COX Metabolism and signalling [291]

Introduction

35

1.6.3.1 Caveolae, caveolins and endocytic processes

The observation that caveolae exist as invaginations of the plasma membrane, as

completely enclosed vesicles, and as aggregates of several vesicles may suggest

that this structures are involved in the endocytosis of macromolecules [292].

Surprisingly, It was revealed that cells predominantly used caveolae for the selective

uptake of molecules as full size proteins [164-165], viruses [293] and even entire

bacteria [294].

The classical endocytosis is associated with clathrin coated vesicles, but it seems

that certain receptors and extracellular macromolecules are exclusively transported

via caveolae rather than clathrin vesicles [99]. This alternative caveolar pathway was

first recognised by the observation that cholera toxin was preferentially bound and

internalized via caveolae (see Fig. 13). Budded caveolae, here known as endocytic

caveolar carriers carrying cholera toxin binding subunit (CTxB), can fuse into the

caveosome, or with the early endosome or be recycled back to the plasma

membrane for reuse. This process is regulated by dynamin, protein kinases or

tyrosine kinases [295-297].

Fig. 13: Caveolae endocytosis [99]. CTxB: cholera toxin binding subunit. Further details please see in text 1.6.3.1.

In endothelial cells, the endocytosed caveolae seem to migrate from the luminal side

to the abluminal side, thereby transferring specific serum molecules to the underlying

tissue and this process is referred to transcytosis [292, 298]. The first direct evidence

Introduction

36

supporting an involvement of caveolin-1 and caveolae in transcytosis was shown by

perfusion with gold-labelled albumin in wild type and caveolin-1 deficient mouse

lungs. The wild type endothelial cells associated with gold-labelled albumin,

internalized and transferred it to the sub-endothelial space through caveolae, but no

uptake or transfer was observed in caveolin-1 deficient mice [89]. Transcytosis can

be both constitutive (fluid-phase transport) or receptor-mediated (the molecule

transported requires the presence of its cognate receptor in caveolae) and many

types of macromolecules such as albumin, insulin, Low-density lipoprotein (LDL)

have been associated with caveolae-mediated transcytosis [89, 270, 299]. The

regulation of endothelial permeability by transcytosis and caveolin has been

introduced in the “vascular permeability regulation” section. In addition, caveolae

have also been implicated in a unique form of solute uptake referred to as

potocytosis in which cells can transport small molecules (< 1 kDa), without

endocytotic vesicles, in caveolae to internal endosomal/lysosomal compartments

[300].

1.6.3.2 Caveolae, caveolins and cholesterol homeostasis

Caveolae are highly enriched in cholesterol relatively to the plasma membrane.

Depletion of cholesterol led to loss of identifiable caveolae [301] and caveolin

deficient mice showed less free cholesterol and cholesterol export [302]. From these

observations, it has been suggested that caveolae and caveolins are involved in

maintaining intracellular cholesterol balance. Cholesterol from de novo production or

extracellular uptake can be transported from endoplasmic reticulum to membrane

caveolae with assistance of caveolin [303-304]. Upon delivery, the cholesterol could

a) remain in caveolae as a functional structure, b) be siphoned into the bulk plasma

membrane, c) be effluxed to serum cholesterol-transporting units like high density

lipoproteins (HDL) [303, 305]. In essence, the caveolins deliver intracellular

cholesterol to a "relay station" wherein the overall fate of cholesterol is determined

[99].

1.6.3.3 Caveolae, caveolins and signal transduction

Caveolae are highly enriched in numerous membrane bound proteins, especially

signalling proteins with lipid modified groups (H-ras, src-family tyrosine kinases,

heterotrimeric G-proteins, eNOS, etc) [258, 306] . It appears that caveolins can bind

and functionally regulate (mostly inhibit) several of these caveolae-localized

Introduction

37

molecules [94, 257, 289, 307-308]. Caveolin scaffolding domain (CSD) mediates this

functional binding process. In addition, caveolae have also been implicated in the

regulation of ion channels and calcium recruitment [309-310].

1.6.3.4 The endothelial nitric oxide synthase, Caveolae and vascular permeability

Nitric oxide (NO) is a multifaceted molecule which plays key roles in many biological

situations [311]. Endothelial nitric oxide synthase (eNOS) is the major NOS isoform

responsible for cardiovascular homeostasis, vessel tonicity and permeability. eNOS

is a calmodulin activated enzyme which consists of an oxygenase and a reductase

domain containing binding sites for a variety of cofactors that promote electron

transfer from one domain to the other, leading ultimately to the conversion of L-

arginine to citrulline and NO [312].

eNOS is found enriched in caveolae where it interacts with caveolins directly. The

interaction between eNOS and caveolins has been well documented. Transgenic

overexpression of Cav-1 inhibited eNOS activation without increasing caveola

formation [313-314]. Further in a mouse inflammation model, delivery of the caveolin-

1 scaffolding domain reduced the vascular permeability response and tissue edema

formation by sequestering eNOS [315]. The interaction between eNOS and caveolins

involves three sites: the oxygenase and reductase domain of eNOS and CSD domain

of caveolin. eNOS enzyme activity is dependent on the calmodulin binding, the

activation of which requires an increase in intracellular [Ca2+]. Binding of eNOS to

caveolins holds it in the inactive state and away from Ca2+. When intracellular Ca2+

increases, eNOS complexes with Ca2+/calmodulin dissociates from caveolin; thereby

it increase eNOS activity and NO production. It appears that calmodulin acts as direct

allosteric competitor promoting the disruption of the heteromeric complex formed

between eNOS and caveolins in a Ca2+ dependent manner [272, 313, 316].

Accordingly, peptides corresponding to the CSD domain were shown to interact

directly with the enzyme and to markedly inhibit eNOS activity [316]. In addition,

eNOS is also regulated by phosporylation mediated by the Ser/Thr kinase Akt [317].

Phosphorylation could bidirectionally increase and decrease eNOS activity:

phosphorylation at serine (Ser1179) increased eNOS activity and sensitivity to

Ca2+/CaM, whereas phosphorylation at threonine (Thr497) negatively regulated eNOS

activity [317-318].

Introduction

38

NO is a ubiquitous cell-permeable intracellular messenger that plays an important

role in the regulation of vascular endothelial barrier homeostasis. It is believed that

decreased NO biogenesis contributes to chronic hypertensive diseases, whereas

increased synthesis contributes to inflammation and vascular disorders [319-320].

There are findings that increased NO production might lead to increased transcytosis

and vascular permeability in mice challenged with the bacterial endotoxin LPS [100].

Consistent with this outcome, sequestering of eNOS by conjugated caveolin-1

scaffold domain (CSD) peptide, which decreases NO synthesis, concomitantly

reduced the vascular permeability response in a mouse model of inflammation [315].

In contrast, in PAF-treated human mesangial cells in culture, rapid increased

permeability was accompanied with the cessation of endothelial NO synthesis [321].

These findings may seem conflicting, but we believe the more rational explanation is

that both too much and too little NO can increase vascular permeability, so that NO

synthase inhibitors can either attenuate or aggravate pulmonary hyperpermeability,

and that also depends on the time and measurements. This discussion will be

continued discussion section (5.2.2 regulation of vascular permeability by nitric

oxide).

1.6.3.5 Ca2+, TRPC channels and caveolae

Ca2+ signalling in endothelial cells plays a key role in maintaining the barrier integrity.

In the section “endothelial barrier regulation of Ca2+ and ion channel” we have

generally addressed the mechanisms of Ca2+ influx and vascular permeability

regulation. Signalling from inflammatory mediators like thrombin, bradykinin and PAF

might activate canonical transient receptor potential (TRPC) channels mediated by

diacylglycerol (DAG) and inositol trisphosphate (IP3). Ca2+ signalling induces actin

polymerization and myosin light chain (MLC) phosphorylation leading in turn to the

generation of contractile forces through actin myosin cross-bridging [322]. The

generated contractile force results in cell retraction and increased endothelial

permeability [322-323].

Several channels, receptors and mediators have been identified to be associated

with endothelial barrier function and caveolae such as store/receptor operated

channel (SOC/ROC) [275], canonical transient receptor potential (TRPC) [324-325]

and the inositol 1,4,5-trisphosphate receptor (IP3R) [285, 326]. These channels or

receptors are reported to be involved in regulation of endothelial barrier function and

Introduction

39

permeability [54, 323]. It is important to note that both IP3R and Ca2+-ATPase have

been described in the plasma membrane and especially in caveolae [81, 284-285]. In

many reports it is also described that IP3R and TRPC channels are found in a multi-

complex with caveolin-1 in membrane microdomains [327-329]. TRPC channels form a subfamily of channels in human most closely related to

drosophila TRP channels. These channels are permeable to cations, with a

selectivity of Ca2+ over sodium variable among the different members. The seven

TRPC channels can be divided into four subgroups based on their structure and

functional similarities: a) TRPC1; b) TRPC2; c) TRPC3, TRPC6, and TRPC7; and d)

TRPC4 and TRPC5 [330-331]. The fluorescence resonance energy transfer (FRET)

and co-immunoprecipitation studies [332] imply that TRPC2 does not interact with

any other TRPC proteins [333]; TRPC1 has the ability to form channel complexes

together with TRPC4 and TRPC5 and these three channels are sometimes

considered to represent a store operated calcium channel (SOC) [323]; All other

TRPCs assemble exclusively into homo- or heterotetramers working as (receptor

operated calcium channel) ROC, sharing a high degree of amino acid identity (70–

80%) and functional, pharmacological, and regulatory similarities (e.g.TRPC3/6/7)

[323].

In this thesis, we have focused on the role of the signals regulating the entry of Ca2+

through the TRPC channels and the response to endothelial barrier integrity as well

as vascular permeability.

1.7 The isolated perfused lung model In the studies of whole animals, there is an elaborate exchange of information and

cross-talk between the lung and other organs [334]. It is necessary to eliminate or

simplify the influence from other organs to study lung functions independent from the

systemic complexity. One physiologically relevant model that can be used to study

lung functions especially the edema formation is the isolated perfused rat lung (IPL)

[335-336]. It is a distinct advantaged model that studies are performed in an isolated

intact organ that stands in between experiments in vivo with whole animals and in

vitro with cultured cells [334]. This model allows examining physiological responses

of the lung which in four categories: respiratory mechanics, vessel mechanics, gas

exchange and edema formation. Since the study can be performed in an intact lung,

the physiological parameters can be determined with established cell-to-cell contacts

Introduction

40

and native intracellular matrixes [334].in our own hands, some substances known to

induce edema in vivo or in isolated perfused lung like PAF have no effect on

permeability in endothelial cell monolayer experiments [274, 337]. Unlike the whole

animal experiments, parameters like ventilation / perfusion pressure, perfusion flow

velocity, perfusate composition can be controlled and most of the information of lung

physiology like airway and vascular resistance, tidal volume, pulmonary compliance

and lung weight can be continuously monitored. It is also possible to distinguish

hydrostatic edema from permeability edema, by using constant pressure perfusion. In

addition, luminal plasmalemma of the lung endothelium can be isolated and purified

by a particular perfuse protocol using silica beads [284].

Real-time fluorescence imaging (RFI), combining microscopic imaging of the isolated

perfused lung, have enabled the investigation of lung cellular and molecular

responses in situ [338]. This method provides an opportunity for obtaining optical

data specifically from intact epithelial and endothelial cells of alveoli and

microvessels. By designed fluorescent indicators, it allows us to detect the diatomic

free-radical NO and cytosolic Ca2+ levels in individual endothelial cells in intact

capillaries [339-340]. In this thesis, our cooperating group of Prof. W. Kuelber would

perform the Real-time fluorescence imaging to examine the endothelial NO and

cytosolic Ca2+ level in isolated perfused rat/mouse lungs.

The aim of the study

41

2 The aim of the study Vascular permeability is a hallmark of pulmonary inflammation. In the lungs increased

vascular permeability leads to Acute Lung Injury (ALI) and the Acute Respiratory

Distress Syndrome (ARDS), clinical conditions for which mortality remains high. One

important mediator that plays a critical role in many experimental models of ALI is the

platelet-activating factor (PAF). PAF increases vascular permeability by activation of

two lipid modifying enzymes: cyclooxygenase leading to production of PGE2 followed

by activation of EP3 receptors and acid sphingomyelinase (ASM) leading to formation

of ceramide. The mechanisms by which ASM-metabolites regulate vascular

permeability were unknown and were subject of the present studies.

The major aim of this work was to investigate the mechanisms by which ASM

regulates the PAF-induced increased pulmonary endothelial permeability.

Particularly, we focused on the role of NO synthesis and Ca2+ influx in response to

PAF. Since caveolae are particularly rich in sphingolipids, we asked whether PAF

acts in the realm of caveolae and which role they play in PAF-induced edema

formation. Therefore, it was another goal of this thesis to establish a method for the

isolation of caveolae from intact endothelial cells of isolated perfused rat lungs and to

characterise these caveolae in PAF-treated lungs.

This work was supported by DFG grants Uh 88/8-1 and Ku 1218/5-1 within the DFG

Priority Programme 1267 “Sphingolipids – Signal and Disease”. In this cooperation, in

Aachen I have performed the experiments on edema formation in isolated perfused

rat lungs, the measurement of acid sphingomyelinase activity, the purification of

caveolae from the pulmonary endothelium from intact lungs and the characterization

by immunoblotting. Our cooperating group of Prof. W. Kuelber in Berlin has

performed in situ fluorescence microscopy for imaging of endothelial calcium

transients and NO production.

Material and methods

42

3 Material and Methods 3.1 Materials 3.1.1 Animals Male Sprague-Dawley rats (350 to 450 g for in situ fluorescence imaging), female

C57 mice (18-22g for isolated perfused lung) and female Wistar rats (weight 220 to

250g for isolated perfused lung) were kept under controlled conditions (22°C, 55%

humidity and 12 h day/night rhythm) and a standard laboratory chow (V1534-000 sniff

R/M; Ssniff Spezialdäten GmbH, Soest, Germany) and water ad libitum.

In addition, the TRPC6 knockout mice were from cooperating group of Prof.

Alexander Dietrich, Institute of pharmacology, Philipps-Universität Marburg and Prof.

Thomas Gudermann, Institute of pharmacology and Toxicology, Ludwig-Maximilians-

Universität München. ASM knockout mice were from cooperating group of Prof.

Dieter AdamA, institute for immunology, Christian-Albrechts-Universität Kiel.

All animals received care in accordance with the Guide for the Care and Use of

Laboratory Animals. The study was approved by the local animal care and use

committee of the local government authorities. Pentobarbital sodium (Nacoren, 400

µL/kg) was purchased from the Wirtschaftsgenossenschaft Deutscher Tieräzte

(Hannover, Germany).

3.1.2 Substances and chemicals Table 5: Overview of inhibitors, substances and suppliers

Substances Concentration Supplier

Platelet-activating factor (PAF,1-O-alkyl-2-acetyl snglycero-3-phosphocholine)

5 nmol/IPL ~50 nM

Sigma (Taufkirchen, Europe)

[N-methyl-14C] Sphingomyelin

472.7 nM GE Healthcare

D609 (Tricyclodecan-9-ylxanthate potassium Salt)

300 µM Sigma (Taufkirchen, Europe)

Imipramine (hydrochloride)

10 µM Sigma (Taufkirchen, Europe)

L-NAME (L-N-Nitroarginine methylester (hydrochloride) )

250 µM Sigma (Taufkirchen, Europe)

SKF-96365, (hydrochloride)

30 µM Sigma (Taufkirchen, Europe)

Material and methods

43

PAPA NONOate

100 µM AXXORA Germany

ARC39 1 µM Generous gift from Prof. Christoph Arenz Humboldt university, Berlin

Aluminium chlorohydroxide

105 g/L PFATZ & BAUER Inc, USA

Nycodenz

70%-55% Axis-Shield PoC AS, Norway

Dexamethasone

10 µM Sigma (Taufkirchen, Europe)

Silica beads (diameter of 5 µm) 10% Bangs Laboratories Inc, USA

Ruthenium red 1 µM Fluka (Germany, Europe)

3.1.3 Other Buffers and chemical solutions TBS: Tris-Buffered Saline buffer contains 10 mM Tris, 150 mM NaCl (pH 7.6).

TBS-T: Tris-Buffered Saline buffer contains 10 mM Tris, 150 mM NaCl and 0.1%

Tween 20.

MBS: MES-Buffered Saline solution contains 125 nM NaCl and 20 mM MES (pH 6).

HBS: HEPES-Buffered Sucrose solution contains 0.25 mM Sucrose, 25 mM HEPES

(Sigma, Taufkirchen, Germany) and 2mM PEFA-Bloc as a protease inhibitor (Roche,

USA).

Krebs-Henseleit buffer: 2% bovine serum albumin (Serva, Heidelberg, Germany)

solution contains 0.1% glucose and 0.3% HEPES.

NO-sensitive fluorescence dye 4-amino-5-methylamino-2’,7’-difluorofluorescein

diacetate (DAF-FM) and the NO donor S-nitroso-N-acetylpenicillamine (SNAP) were

obtained from Molecular Probes (Eugene, OR, USA).

Electrophoresis Buffer: Tris-Glycine SDS Buffer.

Immunoblotting Buffer: Tris-Glycine Buffer contains 20% methanol.

Destilled water.

Material and methods

44

3.1.4 Antibodies

Table 6: Overview of antibodies for Immunoblotting

Primary Antibodies Dilution Secondary

antibodies

Supplier

Goat anti rat ACE 1:1000 Alexa-Fluor 800nm Donkey

anti goat Santa Cruz

Biotechnology, USA

Mouse anti rat Caveolin-1 1:1500 Alexa-Fluor 680 nm Goat

anti mouse BD Transduction

Laboratories, Europe

Mouse anti rat Flotillin-1 1:500 Alexa-Fluor 680 nm Goat

anti mouse BD Transduction

Laboratories, Europe

Goat anti rat LAMP-1 1:1000 Alexa-Fluor 800nm Donkey

anti goat Santa Cruz

Biotechnology, USA

Rabbit anti rat eNOS 1:1000 Alexa-Fluor 700 nm Goat

anti rabbit BD Transduction

Laboratories, Europe

Mouse anti rat PeNOS 1:1000 Alexa-Fluor 680 nm Goat

anti mouse BD Transduction

Laboratories, Europe

Mouse anti rat TRPC-6 1:500 Alexa-Fluor 680 nm Goat

anti mouse Generous gift from

Prof. Veit Flockerzi University Saarlandes,

Germany

Secondary antibodies, Alexa-fluorochrome labelled were obtained from Moleculare

Probes (Eugene, USA). It was used in a dilution of 1:10000~1:15000.

3.1.5 Equipment Isolated perfused rat lung from Hugo Sachs Electronics (March-Hugstetten,

Germany).

LE-80K Ultracentrifuge, SW55Ti rotor and SW28 rotor from Beckmann (Palo Alto,

USA).

Polytron PT 1200 from Kinematica AG (Littau Luzern, Switzerland).

Hoefer SE600 Electrophoresis system (Germany).

TE 77 PWR blotting system from Amersham biosciences (GE, USA).

Material and methods

45

3.2 Methods 3.2.1 Isolated perfused lung To study the edema formation and the role of membrane microdomains (caveolae) in

pulmonary response to PAF, we used the isolated perfused rat lung preparation.

3.2.1.1 Preparation of isolated, ventilated and perfused rat lungs Rat lungs were prepared and perfused essentially as described [334-335]. Briefly,

lungs were perfused through the pulmonary artery at a constant hydrostatic pressure

(12 cm H2O) with Krebs-Henseleit-buffer. Perfusate buffer contained 2% albumin,

0.1% glucose and 0.3% HEPES. Edema formation was assessed by continuously

measuring the weight gain of the lung.

3.2.1.2 Experimental design of the perfused lung studies

After preparation and 40 minutes perfusion, 5 nmol PAF (dissolved in ethanol) was

injected as a bolus directly into the perfusate. Imipramine (10 µM), ARC39 (1 µM), L-

NAME (100 µM), dexamethasone (10 µM) and PAPAnonoate (100 µM) were

prepared in perfusate buffer and were added into the buffer reservoir 10 min prior to

PAF administration. In ASM sole perfusion, ASM (1 U/mL in perfuse buffer) was

continuously infused for 30 min into venular capillaries of isolated lungs via a venous

microcatheter

3.2.1.3 Lung filtration coefficient measurement

The lung filtration coefficient (Kf) was measured as described [341]. Briefly, the lung

filtration coefficient was determined by suddenly raising or lowering the perfusion

pressure by 5 cm H2O and analysing the resulting weight transients by bi-exponential

regression. The measurement was done before and after the stimulation of PAF or

PAF plus inhibitors and the differences of Kf in their two measurements was

calculated.

3.2.1.4 Coating of silica-beads with aluminium hydroxide (chlorohydrol)

The coating of silica particles was accomplished essentially as described by

Jacobson et al [284]. Silica beads, with a diameter of 0.5 µm, were coated with

aluminium hydroxide to generate a positively loaded surface. 10.5 g chlorohydrol was

mixed with 90 mL distilled water and blended with 1.5 g beads for 2 min in a high

speed blender. The mixture was then stirred manually and incubated in a water bath

Material and methods

46

at 80°C for 30 min. Silica beads were chilled down to room temperature (RT) for 16h.

After cooling the pH was adjusted to pH 5.0 with 1 M NaOH. Beads were incubated

again for 24h and the pH was once more adjusted to pH 5.0 with 1M NaOH. Silica

bead suspensions were stored at 4°C. Before the coated beads were used for

perfusion, the bead solution was diluted to 10% silica with attachment buffer (140 mM

sorbitol, 20 mM MES) and centrifuged for 5 min at 800 x g. The beads cake was

washed and resuspended in MBS-buffer and centrifuged for 5 min at 800 x g for

three times. The washed beads were then used for purification of caveolae from

intact pulmonary endothelial cells.

3.2.1.5 Perfusion of rat lung with cationic colloidal silica beads

Silica perfusion was accomplished by a method, described by Schnitzer et al [284].

The method was slightly modified. After a 40 minutes perfusion, 5 nmol PAF (final

concentration 50 nM) was injected as a bolus directly into the perfusate. After 10

minutes the flow rate was reduced from 20 mL/min to 2-3 mL/min and the lungs were

perfused with the following solutions: MBS buffer for 90 sec at room temperature and

1 % colloidal silica beads in MBS buffer (10°C); MBS buffer for 90 sec (10°C), to

clear free silica beads from the lungs; 1% sodium polyacrylate in MBS buffer for 90

sec (10°C), to crosslink and shield membrane-bound silica; and finally with 8-10 mL

HBS buffer (10°C).

3.2.1.6 Purification of endothelial cell membrane

After silica perfusion lungs were removed and immersed in cold HBS, the lung was

minced with a cutter to small pieces on ice and then transferred to 10 mL HEPES

buffer. Minced lung pieces were homogenized on ice using a Polytron PT 1200 and a

Teflon pestleglass homogenizer (15 strokes) with a high speed motor running at 425

x g. After filtration through a 0.65 μm and 0.45 μm Nytex net (GE Osmononics Inc,

Minnetoka, USA) the homogenate was mixed with an equal volume of 1.02 g/mL

Nycodenz containing 20 mM KCl and then layered over 0.5-0.7 g/mL Nycodenz

containing 60 mM sucrose in a centrifuge tube. After centrifugation (72128 x g, 30

minutes, 4°C in a Beckman SW 28 rotor) the floating tissue debris were removed and

the pellet, which contained the silica-coated endothelial membranes fragments was

resuspended with 1 mL MBS buffer. Subsequently, the suspension was

homogenized by Polytron PT 1200.

Material and methods

47

3.2.1.7 Isolation of caveolae from silica-coated endothelial cell membrane

The caveolae from purified pulmonary endothelial cell membrane were isolated as

described by Melkonian et al [342], and Arni et al [343]. The pellet containing the

silica-coated endothelial membranes fragments was resuspended with 1 mL MBS (20

mM MES, 150 mM NaCl, pH 6.0) and 10% Triton-X-100 (final concentration of 1%)

for 60 minutes at 4°C. After incubation the suspension was homogenized and the

homogenate was mixed with 80% sucrose to achieve a 40% membrane-sucrose-

solutions. A 30-5% sucrose gradient was layered over the samples in a Beckman

SW55Ti rotor tube and centrifuged at 4°C overnight at 109368 x g for 16-18 h.

Fractions of 3x150 µL were removed from the top to the bottom and collected as six

membrane fractions (0 and A-E); the pellet was dissolved in 150 µL MBS (pellet-

fraction, P). Figure 14 summarized the procedure for isolation of caveolae from intact

pulmonary endothelial cells as described above.

Fig. 14: Experimental procedure for isolation of the endothelial caveolae from isolated perfused rat lungs.

Material and methods

48

3.2.2 Determination of protein concentration by bicinchoninic acid protein assay (BCA assay) The protein concentrations of all membrane fraction samples were determined with a

BCA assay (Pierce, Rockford, USA). A volume of 25 µL from each sample was mixed

with 200 μL BCA working solution. The working solution contain BCA-reagent A (1

mg sodium bicinchoninate, 2 mg sodium carbonate, 0.16 mg sodium tartrate, 0.4 mg

NaOH, and 0.95 mg sodium bicarbonate) and 2 volumes BCA-reagent B (0.4 mg

cupric sulfate (5 x hydrated) in 10 mL distilled water). Samples were incubated 30

minutes at 37°C. The amount of protein present in the solution was quantified by

measuring the optical intensity at 562 nm that was referenced by linear regression to

protein solutions with known concentrations.

3.2.3 Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) Equal amounts of protein (5 µg) were separated by SDS-poly acrylamide gel

electrophoresis (12% for caveolin-1, 8% for eNOS). Samples were added to the

stacking gel and run by 100 V for 40 minutes. To separate the proteins in the

resolving gel the voltage was increased to 200 V for 100 min. The SDS-PAGE was

performed of room temperature in 1 x electrophoresis buffer solution.

3.2.4 Immunoblotting After SDS-PAGE separation, proteins were transferred to nitrocellulose membranes

(Schleicher&Schuell, Marienfeld, Germany). The protein transfer on nitrocellulose

membranes was performed with the TE 77 PWR blotting system at 0.8 mA/cm² for 75

minutes. The blots were washed with 1 x TBS-T buffer (1 min) and blocked by Roti®

Block (Roth, Karlsruhe, Germany) for 1 h at RT. The nitrocellulose membranes were

incubated overnight at 4°C with different primary antibodies. After washing,

nitrocellulose membranes were incubated with a secondary infrared fluorochrome

labelled antibody for 1 h at RT and protein bands were detected at 680 or 800nm

wavelength with the Odyssey® Infrared Imaging system (LI-COR, Bad Homburg,

Germany). All specific protein bands were quantified with the Odyssey® imaging

software (LI-COR, Bad Homburg, Germany).

All gels were loaded with exactly the same amount of protein and all membranes

were scanned with exactly the same settings (e.g. background correction, contrast,

channel settings, etc.). Fig. 15 shows the samples from a control and a PAF lung that

Material and methods

49

were analyzed on the same gel and that similarly shows increased Cav-1 in the

caveolar fractions from PAF treated lungs.

Fig. 15: Pooled immunoblots showing Cav-1 distributions from untreated control condition and PAF-treated (10 min after bolus injection of 5 nmol PAF) isolated perfused rat lungs from fraction A–E in same gel.

3.2.5 Acid sphingomyelinase activity Acid sphingomyelinase (ASM) activity was determined by using 14C-labelled

sphingomyelin [344]. For all samples, 10 µg protein diluted to 10 µL was incubated at

37°C for 2h with 40 µL substrate (73 nmol 14C-labelled sphingomyelin + 400 nmol

sphingomyelin). Lipids were separated by chloroform/methanol extraction, 4 mL

scintillation liquid was added and radioactivity counted in a ß-counter.

3.2.6 Electron microscopy Lungs were fixed by perfusion with 250 mL Caco-buffer (100 mM Na-cacodylate, 100

mM NaCl, 2% formaldehyde, 2% glutaraldehyde and 2 mM CaCl2). Lungs were

removed and cut into pieces of about 5 x 5 x 5 mm and immersed in Caco-buffer over

night. After washing 4x15 min in PBS, the tissue pieces were immersed in PBS

containing 1% OsO4 followed by washing 4 x 15 min in aqua bidest. Dehydration was

performed by a graded EtOH-series and embedded in Epon. Thin sections were cut

and stained with uranyl acetate and lead citrate. Electron microscopy pictures were

Material and methods

50

digitally recorded using a Zeiss EM10. These experiments were done by cooperating

group of Prof. W. Baumgartner, RWTH Aachen.

3.2.6.1 In situ fluorescence microscopy In situ imaging of endothelial NO production was performed by cooperating group of

Prof. W. Kuelber, Institute for Physiology, Charité – Universitätsmedizin Berlin as

previously described [345]. In brief, lungs were excised and continuously perfused

with 14 mL/min autologous blood at 37°C. Lungs were constantly inflated with a gas

mixture of 21% O2, 5% CO2, and balance N2 at a positive airway pressure (PAW) of 5

cm H2O. Left atrial pressure (PLA) was set to 3 cm H2O, yielding pulmonary artery

pressure (PPA) of 10±1 cmH2O. PAW, PLA, and PPA were continuously monitored

and recorded (DASYlab 32; Datalog GmbH, Moenchengladbach, Germany). Lungs

were positioned on a custom-built vibration-free microscope stage and superfused

with normal saline at 37°C.

For in situ imaging of endothelial NO production, membrane-permeant DAF-FM

diacetate (5 µM/L), which de-esterifies intracellularly to cell-impermeant, NO-

sensitive DAF-FM was infused for 20 min into pulmonary capillaries via a venous

microcatheter. Intracellular DAF-FM is converted by an NO-dependent, irreversible

reaction to an intensely fluorescent benzotriazole derivative with fluorescence

intensity linearly reflecting NO concentration [346]. Single venular capillaries were

viewed at a focal plane corresponding to maximum diameter (17-28 µm). Endothelial

DAF-FM fluorescence was excited at 480 nm by a near monochromatic beam

generated by a digitally controlled galvanometric scanner (Polychrome IV; TILL

Photonics, Martinsried, Germany) from a 75-watt xenon light source. Fluorescence

emission was collected through an upright microscope (Axiotechvario 100HD; Zeiss,

Jena, Germany) equipped with an apochromat objective (UAPO 40x W2/340;

Olympus, Hamburg, Germany) and dichroic and emission filters (FT 510, LP 520;

Zeiss, Jena, Germany) by a CCD camera (Sensicam; PCO, Kelheim, Germany) and

subjected to digital image analysis (TILLvisION 4.0; TILL Photonics). Exposure time

for each single image was limited to 5 milliseconds. Fluorescence images obtained in

10s intervals were background-corrected and fluorescence intensity (F) was

expressed relative to its individual baseline (F0). Since the conversion of DAF-FM to

the benzotriazole derivative is irreversible, NO production is reflected by changes of

the ratio F/F0 (∆F/F0) over time and was determined in 5 min intervals. At the end of

Material and methods

51

experiments, the exogenous NO donor SNAP (1 mmol/L) was added to test whether

endothelial cells still contained unconverted DAF-FM.

3.2.7 Measurement of alveolar fluid influx and reabsorption Fluid fluxes into and out of the alveolar space were quantified by a double-indicator

dilution technique by cooperating group of Prof. W. Kuelber, Institute for Physiology,

Charité – Universitätsmedizin Berlin as previously reported [347]. Briefly, a high-

molecular-weight fluorescence tracer, texas red dextran (70 kDa; Molecular Probes,

Eugene, OR), was instilled into the alveolar space for determination of alveolar net

fluid shift while a low-molecular-weight tracer, Na+ fluorescein (376 Da; Sigma-

Aldrich, Taufkirchen, Germany), was added to the perfusate to allow for

differentiation between alveolar fluid influx and alveolar fluid reabsorption. At time -10

min, 0 min, and 60 min, samples were drawn from both compartments, and alveolar

fluid reabsorption, alveolar fluid influx, and net fluid shift were calculated as

previously described assuming a two-compartmental distribution model [347].

3.2.8 Statistics In case of heteroscedasticity, data were transformed by the Box-Cox transformation

prior to analysis. Data were analysed by an unpaired t-tests or by the Dunnett test

(JMP1 statistical software version 7; JMP Germany, Böblingen, Germany).

Fluorescence data were analysed by the Kruskal–Wallis and Mann–Whitney U-test. If

required, p-values were corrected for multiple comparisons according to the false-

discovery rate procedure using the ‘‘R’’ statistical package (R Foundation for

Statistical Computing, Vienna, Austria)

Results

52

4 Results

We have performed most of our experiments in isolated perfused rat and mouse

lungs. This model was selected for following reasons: a) In isolated lungs the indirect

effects of PAF like activation of neutrophils and increasing hydrostatic pressure can

be excluded by perfusion with blood-free buffer and by keeping hydrostatic pressures

constant. Thus, this model allows studying exclusively the direct effects of PAF on

lung vascular permeability. b) PAF does not increase vascular permeability in

endothelial cell monolayers in culture [274, 337]. Therefore, at present the effects of

PAF on vascular permeability are best studied in isolated perfused lungs. Further

more, this method is suited for the measurement of following parameters that would

be extremely difficult to measure in vivo: The vascular filtration coefficient [341], the

quantification of alveolar fluid clearance and secretion [347], and the in situ

fluorescence imaging of nitric oxide production [345] and Ca2+ concentration [348] in

endothelial cells.

4.1 PAF induced edema formation Isolated rat lungs were perfused for 40 minutes under control conditions before 5

nmol of PAF was injected into the artery. This resulted a rapid and brief increase in

lung weight. The ASM inhibitor Imipramine was given 10 minutes before PAF and

impaired the PAF-induced edema formation about 50%. Figure 16 shows both the

real time curve (a) and summarized weight gain (b) of edema formation from PAF

treated (50 nM) and untreated control lungs.

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53

Fig. 16: PAF-induced edema formation. a) The real time curve and b) The summarized weight gain in lungs perfused under control conditions and PAF-treated (10 min after bolus injection of 5 nmol PAF). Data are presented as mean±SD, n=4. *: p<0.05 versus control.

4.2 PAF induced caveolin recruitment and endothelial nitric oxide synthase (eNOS) distribution 4.2.1 Number and size of caveolae Caveolin-1 is the essential molecule of caveolae, thus in cooperation with Prof. W.

Baumgartner, RWTH Aachen, we examined whether PAF-treatment increased the

number or size of caveolae by electron microscopic images of microvascular

endothelial cells from isolated perfused lungs (Fig. 17). It turned out that PAF did not

increase the number of apical, cellular or basal caveolae (Fig. 17, panel c), nor did it

increase the size of caveolae: the maximal diameter at the centre of caveolae was

152±26nm (n=103, from one lung) in control and 148±26nm (n=106, from one lung)

in PAF-treated lungs. Furthermore, in PAF-treated lungs, we repeatedly observed

loosening of the endothelial adherence junctions between cells in the capillaries (Fig.

17, panel b).

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54

Fig. 17: Number and size of caveolae. Electron microscopic images of endothelial cells showing junctions and caveolae in a) control and b) PAF treated lungs. c) Number of apical, basal and intracellular caveolae in endothelial cells from control and PAF treated lungs. 95–106 apical, basal and intracellular caveolae were counted from one control and one PAF-treated lung. These experiments were performed by cooperating group of Prof. W. Baumgartner.

4.2.2 Preparation and characterisation of caveolae Since sphingolipids, eNOS and other signalling proteins are enriched in caveolae, we

investigated whether PAF alters the composition of caveolae in pulmonary

endothelial cells. Therefore we established a method originally described by

Schnitzer et al [284]. This protocol allowed us to isolate caveolae from the endothelial

cells of isolated perfused rat lungs.

Lungs were perfused for 50 min before perfusion with the colloidal silica beads was

started in order to prepare endothelial cell membrane fractions. Immunoblotting of the

resulting fractions shows a typical distribution for Cav-1: two bands are typical for rat

Cav-1 representing Cav-1α and Cav-1ß [261]. To increase the amount of material

available for subsequent analyses, we pooled the 17 fractions into five fractions

labelled 0 and A–E. We also found the flotillin-1 (another lipid raft marker) in fractions

B-E (data not shown), meanwhile the angiotensin converting enzyme (ACE, a non-

caveolar plasma membrane protein, Fig. 18), lysosomal-associated marker protein-1

(LAMP-1, a lysosomal marker, data not shown) and transferrin receptor (non-raft

membrane marker, data not shown) were only found in highest density fractions. In

Results

55

line with the sucrose concentrations, the presence of Cav-1 and the absent or weak

signals for ACE and other non-caveolar markers, we consider fractions B and C to

represent caveolae, while fraction D may represent a mixture of caveolar and non-

caveolar structures.

Fig. 18: Caveolin-1 identification of pulmonary endothelial cell plasma membrane fractions. a) Immunoblots showing the Cav-1 distribution in pulmonary endothelial cell plasma membrane fractions from untreated control and PAF-treated (10 min after bolus injection of 5 nmol PAF) isolated perfused rat lungs. b) Immunoblots showing the angiotensin converting enzyme (ACE) distribution in pulmonary endothelial cell plasma membrane fractions from control and PAF (5 nmol) treated isolated perfused rat lungs. 4.2.3 PAF recruits caveolin-1 to caveolar fraction PAF caused an increase in the amount of Cav-1 in several fractions from pulmonary

endothelial cells (Fig. 19) and the effect of PAF was clearly noticeable in the pooled

fractions. The strongest increase in Cav-1 was noted in the fractions B, C and D. To

demonstrate that Cav-1 was recruited to the caveolae, we measured the abundance

of Cav-1 in whole cell lysates, in the pooled fractions B/C representing the caveolae

and in the remainder of the cells (fractions A, D, E and pellet) representing the non-

caveolar fractions. Our findings show that the total amount of Cav-1 was unchanged

in the total endothelial cell lysate but that it was increased in the caveolar fractions

and decreased in the remaining fractions.

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56

Fig. 19: PAF increases caveolin-1 in caveolae. a) Pooled immunoblots showing Cav-1 distributions from untreated control condition (n=4) and PAF-treated (10 min after bolus injection of 5 nmol PAF, n=4) isolated perfused rat lungs from fraction A–E. b) Immunoblots showing Cav-1 in fractions from whole endothelial cells (n=3), from the pooled fractions B and C (n=3) and from the remainder of the endothelial cells without the fractions B and C (n=3). The latter two fractions (B/C and the remainder) were obtained from the same preparation. Data are presented as mean±SD. *: p <0.05 versus control.

4.2.4 Role of endothelial caveolar eNOS and NO production It is known that Cav-1 binds and thereby blocks eNOS activation [307, 349], we

investigated the effects of PAF on the abundance of eNOS inside caveolae and on

endothelial NO production. As seen in Fig. 20, PAF increased the amount of eNOS

inside the caveolar fractions B and C.

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57

Fig. 20: Effect of PAF on caveolar eNOS. Immunoblots showing the eNOS distribution in pooled pulmonary endothelial cell plasma membrane fractions from untreated control condition and PAF-treated (10 min after bolus injection of 5 nmol PAF) isolated perfused rat lungs. Data are presented as mean±SD, n=4. *: p<0.05 versus control.

Our cooperating group of Prof. W.M. Kuebler used the DAF-FM loaded lung capillary

endothelial cells to measure the NO production in situ. As seen in Fig. 21, PAF

treatment markedly reduced basal endothelial NO production. Comparatively, NO

production remained stable and continuous in untreated control lungs and was

completely blocked after addition of the NO synthase inhibitor L-NAME.

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58

Fig. 21: Effect of PAF on endothelial NO production. Endothelial NO production as determined by in situ real-time fluorescence microscopy shown as 5 min averages in untreated control condition, under PAF-treated (bolus injection of 5 nmol PAF after baseline recording) and L-NAME (250 µM) treated lungs. NO production was quantified as increase in fluorescence of DAF-FM loaded endothelial cells over 5 min intervals relative to baseline Δ (F/F0). Data are presented as mean±SEM, n=5. *: p<0.05 versus control. These experiments were performed by cooperating group of Prof. W. Kübler.

4.2.5 Effects of NO on PAF-induced edema formation In order to analyse the effects of NO on PAF-induced edema formation, we used the

NO donor PAPA-NONOate to increase the exogenous NO level and L-NAME to

decrease the NO concentration by blocking the eNOS activity. Both agents did not

alter lung weight by sole perfusion. By perfusing lungs together with PAF, PAPA-

NONOate attenuated PAF-induced edema formation, while L-NAME had no effect.

Results

59

Fig. 22: Effect of NO on PAF-induced edema formation. Weight gain in lungs perfused under untreated control condition (n=11), with PAF treated (10 min after bolus injection of 5 nmol PAF, n=12), NONOate/PAF (100 µM NONOate, n=7), L-NAME/PAF (250 µM L-NAME, n=8), NONOate (n=3) or L-NAME (n=3). The pharmacological agents were added after 30 min of perfusion and PAF after 40 min of perfusion. Data are presented as mean±SD. *: p<0.05 versus control; #: versus PAF. 4.2.6 The filtration coefficient measurement The filtration coefficient (Kf), proportionality constant represents the hydraulic

conductance of the pulmonary endothelium. To complement the weight gain

experiments, we measured the Kf in isolated perfused rat lungs with an established

method [341]. Panel a in Fig. 23 illustrates the typical Kf measurement manoeuvre

demonstrating the weight transient induced by the pressure jump and that was

reversible even after PAF administration, which is critical requisite for the Kf

measurement. As seen in panel b, the NO donor PAPA-NONOate did not affect the

Kf, whereas L-NAME increased the Kf to approximately half the value as PAF did.

Given 10 min before PAF, PAPA-NONOate attenuated the PAF-induced Kf increase,

whereas L-NAME had no effect.

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60

Fig. 23: The filtration coefficient measurement. a) Weight gain curve in a typical experiment with administration of PAF and determination of the filtration coefficient. For the Kf measurement perfusion pressure was raised by 5 cm H2O for 15 min and reduced back to baseline. Both weight transients (up, down) were used to calculate the Kf. b) Kf determined as the difference in Kf before and 10 min after PAF administration. Kf in lungs perfused under control condition (n=3), with PAF treated (10 min after bolus injection of 5 nmol PAF, n=4), NONOate/PAF (100 µM, n=3), L-NAME/PAF (250 µM, n=3), NONOate alone (n=3) or L-NAME alone (n=3). The pharmacological agents were added after 30 min of perfusion and PAF after 40 min of perfusion. Data are presented as mean±SD. *: p<0.05 versus control; #: versus PAF. 4.2.7 Alveolar fluid influx, alveolar fluid absorption As NO has been known to be involved in the regulation of the epithelial barrier

functions [347], it was important to know whether the intravascular application of PAF

would alter alveolar fluid influx or absorption. Using a double-indicator technique and

assuming a two-compartmental distribution model [347], our cooperating group found

that PAF did not alter the alveolar barrier properties (see Fig. 24).

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61

Fig. 24: Alveolar fluid influx and alveolar fluid absorptio. a) Alveolar fluid influx. b) Alveolar fluid absorption in untreated control lungs, in PAF-treated (5 nmol) lungs and in lungs exposed to 20 cm H2O hydrostatic pressure for 60 min. Data are presented as mean±SEM, n=3. *: p<0.05 versus control. These experiments were performed by cooperating group of Prof. W. Kübler.

4.3 PAF activates ASM to decrease endothelial NO production 4.3.1 PAF increased ASM activity After 10 minutes perfusion with PAF, ASM activity was increased in the fractions A–

C. Imipramine which could prevent the PAF-induced activation of ASM [111]

evidently impaired the PAF induced increase in ASM activity in the membrane

fractions (Fig. 25).

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62

Fig. 25: Acid sphingomyelinase (ASM) activity in pulmonary endothelial cell plasma membrane fractions from isolated perfused rat lungs. Membrane fractions in lungs perfused for 50 min are shown under control condition, with PAF treated (10 min after bolus injection of 5 nmol PAF) and imipramine/PAF (100 µM imipramine). Data are presented as mean±SD, n=4. *: p<0.05, **: p<0.01 versus PAF.

4.3.2 Role of ASM on PAF-induced edema formation and Cav-1 recruitment Imipramine and D609, two structurally different inhibitors of the ASM pathway [156]

were perfused 10 minutes before PAF administration. These two ASM inhibitors

significantly prevented the PAF-induced edema formation (Fig. 26) and recruitment of

Cav-1 to caveolar membrane fractions (Fig. 27)

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63

Fig. 26: Effects of acid sphingomyelinase (ASM) pathway inhibitors on PAF-induced edema formation. Weight gain in lungs perfused under control conditions, with PAF (10 min after bolus injection of 5 nmol PAF), imipramine/PAF (100 mM imipramine), D609/PAF (300 mM D609) or ARC39/PAF (1 µM ARC39). The pharmacological agents were added after 30 min of perfusion and PAF after 40 min of perfusion. Data are presented as mean±SD, n=4 *: p <0.05 versus PAF.

Fig. 27: Effects of acid sphingomyelinase (ASM) pathway inhibitors on caveolin-1 recruitment. Immunoblots showing the Cav-1 distribution in pooled pulmonary endothelial cell plasma membrane fractions prepared from lungs treated with PAF (10 min after bolus injection of 5 nmol PAF), imipramine/PAF (100 mM imipramine), D609/PAF (300 mM D609) or ARC39/PAF (1 µM ARC39). The pharmacological agents were added after 30 min of perfusion and PAF after 40 min of perfusion. Data are presented as mean±SD, n=4. *: p <0.05 versus PAF.

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64

4.3.3 Role of ASM on eNOS distribution Coincident to Cav-1, the ASM inhibitors imipramine and D609 also significantly

prevented the PAF-induced level of eNOS in caveolar membrane fractions (Fig. 28).

Fig. 28: Effects of acid sphingomyelinase (ASM) pathway inhibitors on the caveolar expression of eNOS. Immunoblots showing the eNOS distribution in pooled pulmonary endothelial cell plasma membrane fractions prepared from lungs treated with PAF (10 min after bolus injection of 5 nmol PAF), imipramine/PAF (100 mM imipramine) or D609/PAF (300 mM D609). The pharmacological agents were added after 30 min of perfusion and PAF after 40 min of perfusion. Data are presented as mean±SD, n=4. *: p <0.05 versus PAF.

4.3.4 Role of ASM on NO production In addition to analysis of eNOS distribution in caveloae, our cooperating group also

measured the NO production under PAF and ASM inhibitor administration with In situ

fluorescence microscopy. Imipramine attenuated the PAF-induced decrease in

endothelial NO production (Fig. 29, panel a and b). Imipramine alone had no effect

on basal NO production, as demonstrated by the unaltered NO production rates

during the 10 minutes that imipramine was infused without PAF.

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65

Fig. 29: Effects of ASM on PAF-mediated endothelial NO production. Panel a) represent fluorescence images of DAF-FM loaded lung venular capillaries in isolated perfused rat lungs. Images were obtained at baseline and 10 min after PAF (5 nmol) stimulation in control lungs and in lungs perfused with imipramine (100 µM). The pharmacological agents were added 10 min prior to PAF infusion. Note that due to the irreversible conversion of the fluorescent dye, absence of fluorescence increase as seen in PAF treated controls indicates lack of NO synthesis. Panel b) shows endothelial NO production as determined by in situ real-time fluorescence microscopy. Curves are shown as 5 min averages at baseline and after stimulation with PAF alone and with PAF plus imipramine perfused lungs. NO production was quantified as increase in fluorescence of DAF-FM loaded endothelial cells over 5 min intervals relative to baseline (∆F/F0). Data are presented as mean±SEM, n=5. *: p<0.05 versus PAF. These experiments were performed by cooperating group of Prof. W. Kübler.

4.3.5 Effects of ASM perfusion and ASM depletion on NO production In order to further demonstrate the role of ASM on NO synthesis, our cooperating

group repeated the NO production measurement with ASM perfusion instead of PAF.

As seen in panel a, figure 30, direct perfusion with ASM (1 U/mL) decreased

endothelial NO production to the same extent as PAF did. As opposed to wild-type

mice, PAF did not decrease endothelial NO production in lungs of ASM deficient mice

(panel b, Fig. 30). Together with previous findings, it shows that PAF stimulates the

ASM which in turn leads to the recruitment of Cav-1 and eNOS to caveolae with a

subsequent inhibition of endothelial NO production that promotes lung edema

formation.

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Fig. 30: Effects of ASM perfusion and ASM depletion on NO production. a) ASM perfusion reproduced the PAF response on endothelial NO production Images were obtained at baseline and 10 min after ASM (1 U/mL) stimulation in control lungs. b) Endothelial NO response to PAF in ASM-deficient mice. Data are shown as wild-type (WT) control mice, PAF (5 nmol) treated wild type mice, and ASM deficient mice. Endothelial NO production were determined by in situ real-time fluorescence microscopy of lung venular capillaries in isolated perfused mouse lungs. Data are presented as mean±SEM, n=5. *: p<0.05 versus WT control. These experiments were performed by cooperating group of Prof. W. Kübler.

4.4 PAF activates ASM to increase endothelial Ca2+ levels Previous studies had shown that PAF-triggered edema is partially mediated by Ca2+

release, influx from extracellular and subsequent Ca2+ dependent activation of

myosin light chain kinase (MLCK) in endothelial cells [322-323]. Consequently, PAF-

induced edema formation could be attenuated by the IP3 receptor blocker

xestospongin, the myosin light chain kinase inhibitor ML-7, or removal of extracellular

Ca2+ [350]. Recently, polymodal sensory channels of the transient receptor potential

(TRP) family have emerged as major regulators of lung endothelial barrier function.

Unlike the members of the vanilloid (TRPV) and melastatin (TRPM), canonical

(TRPC) channels wre shown to mediate primarily agonist-induced permeability

changes [63].

In this work, we have tested the hypothesis that PAF may stimulate endothelial Ca2+

signalling and subsequent changes in microvascular permeability by activating TRP

channels via the ASM. We performed experiments in isolated perfused rat and

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mouse lungs to isolate and identify the TRPC channels, measure the cytosolic Ca2+

concentration and analyse the vascular permeability and edema formation.

4.4.1 Role of TRPC channels in PAF-induced edema formation and endothelial [Ca2+]i In order to investigate the role of TRP channels in the PAF response, we used the

SKF96365 as TRPC blocker. As seen in Fig. 31, the SKF96365 attenuated the PAF-

induced edema by about 50% and reduced the [Ca2+]i as well. Since TRPV channels

are also known to promote vascular barrier failure [351-352], it was also necessary to

examine the involvement of TRPV channels in PAF-induced edema formation of

isolated perfused rat lung. We use retheuium red as the TRPV inhibitor and it turned

out retheuium red had no significant effect neither on edema formation nor

endothelial [Ca2+]i increase.

Fig. 31: Effects of TRPC channels blocker in PAF-induced edema formation and endothelial [Ca2+]i. a) Edema formation was assessed by weight gain in lungs under untreated control condition, with PAF-treated (10 min after bolus injection of 5 nmol PAF), SKF96365/PAF (30 µM SKF96365) and retheuium red/PAF (1 µM retheuium red). The pharmacological agents were added after 30 min of perfusion and PAF after 40 min of perfusion. Data are presented as mean±SD, n=4. *: p< 0.05 versus control, #: p< 0.05 versus PAF. b) PAF stimulates endothelial Ca2+ influx via TRPC channels. Endothelial [Ca2+]i was determined by in situ real-time fluorescence microscopy of fura-2 loaded lung venular capillaries in isolated perfused rat lungs. Data are shown as 5 min averages in PAF (5 nmol) stimulated control lungs and PAF stimulated lungs that had been pretreated with SKF96365 (30 μM) or ruthenium red (1 μM). *: p<0.05 versus PAF alone, Data are presented as mean±SEM, n=5. The in situ real-time fluorescence experiments were performed by cooperating group of Prof. W. Kübler.

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4.4.2 PAF induced TRP channels recruitment in caveolae To identify the TRPC channels that mediates the endothelial intracellular Ca2+

concentration ([Ca2+]i) in response to the PAF and ASM administration, we have

systematically probed the abundance of several TRPC channels (TRPC1, TRPC3,

TRPC4, TRPC5, TRPC6) in caveolae of control and PAF treated lungs. By

cooperation with Prof. Veit Flockerzi, we have identified that PAF stimulates the

expression of TRPC6 channels in caveolae (Fig. 33), and the rest of the TRPC

members were not present.

Fig. 33: PAF increases the abundance of TRPC6 channels in caveolar fractions of lung endothelial cell membrane. a) Representative immunoblots show TRPC6 expression in caveolar fractions freshly isolated 10 min from untreated control and after treatment of isolated rat lungs with a bolus of 5 nmol PAF. b). Densitometric measurements of 4 independent experiments. Data are presented as mean±SD, n=4. *: p< 0.05 versus control.

To examine whether TRPC6 channels play a role in the PAF-induced edema

formation, we measured the endothelial [Ca2+]i in isolated lungs from TRPC6 deficient

mice. As seen in Fig. 32, both the PAF-induced increased in weight gain and

endothelial [Ca2+]i markedly reduced in the lungs of TRPC6 knockout mice.

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Fig. 32: PAF-induced edema formation and endothelial [Ca2+]i responses in TRPC6-deficient mice. a) Edema formation was assessed by the real time curve in lungs from wild type and TRPC6 knockout mice with PAF-treated (10 min after bolus injection of 25 nmol PAF). Data are presented as mean±SD, n=4. *: p< 0.05 versus wild type. b) Endothelial [Ca2+]i was determined by in situ real-time fluorescence microscopy of lung venular capillaries in isolated perfused mouse lungs. Data are shown as PAF (10 min after bolus injection of 5 nmol PAF) treated lungs of wild type and TRPC6 deficient mice. Time point of PAF administration is indicated by arrow.*: p<0.05 versus wild-type control, Data are presented as mean±SEM, n=5. The in situ real-time fluorescence experiments were performed by cooperating group of Prof. W. Kübler. 4.4.3 Role of ASM in PAF induced TRPC6 recruitment ARC39 is a newly developed ASM inhibitor [353] that reduced edema formation in

isolated perfused rat lung model (as seen Fig. 26). Here we use ARC39 to analyse

the effects of ASM in PAF induced TRPC6 recruitment. As seen in Fig. 34, ASM

inhibition by ARC39 significantly impaired TRPC6 recruitment into caveolae fractions.

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Fig. 34: Effects of ASM inhibition on PAF induced TRPC6 recruitment. a) Immunoblots showing TRPC6 distributions from ARC39/PAF (1 µM ARC39) and PAF (10 min after bolus injection of 5 nmol PAF) treated isolated perfused rat lungs from fraction A–E. b) TRPC6 distribution in caveolae fractions (summary of pooled fraction B-E). *: p<0.05 versus PAF. Data are presented as mean±SD, n=4. 4.4.4 Role of ASM on endothelial [Ca2+]i In addition to the analysis of TRPC6 distribution in caveloae, our cooperating group

also measured the endothelial [Ca2+]i under PAF and ASM inhibitor administration

with in situ fluorescence microscopy. Imipramine attenuated the PAF-induced

increase in endothelial [Ca2+]i (Fig. 35, panel a and b), but had no effect on basal

Endothelial [Ca2+]i increase in the first 10 minutes when it was given before PAF.

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Fig. 35: Effects of ASM on PAF-mediated endothelial [Ca2+]i. Panel a) shows fluorescence images of fura-2 loaded lung venular capillaries in isolated perfused rat lungs. Images were obtained at baseline and 10 min after PAF (5 nmol) stimulation in control lungs and in lungs perfused with imipramine (100 µM). The pharmacological agents were added 10 min before PAF infusion. Panel b) shows endothelial [Ca2+]i as determined by in situ real-time fluorescence microscopy. Curves are shown as 5 min averages at control, after stimulation with PAF alone and with PAF plus imipramine perfused lungs. [Ca2+]i was quantified as increase in fluorescence of fura-2 loaded endothelial cells over 5 min intervals relative to baseline (∆F/F0). Data are presented as mean±SEM, n=5. *: p<0.05 versus PAF. These experiments were performed by cooperating group of Prof. W. Kübler.

4.4.5 Effects of ASM perfusion and ASM depletion on endothelial [Ca2+]i For further demonstration of the role of ASM in endothelial [Ca2+]i regulation, our

cooperating group repeated the endothelial [Ca2+]i measurement with ASM perfusion

instead of PAF. As seen in panel a, Fig. 36, Direct perfusion with ASM (1 U/mL)

increased endothelial [Ca2+]i to the same extent as PAF. As opposed to wild-type

mice, PAF did not increase endothelial [Ca2+]i in lungs of ASM deficient mice (panel

b, Fig. 36). Together with previous findings, it shows that PAF stimulates the ASM

which in turn leads to the recruitment of Cav-1 and TRPC6 to caveolae with a

subsequent influx of Ca2+ that promotes lung edema formation.

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Fig. 36: Effects of ASM perfusion and ASM depletion on [Ca2+]i. a) ASM perfusion reproduced PAF response of endothelial [Ca2+]i. Images were obtained at baseline and 10 min after ASM (1 U/mL) stimulation in control lungs. b) Endothelial [Ca2+]i response to PAF in ASM-deficient mice. Data are shown as wild-type (WT) control mice, PAF (5 nmol) treated wild type mice, and ASM deficient mice. Endothelial [Ca2+]i were determined by in situ real-time fluorescence microscopy of lung venular capillaries in isolated perfused mouse lungs. Data are presented as mean±SEM, n=5. *: p<0.05 versus WT control. These experiments were performed by cooperating group of Prof. W. Kübler.

4.4.6 Recomposing of PAF response: Hyperforin, sulprostone and L-NAME Our current hypothesis is that the PAF-induced edema formation has composed

three components: a) The activation of cyclooxygenase (COX), production of PGE2

and activation of EP3 receptors [152]. b) The discontinuation of endothelial NO

production induced by ASM activation and caveolin-1 recruitment. c) Endothelial cell

constriction induced by calcium influx though TRPC6 channels in caveolae. Here we

used hyperforin as a TRPC6 agonist [354], and together with the sulprotone which is

a EP1/3-receptor agonist [152] and L-NAME as eNOS inhibitor to further investigate

the role of TRPC6 and EP1/3 receptors in edema formation. As seen in Fig. 37, both

hyperforin (10 µM) and sulprostone (1 µM) perfusion alone did increase weight gain

of the perfused rat lungs where L-NAME (100 µM) did not; perfusion with L-NAME

and hyperforin increased weight gain another 50% above; perfusion of L-NAME

together with hyperforin and sulprostone gave a weight gain of almost 400mg, of

note, this value is close to the 550mg that are achieved by PAF.

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Fig. 37: Role of hyperforin and sulprostone in edema formation. Weight gain in lungs perfused under control conditions (n=4), or with L-NAME (100 µM) alone, with sulprostone (1 µM) alone, with hyperforin (10 µM) alone, with L-NAME plus hyperforin and with L-NAME plus hyperforin and sulprostone. The L-NAME was added after 30 min of perfusion and sulprostone and hyperforin after 40 min of perfusion. Data are presented as mean±SD, n=3 *: p <0.05 versus control, #: p <0.05 versus preceding columns.

4.5 Effects of steroids To elucidate the short-term anti-inflammatory effects of steroids that are still poorly

understood, we studied the effects of dexamethasone and quinine on edema

formation, Cav-1 and eNOS recruitment, NO production. Dexamethasone was

previously shown to prevent the PAF-induced edema formation by down-regulating

ceramide production [111], and quinine was shown to block PAF-induced edema by

affecting intracellular calcium levels [151, 355-356]. As seen in Fig. 38,

dexamethasone perfusion provided a pharmacological approach to block ASM

activity.

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Fig. 38: Acid sphingomyelinase (ASM) activity in pulmonary endothelial cell plasma membrane fractions from isolated perfused rat lungs. Showns are the membrane fractions in lungs perfused for 50 min under untreated lungs and treated with PAF (10 min after bolus injection of 5 nmol PAF) or dexamethasone/PAF (10 µM dexamethasone). Data are presented as mean±SD, n=4. *: p<0.05, **: p<0.01 versus PAF.

Treatment of perfused rat lungs with dexamethasone and quinine both reduced the

edema formation, but only dexamethasone perfusion reduced the abundance of Cav-

1 in the caveolar fractions of PAF-treated lungs (Fig. 39, panel b), whereas quinine

had no effect on the enhanced expression of Cav-1 in PAF-stimulated lungs.

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Fig. 39: Effects of steroids on edema formation and caveolin-1 recruitment. a) Edema formation was assessed by weight gain in control lungs and the lungs treated with PAF (10 min after bolus injection of 5 nmol PAF), with dexamethasone (10 µM), with dexamethasone/PAF (10 µM dexamethasone) or with quinine/PAF (100 µM quinine). b) Immunoblots showing the Cav-1 distribution in pooled pulmonary endothelial cell plasma membrane fractions prepared from lungs perfused under control conditions, with PAF (10 min after bolus injection of 5 nmol PAF), with dexamethasone (10 µM) alone, with dexamethasone/PAF (10 µM dexamethasone) or with quinine/PAF (100 µM quinine). The pharmacological agents were added after 30 min of perfusion and PAF after 40 min of perfusion. Data are presented as mean±SD, n=4. *: p <0.05 versus PAF.

Moreover, dexamethasone attenuated the PAF-induced increase of caveolar eNOS

expression (Fig. 40, panel a), the decrease in endothelial NO production (Fig. 40,

panel b) and lung edema formation (Fig. 39, panel a).

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Fig. 40: Effects of steroids on eNOS distribution and endothelial NO production. a) Immunoblots show the eNOS distribution in pooled pulmonary endothelial cell plasma membrane fractions prepared from lungs perfused under control conditions, with PAF-treated (10 min after bolus injection of 5 nmol PAF), dexamethasone (10 µM)alone, dexamethasone/PAF (10 µM dexamethasone) or quinine/PAF (100 µM quinine). Data are presented as mean±SD, n=4. *: p <0.05 versus PAF. b) Shown is the endothelial NO production as determined by in situ real-time fluorescence microscopy. Curves are shown as 5 min averages at stimulation with PAF (5 nmol) alone and with dexamethasone plus PAF perfused lungs. NO production was quantified as increase in fluorescence of DAF-FM loaded endothelial cells over 5 min intervals relative to baseline (∆F/F0). Data are presented as mean±SEM, n=5. #: p<0.05 versus PAF. The in situ real-time fluorescence experiments were performed by cooperating group of Prof. W. Kübler.

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5 Discussion Platelet-activating factor (PAF) is a potent pro-inflammatory mediator that elevates

vascular permeability and leads to edema formation in pulmonary inflammation.

Recently, it has been shown that PAF-induced edema formation is partly mediated by

acid sphingomyelinase (ASM) [111]. The mechanisms by which ASM alters vascular

permeability are poorly understood.

The presence of sphingolipids in caveolae is well documented, where they contribute

to the enhanced rigidity of membrane, but may also assume signalling functions.

Caveolae are enriched in caveolins and endothelial NO synthase (eNOS), which is

bound and thereby kept in its inactive state by caveolin-1 [81, 83]. Another important

family of proteins present in caveolae are the transient receptor potential (TRP)

channels [357-359]. In this thesis, we have linked PAF-induced ASM activation to the

recruitment of caveolin-1, eNOS and TRPC6 to caveolae leading to decreased

endothelial NO and increased endothelial [Ca2+]i levels, providing novel insights into

the regulation of vascular permeability. In addition, in cooperation with Prof.

Christoph Arenz (Humboldt University, Berlin) we have characterized the novel ASM

inhibitor ARC39.

In particular, we showed that a reduction in endothelial NO formation contributed to

increased pulmonary vascular permeability under pathophysiological conditions

mimicked by PAF. These findings are consistent with previous studies showing

increased vascular permeability in eNOS-deficient mice [360]. Our findings further

indicate that PAF reduced endothelial NO levels by the ASM-dependent recruitment

of Cav-1 to caveolae with subsequent inhibition of eNOS, providing the first

mechanistic explanation for the role of ASM in the development of pulmonary edema.

Our findings may also provide a rationale for the treatment of pulmonary edema with

exogenous NO or NO donors in order to restore normal endothelial NO levels.

Furthermore, it was shown that PAF causes endothelial Ca2+ influx through TRPC6

channels, probably after they have been recruited to caveolae. Thus, the ASM

pathway contributes to PAF-induced edema formation by down-regulation of a

protective factor, i.e. endothelial NO, and by up-regulation of endothelial [Ca2+]I that

actively increases vascular permeability by causing endothelial cell contraction.

Finally, our observations raise the hypothesis that interference with ASM activity

inside caveolae may explain some of the short-term effects of steroids in the

regulation of inflammation.

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5.1 Regulation of caveolin-1 by PAF

5.1.1 PAF induced caveolin-1 recruitment to caveolae Based on a procedure originally developed by Jan Schnitzer and colleagues, we

established and improved a method to isolate caveolae from intact rat lungs in that

we perfused isolated lungs with silica beads to tag the endothelial cells and separate

their plasma membrane fractions in a sucrose gradient. Immunoblotting of the

resulting fractions showed a typical distribution for Cav-1 predominantly in the

fractions B-D which are expected to contain the caveolae (Fig. 18). Our study

showed a rapid increase of Cav-1 in the caveolae fractions after stimulation with PAF

(Fig. 19). The caveolae fractions are expected in the 10-16% sucrose gradient which

roughly corresponds to our fractions B and C, which were largely devoid of

angiotensin-converting enzyme and contained flotillin-1 (another caveolae or lipid raft

marker). While fraction A (8-10% sucrose) and fraction D (20%-30%) may contain

some caveolae, our data do not exclude the possibility that Cav-1 was recruited also

to membrane fractions distinct from caveolae. Indirect evidence for the recruitment of

Cav-1 in response to PAF is provided by the fact that at the same time PAF

decreased endothelial NO production (Fig. 29). Caveolin-1 is well known to inhibit

eNOS activity by a direct interaction with eNOS through its “scaffolding domain,”

within endothelial caveolae [272, 317-318].

As for the source of the Cav-1, our findings indicated that it translocates to the

caveolae in response to PAF. Fig. 19 shows that the total amount of Cav-1 remains

unchanged. The increase in the caveolae (fractions B/C) was matched by a decrease

in the remainder of the cells. As this remainder consists of the entire cell except the

caveolae, it is tempting to speculate that Cav-1 was recruited from inside the cell. In

fact, recruitment of Cav-1 to the plasma membrane has previously been described for

bovine aortic endothelial cells in response to shear stress [361], in models of

coronary ischaemia reperfusion [362], portal hypertension [363] and liver cirrhosis

[364].

5.1.2 ASM and caveolin-1 distribution With respect to the molecular mechanisms that explain the Cav-1 recruitment, the

reduction in NO synthesis, the entrance of calcium and thus the PAF-induced edema

formation, it is of interest to examine the role of acid sphingomyelinase (ASM). The

evidence supporting our conclusion that the PAF-induced redistribution of Cav-1 is

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79

mediated by ASM is as follows: 1) Pharmacological ASM inhibitors (imipramine,

D609, ARC39, steroids) and ASM-deficiency impaired PAF-induced pulmonary

edema, NO production and the endothelial calcium increase (Fig. 26, 27, 29, 30). 2)

While it has previously been shown that PAF increases circulating ASM levels, herein

we demonstrate that PAF increases ASM activity inside endothelial caveolae (Fig.

25). Since no reliable ASM antibodies are available we could not directly examine

whether the increased activity is related to elevated amounts of the enzyme. 3) By

measuring the ASM activity inside caveolar fractions we demonstrated that

imipramine, which is not an enzyme inhibitor but accelerates the proteolytic

degradation of ASM [156], reduced the ASM activity below baseline levels (Fig. 25).

4) Injection of ASM reduced endothelial NO production and increased endothelial

calcium levels, providing further evidence for the ability of ASM to affect NO

production and calcium influx (Fig. 29). Unfortunately, the effect of ASM on Cav-1

and edema formation could not be examined because such measurements had to be

performed in an isolated lung model with 100 mL of recirculating buffer, so that the

required amounts of ASM were beyond feasibility. 5) The reduction of caveolar ASM

activity by imipramine resulted in reduced caveolar levels of Cav-1, eNOS and

TRPC6 despite the presence of PAF, and inhibition of edema formation. The

specificity of imipramine for ASM under these conditions is further supported by the

finding that D609 and ARC39, the ASM-pathway inhibitors which are structurally

completely unrelated to imipramine, also prevented the PAF-induced redistribution of

Cav-1 (Fig. 26, 27) and TRPC6 (Fig. 34). 6) Finally, the same pattern of evidence as

for imipramine could also be shown for dexamethasone. Dexamethasone was

previously shown to prevent the PAF-induced increase in ceramide and edema

formation [111]. Herein we show that dexamathasone prevented the PAF-induced

increase of ASM activity in endothelial caveolae and all its sequelae such as Cav-1

and eNOS recruitment to the plasma membrane, the loss of endothelial NO

production and subsequent formation of lung edema (Fig. 39, 40). These findings are

not only of interest because they corroborate the conclusions of our study, but also

because they suggest a novel hypothesis on how to explain the poorly understood

short term effects of steroids that have already been linked to caveolae [168]. It

seems possible that steroids attenuate the formation of ceramide-rich microdomains,

which are required for many signalling events, by interfering with the activation of

ASM [156]. Other than acting as a signalling mediator, ceramide was suggested to

Discussion

80

form microdomains on the membrane that are crucial for cell signalling [161-162,

168-169]. Specific receptor molecules and signalling proteins like Cav-1, eNOS and

calcium channels cluster within such platforms to exclude potential inhibitory signals,

while initiating and greatly amplifying primary signals [156].

5.2 Endothelial permeability regulation by caveolin and NO in lungs 5.2.1 Role of caveolin in the regulation of endothelial NO production Our caveolae isolation protocol permitted us to study in detail how PAF treatment

alters the composition and the functional properties of the caveolar compartment and

regulates NO production in situ. PAF treatment induced a rapid (<10 min) recruitment

of both Cav-1 and eNOS into caveolae (panel b, c, Fig. 41) in parallel with increased

ASM activity in caveolar fractions (panel d, Fig. 41). Furthermore, inhibition of ASM

activity by ARC39 or imipramine blocked all these caveolar responses and prevented

the PAF-induced cessation of NO production and the edema formation (panel a, Fig.

41). Caveolins are well known to inhibit eNOS, so the increased caveolar levels of

caveolin-1 can explain the impaired endothelial NO production in response to PAF

(Fig. 29). All these findings indicated that the inhibition of endothelial NO production

which contributed to PAF-induced edema was caused by ASM involved Cav-1

recruitment to caveolae.

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Fig. 41: Summary of PAF-induced changes in edema formation, caveolae, and ASM activity. Panel a shows weight gain of isolated perfused rat lungs 10 min after PAF treatment. Panel b: Immunoblot showing caveolin-1. Panel c: Immunoblot showing eNOS. Panel d: Enzymatic activity of acid sphingomyelinase. Lungs were infused with a bolus of 5 nMol PAF and pretreated 10 min before PAF administration with 10 μMol/L imipramine or dexamethasone. mean±SD, n=4. *: p<0.05 versus control. Data represent a summary from Fig. 25- 28, 38-40. 5.2.2 Regulation of vascular permeability by NO Using the isolated perfused lung model we have shown, for the first time, that PAF

rapidly attenuates endothelial NO production in pulmonary endothelium. This

reduction of NO contributes to enhanced vascular permeability, as is illustrated by the

effects of L-NAME and PAPA-NONOate on weight gain and Kf (Fig. 22, 23). This

conclusion is concordant with altered endothelial junctional permeability in eNOS-

deficient or L-NAME-treated mice [365] and with the finding that endothelial NO

synthesis reduces Kf in hydrostatic lung edema [338]. The finding that edema

formation and endothelial NO reduction were prevented by ASM inhibitors imipramine

or ARC39 (Fig. 26, 29) support the conclusion that this drop in endothelial NO

production is regulated by ASM.

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82

It is important to note that L-NAME alone had no effect on lung weight, but did only

increase the Kf (Fig. 22). This is similar to the observation in eNOS-deficient mice

that also showed increased vascular permeability but no lung edema [366]. These

findings suggest that the role of the PAF-induced cessation of NO production is to

amplify the increase in vascular permeability which is caused by another mechanism.

Hence, along the PAF-ASM-axis additional mechanisms are likely to come into play,

such as Ca2+ influx or PGE2 [111, 142].

This conclusion is in accordance with the notion that the PAF-induced edema rests

on different mechanisms, i.e. activation of the cyclooxygenase pathway [152] and

acid sphingomyelinase (ASM)-dependent production of ceramide [111]. Accordingly,

PAF-induced lung edema is attenuated, but not prevented by single agents that

interfere with ceramide synthesis. In section 5.3.3, we will further discuss the

interplay between the various mechanisms that constitute the PAF-induced edema

formation. Here we would like to point the pathophysiological relevance of ASM that

is further highlighted by the fact that several lung diseases are mitigated by genetic or

pharmacological blockade of this enzyme [156]. In addition, elevated ASM activities

are present in a variety of pathological conditions associated with increased vascular

permeability such as models of acute lung injury and sepsis [111] as well as in septic

human patients [367]. Thus, inhibition of ASM activity might be an interesting

pharmacological target in inflammatory disorders.

5.3 Caveolae, calcium influx and permeability regulation 5.3.1 Caveolae as sites of calcium influx The important role of Ca2+ for pulmonary edema formation in general is well

established [322-323]. In previous studies, it was observed that PAF-induced edema

formation is dependent on an increase in intracellular Ca2+ concentration and on

extracellular Ca2+ [350]. The rise of intracellular Ca2+ appears to be controlled by IP3-

dependent Ca2+ release from intracellular stores (e.g. ER). This conclusion is

supported by the finding that inhibition of IP3 activation with L-108 (inhibitor of the PI-

specific phospholipase C) and Xestospongin C (specific IP3R antagonist) attenuated

PAF-induced edema formation [350]. The role of extracellular calcium is supported by

the finding that lanthanum chloride, an unspecific Ca2+-channel blocker [350],

reduced PAF-induced edema. These findings are further corroborated by the

observation that thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase,

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83

which is known to empty intracellular Ca2+ stores and activate SOC, induces edema

formation in isolated rat lungs in a manner dependent on extracellular Ca2+ [368].

Thus, Ca2+ entry through store-operated Ca2+-channels (SOC) [369] or receptor-

operated Ca2+ channels (ROC) [275] in pulmonary edema formation is a well

accepted paradigm. It is important to note that the IP3R has been described in the

plasma membrane and especially in caveolae [81, 284-285]. In many reports it is also

described that IP3R and TRPC channels are found in a multimeric-complex with

caveolin-1 in membrane microdomains [327-329]. These observations support our

assumption that caveolae are involved in PAF-induced vascular hyperpermeability.

In endothelial cells of the rat mesentery, PAF increased cytoplasmic Ca2+

concentrations more than 5-fold within 1-2 min [365]. We now report similar

observations in the pulmonary circulation. In particular, we demonstrated that

perfusion with calcium-depleted media (data not shown) and with the non-selective

TRPC blocker SKF96365 (Fig. 32) attenuated the PAF-induced endothelial [Ca2+]i increase and edema formation, while the unspecific TRPV blocker ruthenium red had

no effect (Fig. 31). This finding led to cooperation with the group of Prof. Veit

Flockerzi, in order to systemically probe the abundance of TRPC channels in

caveolar fractions, which led to the discovery that among the TRPC channels only

TRPC6 channels are recruited into caveolae by PAF (Fig. 33). To demonstrate the

functional relevance of this finding, we studied TRPC6-deficient mice that showed

greatly attenuated endothelial [Ca2+]i responses and edema formation (Fig. 32) in

response to PAF. Again this pathway was linked to the ASM pathway because the

increased endothelial [Ca2+]i was attenuated by genetic and pharmacological

inhibition of ASM (Fig. 35, 36) and ASM perfusion reproduced the PAF response in

edema and endothelial [Ca2+]i (Fig. 36). In summary, our data demonstrate that PAF

increases endothelial Ca2+ levels, a mechanism that accounts for part of the PAF-

induced edema formation. The calcium enters the cells through TRPC6 channels that

were recruited to caveolae in a ASM-dependent fashion (see also Fig. 42).

As noted, regulation of pulmonary vascular permeability by calcium is a commonly

accepted mechanism. In mesenteric venular microvessels [365], hamster cheek

pouch [370] and human umbilical vein endothelial cells [371], PAF or thrombin

increased endothelial permeability and [Ca2+]i in a rapid manner. There are also

evidences from frog, rat and hamster mesenteric venular microvessels showing that

increased endothelial [Ca2+]i increased microvessel permeability [372-373]. Genetic

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84

and pharmacological Ca2+-channels inhibition attenuated the increased endothelial

permeability trigger by agents such as PAF, thrombin, VEGF and ATP [370-371, 373-

374]. It is commonly thought that MLCK and MLC phosphorylation which are in part

regulated by Ca2+ lead to actino-myosin contraction with a subsequent increase in

endothelial barrier permeability [66-67, 350]. In some extra-pulmonary tissues Ca2+ is

also thought to act by activation of eNOS [375-376]; this discrepancy to the current

findings is discussed in chapter 5.4.

5.3.2 Calcium influx and endothelial permeability regulation by TRP channels We demonstrated that TRPC6 channels play a central role in endothelial calcium

influx and the regulation of pulmonary vascular permeability. Our findings are in line

with an increasing number of reports that implicate endothelial permeability with

TRPC and TRPV channels.

In general, TRPC4 and TRPC5 are considered to form store-operated calcium

channels (SOC); TRPC1 may be also involved [323]. On the other hand, TRPC3,

TRPC6 and TRPC7 form store-independent cation channels and are considered to

function as receptor operated calcium channels (ROC) [54, 323, 377]. TRPC1,

TRPC4 and TRPC6 are extensively expressed on endothelial cells, not only in the

lungs but also in extra-pulmonary organs [378-382]. In isolated lungs and lung

vascular endothelial cells from TRPC4 deficient mice, calcium influx induced by

thrombin was drastically reduced, as well as the endothelial cell retraction response

and the filtration coefficient (Kf) [371, 379]. In TRPC4-deficient confluent monolayers,

thrombin-induced decreases of trans-endothelial resistance of cultured endothelial

monolayers (a measure of endothelial permeability) were significantly smaller

compared to wild type controls [383]. In addition, in the mouse aorta of TRPC4-

deficient mice, agonist-activated Ca2+ influx induced by acetylcholine and ATP was

drastically decreased and this was associated with a markedly impaired endothelium-

dependent relaxation of blood vessels.

Interestingly, there is already evidence that caveolin-1 may regulate TRPC channels.

Rat and human in pulmonary artery endothelial cells PAEC express TRPC1, and it

was suggested that TRPC1-mediated Ca2+-influx promotes endothelial cell shape

change [378]. The cell permeant antennapedia-conjugated CSD peptide, which

competes for protein binding partners with caveolin-1, markedly reduced thrombin-

induced Ca2+-influx via SOC [384] suggesting that caveolin-1 regulates calcium influx

Discussion

85

via SOC by interaction with TRPC channels [385]. In another study, TRPC1 were

shown to contribute to the calcium store-operated current (ISOC) which is essential for

endothelial permeability regulation [386]. In addition, anti-TRPC1 antibodies inhibited

the VEGF-induced calcium entry and the increased endothelial permeability in

HUVECs. Finally, TRPC1 over-expression augmented the VEGF-induced calcium

entry [374]. In future studies it will be highly interesting to examine whether caveolin-

1 and TRPC6 channnels can interact in a similar manner as caveolin-1 and TRPC1.

As noted, TRPC-channels may form multimers. For instance, human umbilical

endothelial cells (HUVECs) transfected with the N-terminal fragment of hTRPC3 to

disrupt TRPC assembly, showed an absence of store-operated currents [382].

Clearly, our findings do not exclude the possibility that TRPC6-molecules are part of

a multimeric complex, although by immunoblotting we were unable to detect TRPC3

channels in the caveolae preparations. In our isolated perfused lung model, TRPC6

regulated the calcium influx and hyperpermeability induced by PAF. In PAECs,

TRPC6 knockdown prevented myosin light chain phosphorylation, actin stress fiber

formation as well as inter-endothelial junctional gap formation in response to oleoyl-2-

acetyl-snglycerol or thrombinin [385]. Overexpression of a dominant negative TRPC6

construct inhibited the VEGF-mediated increases in cytosolic calcium, migration,

sprouting, proliferation and hyperpermeability in HUVECs [387]. TRPC6 also

appeared to regulate calcium influx in perfused rana mesenteric microvessels [388].

Thus, our study further supports a critical role of TRPC6 channels in the regulation of

vascular permeability and further suggests that its activity is regulated by the ASM.

Another group of TRP channels that have been implicated in the regulation of

pulmonary vascular permeability are TRPV channels. It was shown that the TRPV4

channels is involved in the regulation of permeability induced by mechanical stress,

[389]. In a study of high peak inflation pressure (PIP)-induced pulmonary

hyperpermeability in isolated mouse lungs, calcium influx occurred though endothelial

TRPV4 channel and TRPV inhibition by ruthenium red or TRPV4 deficiency

attenuated the increase in Kf [390]. In isolated perfused rat/mouse lung model,

elevation of lung microvascular perfuse pressure was shown to increase endothelial

[Ca2+]i via activation of TRPV4 channels and the endothelial [Ca2+]i transient

increased Kf via activation of myosin light-chain kinase [351]. In isolated lungs from

TRPV4 deficient mice subjected to hypertension, hyperpermeability, calcium influx,

and alveolar fluid volume were decreased compared to wild type mice [352]. In

Discussion

86

addition, the TRPV4 activator 4α-phorbol-12,13-didecanoate (4α-PDD) increased

lung endothelial permeability and the permeability response to 4α-PDD was absent in

TRPV4 deficient mice [391-392]. For all these findings, it was important to exclude a

role of TRPV4 channels in our model by showing that ruthenium red failed to affect

the PAF-induced calcium responses and edema formation.

5.3.3 Calcium influx, nitric oxide and cyclooxygenase act in concert The present (Fig. 37) and previous findings show that the PAF-induced edema is

composed of three components, one that is directly related to increased permeability

namely calcium, and two mechanisms that sensitize the cells to this response namely

the reduction in NO (Fig. 30) and cAMP [152]. At the molecular levels, these

responses appear to be mediated the ASM-dependent recruitment of Cav-1 and

TRPC6 channels to caveolae and by the cyclooxygenase-dependent activation of

EP3 receptors (Fig. 37). An important question is how these three events are related

to each other. In the following we will discuss possible mechanisms of how these

mechanisms interact:

Hypothesis 1: PGE2 may regulate the activity of TRPC6 channels. This hypothesis is

supported by the following findings: a) the EP3 receptor that partly mediates PAF-

induced edema formation is exclusively localized to the raft fractions [393]; b) in

eryptosis triggered by PAF, the TRPC6 channel was activated by PGE2 [394-396].

Therefore it appears possible that in PAF-induced edema, the EP3-receptor leads to

activation or sensitization of TRPC6 receptors (Fig. 43).

Hypothesis 2: NO may regulate the activity of the TRPC6 channels. While it is well

known that increased endothelial Ca2+ can promote eNOS phosphorylation and NO

production [397], it is also established that NO can negatively regulate calcium influx

and TRP channels. In particular, the elevated NO level stimulate guanylyl cyclases to

produce cGMP, which activates multiple pathways and which may in fact also inhibit

TRP channels. Both TRPC channels [398-399] and TRPV channels [351] were

reported to be regulated by this negative feedback pathway.

Thus, a negative feedback regulation seems to be in place: Normally the rise in

[Ca2+]i provides a signal to reduce Ca2+-influx through a signal transduction pathway

involving NOS-NO-cGMP-PKG and TRP channels. This inhibition of TRP channels

would serve to protect the cells from the detrimental effects of excessive [Ca2+]i and/or NO [400]. It has been suggested that TRPC6 is inhibited by NO-cGMP-PKG

Discussion

87

related phosphorylation in both a heterologous expression system and vascular

myocytes and that cationic currents due to TRPC6 activation are suppressed by the

NO donor SNAP (S-nitroso-N-acetyl-penicillamine) [400]. A similar extent of

suppression was also observed with a membrane-permeable analogue of cGMP,

8Br-cGMP [398, 401]. The inhibitory effects of SNAP and 8Br-cGMP on TRPC6

channel currents were strongly attenuated by the presence of inhibitors for guanylyl

cyclase and PKG [398, 401]. In another study, in isolated mouse lungs challenged

with elevated perfusion pressure, endothelial NO formation limited the permeability

increase by cGMP-dependent attenuation of [Ca2+]I through TRPV4 channels; these

findings were also confirmed by whole-cell patch-clamp of pulmonary microvascular

endothelial cells [399].

In line with all these, further studies from our group (data not shown) show that cGMP

attenuates the PAF-induced endothelial [Ca2+]i increase and hyperpolarizes

endothelial cells in presence of the TRPC6-agonist hyperforin. All these findings put

our finding that NO-depletion occurs simultaneous with the increase in calcium into

perspective: the cessation of NO production by PAF can be interpreted as a measure

to exclude the NO-dependent feedback on TRPC6-channels.

Fig. 42: Mechanisms of PAF-induced hyperpermeability in the pulmonary vasculature, Summary of our current hypothesis. ASM: acid sphingomyelinase; COX: cyclooxygenase; PGE2: Prostaglandin E2; MLCK: myosin light chain kinase

Discussion

88

5.4 Endothelial permeability regulation in the lungs and in extra-pulmonary organs 5.4.1 Regulation of vascular permeability by NO and calcium In the present work, it was shown that PAF causes a decrease in endothelial NO

production. However, in the non-pulmonary circulation, where PAF alters vascular

permeability and intracellular calcium with a similar time course as in the lungs, it

affects NO production in the opposite direction leading to an increase (Fig. 43).

Accordingly, NO-synthase inhibitors attenuated PAF-induced edema formation in the

systemic circulation, in contrast to the lungs where NO donors were effective (Fig.

43). In this chapter we will discuss these conflicting results

Fig. 43: Effects of PAF on vascular permeability, NO formation and calcium influx. Experiments were performed in the extra-pulmonary organs following topical application of 100 nMol/L PAF (A, C, E in hamster cheek pouch and G in mesentery microvessels) and in isolated perfused rat lungs challenged with 5 nMol PAF which after equilibration corresponds to 50 nMol/L (B, D, F, H). PAF was always added at t=0 or as indicated by the arrows. Panel A: Hamster cheek pouch: Extravasation of FITC-dextran expressed as integrated optical intensity (IOI) [402]. Panel B: Isolated perused rat lung: weight gain in PAF-treated (●) and control lungs (○). Panel C: Hamster cheek pouch: nitric oxide levels measured with a NO-sensitive microelectrode [403]. Panel D: Isolated perused rat lung: endothelial nitric oxide

Discussion

89

production assessed by DAF-FM fluorescence microscopy in control lungs (○), lungs treated with PAF (●) or 250 μM L-NAME (◊) as positive control for inhibited NO production. Panel E: Extravasation of FITC-dextran expressed as integrated optical intensity (IOI) in the hamster cheek pouch treated with PAF ± L-NAME (10 μMol/L) [402]. Panel F: Isolated perused rat lung: change in filtration coefficient (Kf) in lungs treated with PAF ± L-NAME (250 μM). Panel G: Mesentery microvessels: endothelial [Ca2+]i assessed by fura 2-AM fluorescence microscopy in PAF treated (●) microvessels [365]. Panel H: Isolated perused rat lung: endothelial [Ca2+]i assessed by fura 2-AM fluorescence microscopy in PAF tereated lungs. The data from the extra-pulmonary organs are shown as mean±SEM, the data from the rat lung as mean±SD. *: p<0.05 versus control, #: p<0.05 versus PAF. Data in panel D were analyzed by the Kruskal-Wallis test, data in panel F by two-sided t-tests and p values corrected for multiple comparisons according to the false-discovery rate procedure. Data in panel E were analyzed by the Student’s-Newman-Keuls test.

In the isolated perfused lung preparation, PAF caused rapid edema formation and

cessation of endothelial NO synthesis in venular capillaries within less than 5 min

(Fig. 43 B and D); a similar instantaneous reduction in NO has been described in

PAF-treated human mesangial cells in culture before [321]. In striking contrast to the

lungs (Fig. 43, panel C), in the hamster cheek pouch [402-403], in perfused

mesenteric venules [365], and in human umbilical vein endothelial cells (HUVECs)

[404] PAF induced a marked increase in endothelial NO production. In the systemic

microvasculature of the hamster cheek pouch, the increased leakage in response to

PAF is inhibited by nitric oxide synthase (NOS) inhibitors [400, 402], and this

inhibition is overcome by administration of the exogenous NO donor SNAP [400],

indicating that enhanced NO production plays an obligatory role in PAF-induced

leakage of systemic microvessels.

This opinion is supported by studies in rat mesenteric microvessels [405], in the

cremaster muscle of mice deficient in eNOS [406] and in vitro in monolayers of

coronary postcapillary venular endothelial cells in which PAF induced a characteristic

hyperpermeability that was abolished after depletion of endogenous eNOS (Fig. 43,

panel E). In contrast, the present work revealed that PAF-induced edema formation

in the lungs was not impaired by the NOS inhibitor L-NAME, but instead attenuated

by the exogenous NO donor PAPA NONOate (Fig. 43, panel F); L-NAME even

exacerbated the Kf and PAF-induced edema formation in the lungs. PAF caused a

calcium influx and endothelial [Ca2+]i increase both in extra-pulmonary system and

intact lungs (Fig. 43, panel G and H).

Discussion

90

Fig. 44: Regulation of PAF-induced edema formation in different tissues. The abscissa shows putative NO concentrations [407]. The ordinate indicates increasing vascular permeability on an arbitrary scale. In rat and mouse lungs PAF leads to a decrease in NO formation that is related to edema formation. In extra-pulmonary tissues, such as the hamster cheek pouch, the mouse cremaster muscle and the mouse mesenteric venules [365, 405-406, 408] PAF-induced edema is caused by increased endothelial NO production.

Our explanation for these conflicting reports is that vascular barrier function in the

lung and extra-pulmonary organs are maintained only if NO is kept at its proper

physiological level, being neither too much nor too little (Fig. 44). Accordingly, both

too much and too little NO can increase vascular permeability [376, 405-406] and NO

synthase inhibitors can either attenuate [400, 402, 409] or aggravate [366, 410-411]

pulmonary and extra-pulmonary hyperpermeability.

An additional explanation might be the different time points studied and the

numerous and diverse effects of NO on leukocytes and endothelial cells. Our model

of the isolated blood-free perfused rat lung allows circumvention of most of these

confounders. a) In vivo, the PAF-induced edema formation appears to consist of an

early phase (~10 min) caused by a direct effect on the vascular endothelium and a

later phase (~20 min) mediated by PAF-activated leukocytes [412]. The focus on the

first 10 min and the absence of leukocytes in our model [413] allowed us to

exclusively study the early effects of PAF on the pulmonary endothelium. b) blood-

free perfusion allows the exclusion of complications as NO effects on neutrophils.

Another potential concern arises from the observation that NO is involved in the

Discussion

91

regulation of alveolar epithelial barrier functions, and thus that the effect of PAPA-

NONOate might be on the alveolar epithelium rather than on the endothelium.

Therefore, it was important to show that PAF does not cause alveolar edema or alter

alveolar fluid absorption in our model (Fig. 24).

PAF causes a rapid calcium influx and endothelial [Ca2+]i increase in both extra-

pulmonary and pulmonary endothelial cells (Fig. 43, panel G and H). Commonly it is

thought that the increased calcium levels increase lung microvascular permeability

via MLCK-activation and MLC phosphorylation and an actin-myosin contractile

mechanism [66-67]. This mechanism appears to account for the PAF effects in the

lungs, since the MLCK inhibitor ML-7 attenuated PAF-induced edema formation [350].

Contrasting the results in the lungs again, PAF did not cause MLCK phosphorylation

in HUVEC [143] and ML-7 failed to affect PAF-induced hyperpermeability in rat

mesenteric venules [146]. Thus again, a pattern of two differential pathways emerges

such that in the lungs PAF increases microvascular permeability by an MLCK-

dependent mechanism that is facilitated by cessation of endothelial NO production,

while PAF-induced hyperpermeability in many systemic microvessels is independent

of MLCK, but instead requires endothelial NO production. Since Ca2+/calmodulin

promotes eNOS phosphorylation and NO production in a rapid manner [397], this

may explain the role of calcium in edema formation in extrapulmonary vessels.

5.4.2 Role of caveolae, ASM and ceramide The regulation of NO production by ASM and ceramide again appears to differ in

pulmonary and in extra-pulmonary vessels. Ceramide activates eNOS and stimulates

endothelial NO formation in cultured bovine aortic endothelial cells (BAEC) [326,

414], as well as in eNOS expressing chinese hamster ovary (CHO) cells [415-416].

Notably, the ceramide induced stimulation of eNOS in systemic endothelial cells is

independent of Ca2+ regulation since it is unaffected by the intracellular Ca2+ chelator

1,2-bis-o-aminophenoxyethane-N,N,N´,N´-tetraacetic acid (BAPTA) and not

accompanied by a simultaneous [Ca2+]i signal [326]. In addition it also seems to be

independent of the source of ceramide, since it is equally triggered by stimulation of

both ASM and neutral sphingomyelinase (NSM) [417]. Taken together,

sphingomyelinase-dependent ceramide formation causes divergent activation and

inhibition of eNOS in systemic and pulmonary endothelial cells, which is likely to

underlie the differential NO response and may contribute to the different forms of

Discussion

92

hyperpermeability induced by PAF in these cells. The question to be addressed

further concerns the mechanisms underlying the differential regulation of eNOS by

ceramide.

It is commonly accepted that direct interaction of eNOS with the scaffolding domain

of Cav-1 keeps the enzyme in its inactivated, dormant status in the absence of

stimulation. This concept is supported by the observation that administration of a cell

permeable form of this scaffolding domain prevented the PAF-induced increase in

vascular permeability in rat mesenteric venules probably by interception of the eNOS-

caveolin-1 interaction in caveolae [405]. In coronary postcapillary venular endothelial

(CVE) cells and ECV304 cells studies, Sánchez and colleagues showed that eNOS

translocation from caveolae to the cytosolic fraction was associated with increased

NO production and a concomitant increase in endothelial permeability [375-376,

418]. Inhibition of caveolar scission by transfection of the dynamin dominant-negative

mutant dyn2K44A prevented the endothelial hyperpermeability in response to PAF

and eliminated NO production [375-376].

Fig. 45: regulation of NO production by caveolin. a) In isolated rat lungs, PAF causes a cessation of NO production by stimulating the acid sphingomyelinase (ASM)-dependent translocation of eNOS to caveolae. PAF also causes Ca2+-dependent activation of the MLCK that is thought to increase vascular permeability by endothelial cell contraction. b) In bovine CVEC and in ECV304 cells PAF stimulates NO production that depends on internalization of caveolae [408]. MLCK: myosin light chain kinase; CVEC: coronary postcapillary venular endothelial cells.

Discussion

93

Thus internalization of caveolae appears to be necessary for PAF-induced NO

production in CVE cells [375-376]. Internalization of eNOS is considered to allow for

targeted NO delivery to subcellular structures and the subsequent increase in

endothelial permeability by promoting the internalization or inhibiting the arrival of

endothelial junctional proteins [375, 418]. NO driven S-nitrosylation of barrier-

regulating proteins could play a major role in this scenario, since it has been shown

to be associated with increased vascular permeability in pathological conditions such

as cerebral ischemia and reperfusion [419]. The notion that eNOS internalization and

targeted NO delivery are critical for PAF-induced barrier failure in systemic

endothelial cells is supported by the fact that PAF-stimulated NO synthesis does not

cause hyperpermeability in cav1Y14F transfected cells in which caveolae are

anchored to the plasma membrane [375]. In the lungs in contrast, caveolin and eNOS

are recruited to caveolae, but are not internalized in response to PAF. As discussed

this explains the rapid cessation of endothelial NO synthesis by the well-established

interaction of eNOS inactivation with caveolin-1 [307, 317, 420] probably leading to a

reduction in PKG-dependent TRPC6-phosphorylation and increased calcium influx

[351, 358, 398-399, 421].

5.5 Concluding remarks The pulmonary and the systemic circulation differ in many fundamental aspects such

as the blood pressure (low versus high), the arterio-venous oxygen gradient

(oxygenation versus deoxygenation), the response to hypoxia (vasoconstriction

versus vasorelaxation [422]), and the endothelial permeability in response to

histamine (no effect versus increased [423]), to name some of the most striking

discrepancies. Another example of this dichotomy appears to be endothelial NO

production in response to PAF: NO production decreases in lung endothelial cells

and increases in the systemic circulation. Surprisingly, both responses have the

same effect, in that they increase vascular permeability. Therefore, we conclude that

vascular barrier function in both the lung and extra pulmonary organs is only

maintained if NO is kept at its physiological level. A clinical implication of this insight

is that it will be enormously difficult to treat diseases with a wide-spread increase of

vascular permeability, such as sepsis, by NOS-inhibitors or NO-donors, because

these agents will be barrier-protective in one vascular bed, but detrimental in another

Discussion

94

Our conclusions relate specifically to the effects of PAF in dissimilar vascular beds. In

pulmonary endothelial cells from intact lungs, PAF causes barrier failure by activation

of cyclooxygenase and ASM resulting in the formation of PGE2 and ceramide. The

latter increases endothelial permeability partly through activation of MLCK by

stimulation of Ca2+ signalling and a concomitant inhibition of basal NO synthesis

which is brought about by the parallel recruitment of caveolin-1 and eNOS to

caveolae. Conversely, in endothelial cells of the extra-pulmonary system, PAF

increases permeability by MLCK-independent formation. In these cells both PAF and

possibly also ASM/ceramide increase endothelial NO synthesis. The PAF-induced

stimulation of eNOS in the non-pulmonary endothelium is regulated by caveolar

internalization, which simultaneously allows for targeted delivery of NO to subcellular

targets which appears to constitute a prerequisite for the subsequent permeability

increase.

Summary

95

6 Summary Platelet-activating factor (PAF) is a mediator of pulmonary edema in acute lung injury

that increases pulmonary vascular permeability within minutes, partly through

activation of acid sphingomyelinase (ASM). Caveolae are rich in sphingolipids and

caveolin-1 (Cav-1) which binds and blocks eNOS. Caveolae also may act as entry

portals for Ca2+. Therefore, we examined the relationship between ASM, Cav-1,

eNOS and Ca2+. Experiments were performed in isolated perfused rat and mouse

lungs. Caveolae from pulmonary endothelial cells were isolated after tagging them by

perfusion with silica beads and followed by the detergent resistant membrane

fractions preparation. Endothelial NO and Ca2+ levels were determined by in situ

fluorescence microscopy.

PAF treatment induced edema formation and decreased endothelial NO formation. In

caveolae fractions from pulmonary vascular endothelial cells (isolated from perfused

rat lungs), the abundance of caveolin-1 and eNOS increased rapidly after PAF

perfusion. Restoration of endothelial NO levels with exogenous NO donor mitigated

the PAF-induced edema. PAF treatment increased the ASM activity in caveolar

fractions and perfusion with ASM decreased endothelial NO production.

Pharmacological inhibition of the ASM pathway with imipramine, D609 or

dexamethasone blocked the PAF-induced increase of caveolin-1 and eNOS in

caveolae, as well as the decrease in NO production and edema formation.

PAF treatment also increased endothelial [Ca2+]i. This response was abrogated by

Imipramine, the ASM-inhibitor ARC39 and in ASM-deficient mice. Conversely,

endothelial [Ca2+]i was increased by direct perfusion with ASM. Both the PAF-

induced [Ca2+]i increase and edema formation was blocked by the unselective TRPC

inhibitor SKF96365. Screening for a variety of TRPC channels in caveolar fractions

from pulmonary vascular endothelial cells revealed that PAF increased the

abundance of TRPC6 channels, and this response was abrogated ARC39. The PAF-

induced increase in endothelial [Ca2+]i and the edema formation was absent in

TRPC6-deficient mice.

We conclude that PAF causes ASM-dependent enrichment of caveolin-1 and

TRPC6-channels in caveolae of endothelial cells. This leads to decreased NO

production and increased Ca2+ levels. We propose that the decreased NO levels

sensitize endothelial cells for Ca2+-dependent mechanisms of endothelial

permeability.

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125

Curriculum vitae

Personal information: First name: Yang

Family name: Yang

Birthday: 21.08.1982

Nationality: Chinese

Qualifications: 1998-2001: QingHai HuangChuan high school, QingHai, China.

2001-2005: School of Life Science, Fudan University, ShangHai, China. Bachelor

of Science.

2005-2007: Martin-Luther-Universität Halle-Wittenberg, Halle, Germany. Master of

biomedical engineering, Master Thesis by Prof. Dr. H. Foth:

"Inhalation system for long term nano particle exposure" (note: 1.0).

Since 2007: Promotion by Prof. Dr. S. Uhlig. Institute of Pharmacology and

Toxicology, RWTH Aachen, Germany.