Linköping University Medical Dissertations No. 1203
Platelets and airway remodeling Mechanisms involved in platelet-induced fibroblast and airway smooth muscle cell proliferation in vitro
Ann-Charlotte Svensson Holm
Division of Drug Research Department of Medical and Health Sciences Linköping University, Sweden
Linköping 2010
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During the course of the research underlying this thesis, Ann-Charlotte Svensson Holm was enrolled in Forum Scientium, a multidisciplinary doctoral program at Linköping University, Sweden
©Ann-Charlotte Svensson Holm, 2010
Printed in Sweden by LiU-Tryck, Linköping, Sweden, 2010
ISBN 978-91-7393-324-7 ISSN 0345-0082
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Till min lilla solstråle Alva
Det finns inga problem, bara lösningar (Ronny Svensson, 1946-1996)
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ABSTRACT Airway remodeling is a contributing cause to the pathological structural changes, such as increased cell proliferation, observed in asthma. Platelets have been found in autopsy lungmaterial obtained from asthmatic patients and are well known to induce proliferation in vitro of a variety of cells. However, the role of platelets in airway remodeling is far from understood. This thesis aims to clarify the involvement of platelets in fibroblast and airway smooth muscle cell (ASMC) proliferation in vitro and to elucidate the importance of HA, FAK, eicosanoid and ROS dependent signaling. The results demonstrate that platelets induce ASMC proliferation through NADPH-oxidase and 5-LOX dependent mechanisms. In addition, platelets induce a 5-LOX dependent fibroblast proliferation. Morphological analysis suggests that platelets bind to the extracellular matrix component HA through its receptor CD44 and thereby induce a FAK dependent ASMC proliferation. Taken together, the results obtained in this thesis suggest that platelet/HA interaction mediated through CD44 is of importance for platelets ability to induce cell proliferation. Moreover, the results propose that platelet-induced fibroblast proliferation is 5LOX dependent and that platelets induce a HA, CD44, FAK, 5-LOX, and ROSmediated ASMC proliferation. This action of platelets represents a potential important and novel mechanism that may have an impact on the remodeling process and in the development of new pharmacological strategies in the treatment of inflammatory respiratory disease such as asthma.
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POPULÄRVETENSKAPLIG SAMMANFATTNING I lugnvävnaden hos personer med inflammatorisk lungsjukdom som t.ex. astma sker strukturella förändringar (airway remodeling) i form av ökad cellmassa genom att celler blir större och fler. Trombocyter (blodplättar), vars främsta roll är att förhindra blödningar, har identifierats i lungvävnad från avlidna personer med astma men trombocyternas betydelse i denna sjukdom är okänd. Det finns många olika faktorer som kan leda till ökad tillväxt av celler, och några av dem är arakidonsyrametaboliter, s.k. eikosanoider, och reaktiva syreradikaler. Vidare så vet man att glattmuskelceller producerar bindväv som är en samling av olika protein och kolhydrater, t.ex. hyaluronsyra (HA), essentiella för cellers normala funktion. Huvudsyftet med denna avhandling har varit att studera hur trombocyter reglerar tillväxten av två vanligt förekommande celltyper i luftvägarna, fibroblaster och glattmuskelceller. En viktig del har varit att identifiera betydelsen av eikosanoider och reaktiva syreradikaler. Resultaten visar att i närvaro av trombocyter så ökar tillväxten av både fibroblaster och glattmuskelceller och att denna ökade tillväxt är beroende av eikosanoider. Vidare så framgår det att reaktiva syreradikaler krävs vid trombocyt-reglerad glattmuskelcellstillväxt. Våra resultat visar att glattmuskelcellerna bildar HA och att interaktionen mellan trombocyter och HA är viktig för trombocyters förmåga att framkalla glattmuskelcellstillväxt. Även focal adhesion kinase (ett protein viktig för interaktionen mellan ytstrukturer och bindväv) identifierades som en nyckelmolekyl i den glattmuskelcellstillväxt som orsakas av trombocyter. Påvisandet av en aktiv roll för trombocyter vid inflammatoriska lungsjukdomar och identifiering av specifika signalvägar kan leda till utveckling av nya behandlingsmetoder och mer effektiva läkemedel.
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TABLE OF CONTENTS ABBREVIATIONS ...................................................................................................... 1 LIST OF PAPERS ....................................................................................................... 2 INTRODUCTION....................................................................................................... 4 AIRWAY REMODELING..................................................................................................... 4 Epithelium disruption ............................................................................................ 5 Goblet cell proliferation and mucous production .................................................. 6 Angiogenesis ......................................................................................................... 6 Fibroblasts and subepithelial fibrosis .................................................................... 6 Airway smooth muscle cells and proliferation....................................................... 7 PLATELETS AND AIRWAY REMODELING ............................................................................. 12 MEDIATORS INVOLVED IN CELL PROLIFERATION .................................................................. 17 Hyaluronic acid.................................................................................................... 17 Focal adhesion kinase ......................................................................................... 18 Eicosanoids.......................................................................................................... 21 Reactive oxygen species ...................................................................................... 24 AIMS ..................................................................................................................... 28 METHODS ............................................................................................................. 30 CELL CULTURE ............................................................................................................. 30 Media .................................................................................................................. 30 Airway smooth muscle cells and fibroblasts ........................................................ 30 PREPARATION OF PLATELETS, PLATELET MEMBRANES, PLATELET LYSATE AND SUPERNATANT........ 30 CELL PROLIFERATION .................................................................................................... 32 The CellTiter96®Aqueous One Solution Cell Proliferation Assay .......................... 32 3
H-thymidine incorporation ................................................................................. 32
Cell counting and DNA-measurements................................................................ 33
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MICROSCOPIC EXAMINATION OF PLATELET-ASMC/FIBROBLAST INTERACTION ......................... 33 WESTERN BLOT OF FOCAL ADHESION KINASE AND 5-LIPOXYGENASE ....................................... 33 REVERSE TRANSCRIPTASE-MEDIATED PCR......................................................................... 34 SOLID PHASE EXTRACTION AND HPLC .............................................................................. 34 MEASUREMENT OF INTRACELLULAR REACTIVE OXYGEN SPECIES ............................................. 35 STATISTICAL ANALYSIS ................................................................................................... 36 RESULTS AND DISCUSSION .................................................................................... 38 PLATELETS INDUCE FIBROBLAST AND ASMC PROLIFERATION ................................................ 38 PLATELETS BIND TO HYALURONIC ACID THROUGH CD44 AND INDUCE A FOCAL ADHESION KINASE DEPENDENT ASMC PROLIFERATION................................................................................. 40
THE MITOGENIC EFFECT OF PLATELETS IS MAINLY DUE TO MEMBRANE-ASSOCIATED FACTORS ...... 45 PLATELET-INDUCED ASMC/FIBROBLAST PROLIFERATION IS 5-LIPOXYGENASE DEPENDENT .......... 47 PLATELET-INDUCED ASMC PROLIFERATION IS DEPENDENT ON REACTIVE OXYGEN SPECIES .......... 51 SUMMARY AND CONCLUSIONS ............................................................................. 56 TACK ..................................................................................................................... 58 REFERENCES .......................................................................................................... 62
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ABBREVIATIONS AA
arachidonic acid
ACD
acid-citrate dextrose solution
ASMC
airway smooth muscle cells
BALF
bronchoalveolar lavage fluid
COX
cyclooxygenase
DCFDA
2´-7´-dichlorodihydrofluoroscein diacetate
DMEM
Dulbecco´s modified eagle medium
DPI
diphenyleneiodonium chloride
ECM
extracellular matrix
FBS
foetal bovine serum
5-LOX
5-lipoxygenase
FAK
focal adhesion kinase
HA
hyaluronic acid
HPLC
high performance liquid chromatography
KRG
Krebs-Ringers glucose
LTC4 , LTD4…
Leukotriene C4, Leukotriene D4…
MAPK
mitogen activated protein kinase
PBS
phosphate buffered saline
PDGF
platelet-derived growth factor
PFA
paraformaldehyde
PI3K
phosphatidylinositol 3-kinase
PLA2
phospholipase A2
PMP
platelet microparticles
ROS
reactive oxygen species
RTK
receptor tyrosine kinase
RT-PCR
reverse-transcriptase PCR 1
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LIST OF PAPERS This thesis is based on the following papers, which will be referred to by their roman numerals I
Berg C, Hammarström S, Herbertsson H, Lindström E, Svensson A-C, Söderström M, Tengvall P, and Bengtsson T. Platelet-induced growth of human fibroblasts is associated with an increased expression of 5-lipoxygenase. Thromb Haemost 96: 652-659, 2006.
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Svensson Holm A-C B, Bengtsson T, Grenegård M and Lindström E G. Platelets stimulate airway smooth muscle cell proliferation through mechanisms involving 5-lipoxygenase and reactive oxygen species. Platelets 19 (7): 528-536, 2008.
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Svensson Holm A-C B, Bengtsson T, Grenegård M and Lindström E G. Platelet membranes induce airway smooth muscle cell proliferation. In Press Platelets 2010; doi: 10.3109/09537104.2010.515696
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Svensson Holm A-C B, Bengtsson T, Grenegård M and Lindström E G. Platelet bind to hyaluronic acid through CD44 and induce a focal adhesion kinase dependent airway smooth muscle cell proliferation. Submitted
Papers are reprinted with permission from Schattauer (Paper I) and Informa Healthcare (Paper II-III). 2
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INTRODUCTION The opinion regarding the underlying cause of asthma has changed a lot during the years, e.g. for 50 years ago asthma was considered being a result of abnormal contractility of smooth muscle, while 20 years ago inflammation was in focus. Standard treatment for asthma today is inhibition of the inflammation using corticosteroids and/or inhibition of the airway obstruction using β2 adrenergic agonists [1]. Patients with severe asthma sometimes require other medications e.g. leukotriene receptor antagonists that also have been shown to induce relaxation of smooth muscle [2]. Treatment with inhibitors directed against phosphodiesterases (PDE) is another alternative since they elevate the levels of cAMP and cGMP leading to smooth muscle relaxation and inhibition of inflammation [3]. However, during the last decades it have been shown that not all asthmatic patients are controlled using recommended treatment indicating that today´s asthma therapy has to find new alternative strategies. Research conducted recent years has focused on structural changes in the airways, called airway remodeling. Airway remodeling Airway remodeling is caused by a repair process in response to airway injuries resulting from chronic inflammation. Current asthma therapies do not specifically prevent the airway remodeling process and therefore identificantion of crucial targets in this process might lead to novel asthma therapies [4, 5]. Structural changes observed in the airway wall of asthmatic patients are epithelium disruption, increased goblet cell proliferation, angiogenesis, subepithelial fibrosis and increased mass of smooth muscle cells (Figure 1) [6].
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Th2-cell Increased goblet cell proliferation and mucous production
Eosinophil Epithelial disruption
Lamina densa Subepithelial fibrosis Myofibroblast
Fibroblast
Angiogenesis
Increased airway smooth muscle cell mass
Figure 1. A schematic picture of the remodeling process observed in asthmatic airways. Structural changes caused by chronic inflammation, such as epithelial disruption, increased goblet cell proliferation and mucous production, subepithelial fibrosis, angiogenesis and increased smooth muscle cell mass are all included in the term airway remodeling. In addition, infiltration of eosinophils and Th2-cells has also been observed.
Epithelium disruption One of the main characteristics of airway remodeling observed in asthmatic patients is the damaged airway epithelium and cells that have detached from their basal membrane [6]. In addition, airway epithelial cells have been found to release higher levels of proinflammatory cytokines and growth factors [7, 8]. Epithelium disruption together with the cell repair process is therefore thought to be one of the main initiators of the remodeling process [8].
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Goblet cell proliferation and mucous production Mucous, secreted either from mucous glands or produced by goblet cells (specialised epithelial cells), is normally released into the airways for protecting the lung against foreign particles. Increased proliferation of goblet cells with enhanced mucous production has been found in asthmatic airways, although it is not a consistent finding among all asthmatic patients [6]. Vameer et al. showed in an in vitro study that IL-9 is able to induce goblet cell proliferation [9]. However, enlargement of mucous glands and thereby an increased mucous secretion is a well known phenomena observed among asthmatic patients with occlusion of the airways as a consequence. The volume of the mucous glands has been found to be twice as big in asthmatic patients compared to healthy controls [10, 11]. Angiogenesis Another structural change observed in asthmatic patients is increased number and size of vessels [12-14]. Vascular endothelial growth factor (VEGF) is one key mediator involved in the increased vascularity and permeability observed in airway disease [15, 16]. In addition, inflammatory cells such as eosinophils, and Th2-cells enter the lung during inflammatory airway disease through the pulmonary vessels [14, 17, 18]. Interestingly, it was recently shown in a mouse model that platelets are able to migrate out of vessels and localise in the lung tissue [19]. Fibroblasts and subepithelial fibrosis Thickening of the lamina reticularis is one of the early features in asthmatic airways and is termed subepithelial fibrosis [20-22]. The lamina reticularis has been shown to increase almost two-fold in asthmatic patients and contains the extracellular matrix (ECM) molecules collagen I, III and V and fibronectin [6, 6
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20, 23, 24]. Fibroblasts, by producing ECM molecules, and eosinophils, by producing e.g. TGF-β, are two of the main cell types involved in subepithelial fibrosis [23, 25, 26]. Fibroblasts include a number of celltypes e.g. circulating fibroblast progenitors called fibrocytes and the ECM protein producing myofibroblasts that have the ability to contract [27, 28]. Fibroblasts that are located in the airways have been shown to be in close proximity with the epithelial cells and differentiate upon stimulation into myofibroblasts followed by secretion of ECM proteins. In addition, fibroblasts and/or myofibroblasts are found located in and beneath the thickened lamina reticularis generated during airway remodeling and a correlation between production of ECM molecules in the airways and the number of fibroblasts has been observed [6, 23, 29]. Airway smooth muscle cells and proliferation Airway smooth muscle cells (ASMC) are well known for their ability to contract and thereby regulate airway resistance but were for a long time considered to play a passive role as structural cells. The research regarding the role of ASMC in chronic airway inflammation increased considerably when it was possible to use cultures of ASMC and the view of ASMC in pathophysiological changes of asthma has changed a lot over the last decades [30]. ASMC are now proven to be important and active participants in the inflammation and the allergic events that occur in asthma [31, 32].
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ASMC have for example been shown to: • undergo proliferation in response to many different growth factors, e.g. thrombin and platelet-derived growth factor (PDGF), which results in increased muscle volume and airway narrowing [33, 34]. • produce and release a number of cytokines e.g. IL-6 that has been shown to induce mucous secretion, T-cell activation and differentiation [35-38]. • express adhesion molecules on their surface that attract inflammatory cells e.g. CD40 and CD44 [39, 40]. • produce extracellular matrix proteins that affect muscle cell function e.g. collagen I, III and V, fibronectin, hyaluronic acid and laminin [20, 41].
ASMC have been shown to undergo reversible phenotype switching in vitro, also
called
phenotype
plasticity,
between
a
contractile
and
a
proliferative/synthetic phenotype (Figure 2). This means that ASMC are able to control the airway diameter by changing into a contractile phenotype acutely or into the proliferative/synthetic phenotype when needed during the inflammatory process [42].
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Contractile
Proliferative/Synthetic
Maturation: • ECM • TGF-β, insulin • Confluent cells
Modulation: • Mitogens • ECM • non-confluent cells
Figure 2. Schematic picture showing phenotype switching in ASMC in vitro. ASMC are able to switch between two different phenotypes; the proliferative/synthetic and the contractile phenotype. Switching from a proliferative/synthetic to a contractile phenotype is called maturation and is initiated by ECM proteins, TGF-β, insulin and when ASMC becomes confluent. Switching from a contractile to a proliferative/synthetic phenotype is called modulation and is induced by mitogens, ECM proteins and when ASMC are non- or sub-confluent.
Contractile ASMC have been shown to undergo a phenotype switch from the contractile to the proliferative/synthetic phenotype (modulation) when they are seeded into sub-confluence in the presence of mitogens [43-45]. The modulation process results in increased quantity of mitochondria and organelles [42]. When ASMC in culture becomes confluent they switch back to the contractile phenotype and this process is called maturation and results in increased levels of e.g. cytoskeleton-associated proteins and reduced quantities of organelles for protein and lipid synthesis [46, 47]. The difference in protein expression between the two different phenotypes are often used for characterisation [43]. 9
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Increased mass of ASMC, another feature of airway remodeling, is found in both small and large airways and may lead to decreased airway diameter and amplify airway constriction [11, 48-53]. The first detailed report about this phenomenon was described in fatal asthma and published in 1922 by Huber et al [54]. Increased ASMC mass is thought to enhance bronchoconstriction and airway hyperresposiveness [55]. However, there are some uncertainties to whether the increased ASMC mass is due to size enlargement or increased number of ASMC. Studies indicate both, although with a preponderance of increased ASMC proliferation [52, 56-60]. Inflammatory mediators have been found to increased ASMC proliferation in healthy humans and in a variety of animal models. In addition, studies have shown that there is a difference in the cell cycle, cytokine production and proliferation of ASMC obtained from non-asthmatic after stimulation with serum or bronchoalveolar lavage fluid (BALF) from asthmatic patients [61-63]. However, the first study where a difference in cell proliferation was found between ASMC obtained from asthmatic patients compared to ASMC from controls was published in 2001 by Johnson et al [57]. A difference in intracellular signaling pathways when comparing ASMC from asthmatic patients with ASMC obtained from healthy individuals has also been found [56]. ASMC proliferation is stimulated through mitogens that bind to four different receptor systems; tyrosine kinase linked receptors (RTK) that bind e.g. PDGF, cytokine receptors that bind cytokines such as TGF-β and IL-6 and G-protein coupled seven transmembrane receptors (GPCR) that bind contractile agonists such as leukotrienes. The binding of these mitogens activates the small guanidine triphosphate (GTPase) binding protein p21ras that, in its GTP bound 10
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state, will interact with downstream mediators [64]. In addition, ECM proteins such as collagen and fibronectin have been shown to bind to different types of integrins resulting in activation of the non-receptor protein tyrosine kinase focal adhesion kinase (FAK) [65]. Mitogen binding to its receptor will finally activate mitogen activated protein kinase (MAPK) [66-68] and/or phosphatidylinositol 3kinase (PI3K) [33, 69] resulting in increased ASMC proliferation (Figure 3). The function and intracellular signaling pathways regulating FAK will be described in more detail later on in the introduction. RTK
Cytokine receptor
GPCR
Integrin
FAK
MAPK p21ras PI3K
ASMC proliferation
Figure 3. Schematic presentation of signal transduction mechanisms that regulate ASMC proliferation. ASMC mitogens act via tyrosine kinase linked receptors (RTK), G-protein coupled seven transmembrane receptors (GPCR), cytokine receptors or integrins to activate mitogen activated protein kinase (MAPK) or phosphatidylinositol 3-kinase (PI3K) resulting in increased ASMC proliferation. Focal adhesion kinase (FAK) activates MAPK and PI3K.
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Platelets and airway remodeling Platelets are the smallest components of the human blood with a diameter between 2-5 µm and 0.5 µm in thickness [70]. Platelets are derived from giant haematopoietic precursor cells called megakaryocytes and are therefore not provided with a nucleus. The exact mechanism of platelet formation is not understood, however three different processes have been suggested; budding, cytoplasmic fragmentation and proplatelet formation [71]. Megakaryocytes are produced in the bone marrow and one megakarocyte can shed of thousands of platelets that stay in the circulation for 7-10 days and they are thereafter degraded in the liver or spleen [72]. Megakaryocytes are able to migrate into the blood stream and are found in e.g. the lung [71, 73, 74]. About 250 000 megakaryocytes reach the lung every hour and are 10 times more concentrated in blood collected from the pulmonary arteries than from the aorta [75]. The resting platelet contains a band of microtubules that serve to maintain its discoid shape and upon activation platelets undergo a shape change to a spherical form with pseudopodia. Platelet plasma membrane expresses many receptors that recognise e.g. collagen, thrombin, von Willebrand factor and ADP [72]. Platelets possess three types of cytoplasmatic secretory granule with different molecular content: dense granules (e.g. ADP, ATP and calcium), alpha granules (e.g. von Willebrand factor, P-selectin and PDGF) and lysosymes (acid hydrolases, cathepsin D and E and LAMP-2) [70, 76]. Platelets role in haemostasis is to prevent blood loss at sites of vascular injury. When a vessel is injured, substances that are present in the subendothelium, such as collagen and von Willebrand factor, are exposed and platelets, through receptors for these substances, are able to bind and adhere to the wounded area. On the damaged vessel surface activated platelets continue to spread and adhere 12
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and thereby losing its discoid form and develop pseudopodia, resulting in a more efficient platelet-platelet contact and adhesion. Activated platelets also bind to fibrinogen via the integrin αIIbβ3 which leads to formation of a platelet plug. In addition, platelet activation involves secretion of granular substances which lead to a further recruitment and activation of platelets and other cells [72]. Platelets are specialised for haemostasis but they are also suggested to be involved in the inflammatory response by generating a variety of inflammatory mediators e.g. P-selectin and RANTES and interacting with leukocytes and endothelial cells [17, 18, 72, 77]. In fact, it has been suggested that platelets have the capacity to undergo both chemotaxis and phagocytosis [78-80]. Platelets have been found in bronchial biopsy materials from asthmatic patients, in the extra-vascular compartment and on the surface of damaged airway epithelium demonstrating that platelets are able to reach the lung [81, 82]. Platelets
are
suggested
to
contribute
to
hyperresponsiveness
and
bronchoconstruction by secretion of various substances [83-86] (Figure 4). However, platelets are also considered as important players in asthma through their role in inflammation e.g. increased levels of the α granule component RANTES have been found in plasma obtained from asthmatic patients [87, 88]. Platelets might also be involved in other respiratory diseases such as chronic obstructive pulmonary disease and cystic fibrosis [89-93]. However, the involvement of platelets in these diseases is less well studied, compared to their role in asthma.
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De Sanctis and coworkers found in 1997 that P-selectin deficient mice, challenged to ovalbumin (OVA), had fewer eosinophils and lymphocytes in the BAL fluid compared to control mice [94]. In addition, Moritani and colleagues showed that human asthmatic patients had increased number of circulating platelets expressing P-selectin compared to healthy control subjects [77]. It has also been shown that blood from asthmatic patients possess a P-selectin dependent eosinophil clustering [95]. Pitchford and coworkers observed increased numbers of platelet-leukocyte aggregates in blood from asthmatic patients and a P-selectin and platelet dependent leukocyte infiltration in allergic mice [17]. They also found that P-selectin is a prerequisite for pulmonary eosinophil and lymphocyte recruitment [18]. These studies suggest that platelets, by expressing P-selectin, are important in the recruitment of inflammatory cells from the circulation into the lung in patients with asthma. Platelets have been found to induce repair and remodeling in other organs and are therefore suggested to contribute in the airway remodeling process [96-101]. Platelets might affect airway remodeling either, as described above, as being an important mediator involved in infiltration of inflammatory cells or by affecting cell proliferation e.g. through secretion of mitogenic growth factors such as PDGF. Pitchford and coworkers published in 2004 a paper about the role of platelets in airway remodeling using a mouse model where the mice were repeatedly challenged with OVA for 8 weeks, this to mimic the processes observed in asthmatic patients. They found that OVA treated mice possessed significant thicker smooth muscle layer and also increased subepithelial fibrosis, structural changes that were not present in the control mice. The impact of platelets in their model was tested using anti platelet serum or busulfan. Busulfan is an old chemoterapeutic treatment for chronic myeloid leukaemia that have been found to decrease platelet levels [102]. They demonstrated that 14
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platelet depletion using either of these two treatments reduced airway remodeling observed in the OVA-sensitised mice [103]. This study revealed that platelets are essential for airway remodeling in mice induced by an allergen and suggest that platelets also might play an important role in structural changes observed in human airways (Figure 4).
Inflammatory cell Lumen of the lung
Airway inflammation: Via enhanced recruitment of inflammatory cells
Pulmonary capillary lumen
platelets
Bronchoconstriction: Via release of constricting agents
Airway remodeling: Stimulation of ASMC and fibroblast proliferation
Hyperresponsiveness: Via release of hypersensitising mediators resulting after platelet interaction with inflammatory cells
Figure 4. A schematic picture showing possible roles for platelets in asthma. Experimental and animal studies suggest that platelets influence the pathology of asthma by affecting the inflammation via infiltration of inflammatory cells from the blood, the hyperresponsiveness and contraction of ASMC by releasing bronchoconstrictors and airway remodeling by stimulating ASMC and fibroblast proliferation.
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When platelets become activated, e.g. by thrombin, they release two different types of microparticles, either budded from the plasma membrane or released from α-granules [104]. Platelet microparticles (PMP) vary in size and the largest microparticles are those budded from the plasma membrane and their size varies between 0.1 and 1 µm in diameter, i.e. almost the same size as resting platelets. PMP have been found to express some of the glycoproteins that intact platelets do, e.g. GpIb (CD42b), CD31, CD62P and GpIIb/IIIa, and are very important for the coagulation cascade and for adhesion of platelets to the subendothelium matrix [105-107]. Studies have shown that cell proliferation induced by both platelet membranes and PMP is PDGF-independent [108, 109]. Furthermore, unstimulated platelets stored either in room temperature or -80 °C have been shown to posses greater wound healing properties on diabetic wounds than plasma [110]. In addition, both freeze dried platelets and freeze dried plateletrich plasma posses wound healing properties suggesting that released growth factors do not solely explain platelet-induced proliferation [111, 112]. Interestingly, PMP have also been shown to induce proliferation of hematopoietic cells and vascular smooth muscle cells [109, 113, 114]. A number of studies have shown that platelets from asthmatics often behave abnormally e.g. with reduced activity [84, 115-118]. In addition, platelets from asthmatics have abnormal arachidonic acid metabolism and intracellular levels of different second messenger molecules, e.g. calcium [119-121]. Increased bleeding time has also been observed in asthmatics, an effect that was normalised after treatment with glucocorticoids, suggesting that inflammation might be involved [115, 116, 122].
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Mediators involved in cell proliferation There are several different mediators (e.g. mitogens, cytokines and extracellular matrix components) which have been shown to affect the pathological structural changes, e.g. increased ASMC mass, observed in airway remodeling. In the papers included in the present thesis we have focused on the following mediators: hyaluronic acid, focal adhesion kinase, eicosanoids and reactive oxygen species. Hyaluronic acid Subepithelial fibrosis in airway remodeling is partly due to increased levels of extracellular matrix (ECM) components such as collagens, fibronectin, glycoproteins and proteoglycans. Fibroblasts, myofibroblasts and ASMC have been shown to secrete different ECM and considered as important contributors to the increased ECM mass [28, 123]. Hyaluronic acid (HA), one of the main components building up the ECM, is a glycosaminoglycan composed of repeating units of GlcNAc-β(1-4)-GlcUA- β(13) produced by most cell types by three different HA synthases (HAS 1-3). HAS uses UDP-GlcUA and UDP-GlcNac as substrates and HA is expressed throughout the body including the lung [124, 125]. However, the amount of HA in the respiratory tract during inflammatory airway disease has been shown to either increase or decrease [126-128]. HA is mostly expressed in areas surrounding
proliferating
and
migrating
cells
and
especially
during
inflammation and tissue repair [129] and is thus believed to play an important role in regulating both proliferation and migration of cells [130, 131]. HA has a molecular mass ranging from 1-10 000 kDa and the large 500- 10 000 kDa HA fragments are called high molecular weight HA (HMW-HA) and the smaller fragments, 1-500 kDa, are called low molecular weight HA (LMW-HA) [132]. 17
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HMW-HA is produced by HAS 1-3 while LMW-HA mostly is produced by enzymatic degradation of HMW-HA by hyaluronidases (HYAL) or oxidative hydrolysis by reactive oxygen species of HMW-HA [132, 133]. HMW-HA has anti-proliferative effects while LMW-HA are pro-proliferative [132]. It was recently found that platelets possess HYAL-2 and is therefore able to cleave HA into small fragments [134]. HA and HA fragments have recently been shown to bind to different receptors, e.g. the RHAMM receptor that is mainly involved in cell motility [135] and also to HARE, the receptor responsible for endocytosis of HA [136, 137]. However, the best known and studied receptor for HA and HA fragments is CD44, which recently was associated with both the anti-proliferative and the pro-proliferative effect of HA [137]. However, other ECM components have also been shown to bind CD44, e.g. collagen, laminin, fibronectin and osteopontin [133, 138]. CD44 is a type 1 transmembrane receptor expressed on the surface of most cell types, e.g. smooth muscle cells and platelets, and mediates both cell adhesion and cell growth [139]. CD44 exists in many different isoforms that all have HA binding properties [140] and regulates several signaling pathways including Src family kinases, Rho family GTPases, extracellular signal-regulated kinases and MAPK [141, 142]. CD44 has a structural role in linking the ECM with the cytoskeleton and thereby regulate cell shape and motility [143-145]. Focal adhesion kinase When cells interact with ECM proteins, different intracellular signals are generated that are important for cell growth, survival and migration. Intergrins are a group of transmembrane receptors linking ECM proteins with the actin cytoskeleton leading to regulation of e.g. cell shape. Integrins have been shown to cooperate with RTK and thereby mediate signaling pathways stimulated by 18
27
growth factors [146, 147]. In addition, several non-receptor tyrosine kinases, e.g. the 120 kDa protein focal adhesion kinase (FAK), are activated upon interaction between integrins and the ECM. FAK is in adherent cells colocalised to integrins at focal adhesions (signaling proteins and structural proteins associated with the actin cytoskeleton) and recruited at an early stage of the signal transduction to focal adhesions and mediates many downstream responses. FAK consists of a large central catalytic domain containing 6 different phosphorylation sites and is expressed in most cell types, including smooth muscle cells and fibroblasts. FAK is an adaptor for protein-protein interaction, and transmits thereby adhesion and growth factordependent signals into the cell [148]. FAK has been shown to regulate many different signaling pathways by affecting G-protein linked receptors and other transmembrane receptors for different growth factors [149]. Moreover, pulmonary artery smooth muscle cells and glioma cell proliferation is inhibited by antisense oligonucleotides directed against FAK, suggesting role for FAK in cell proliferation [150, 151].
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One of the well known signaling pathways involved in FAK-mediated regulation of cell proliferation is initiated by autophosphorylation of tyrosine 397 on FAK, forming a binding site for Src, which in turn phosphorylates tyrosine 576/577 leading to activation of protein kinase C and PI3K [152]. The FAK/Src complex also phosphorylates FAK at tyrosine 925, which forms an adaptor Grb-2 docking site and induces MAPK activation [153]. PI3K, PKC and MAPK are all involved in signaling pathways regulating cell proliferation (Figure 5).
phosphoTyr 397
Integrin
Integrin
FAK
FAK
Src SHC
phosphoTyr 576/577
PKC
PI3K
phosphoTyr 925
Src
GRB-2
SOS
MAPK
Increased cell proliferation
Figure 5. FAK´s signaling pathways and cell proliferation. Phosphorylated FAK activates PI3K, protein kinase C (PKC), and MAPK and thereby affects cell proliferation
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29
Platelets also express FAK and several studies have demonstrated that ligand binding to both the fibrinogen receptor αIIbβ3 and the collagen receptor α2β1 is necessary for FAK to get activated [154-157]. FAK is important for the production of megacaryocytes and for platelets ability to spread on fibrinogen surfaces [158]. Platelet spreading on fibrinogen, as well as release of intracellular calcium, dense granule secretion and aggregation are blocked by the FAK specific inhibitor PF 573228 [159]. Eicosanoids Arachidonic acid (AA) is the common precursor of eicosanoids (prostaglandins, leukotrienes, and tromboxanes), found in both the membrane and in the cytosol [160]. In resting cells, AA is stored in the plasma membrane and released predominately by the calcium-dependent enzyme phospholipase A2 (PLA2), but also by phospholipase C and phospholipase D. Free AA has different fates: it can interact with and regulate several target proteins such as ion channels and enzymes, it may diffuse outside the cell, be incorporated into the plasma membrane or further metabolised [161]. Prostaglandins and tromboxanes are formed by the enzyme cyclooxygenase (COX) while leukotriens and 5-HETE are formed by 5-lipoxygense (5-LOX) via 5-hydroperoxyeicosatetraenoic (5-HPETE). 5-LOX binding protein (FLAP) is involved in the 5-LOX pathway by presenting AA to 5-LOX, thereby enabling this enzyme to efficiently produce oxidised lipid products. AA can also be metabolised by 12- and 15-lipoxygenase (12- and 15-LOX) to 12- and 15hydroxyeicosatetraenoic hydroperoxyeicosatetraenoic
acid
(12acid
and
15-HETE)
(12-HPETE)
-
via and
1215-
hydroperoxyeicosatetraenoic acid (15-HPETE) (Figure 6). All of these eicosanoids may trigger a variety of receptor mediated effects [160, 161]. 21
30
PLA2
COX Prostaglandins and Tromboxanes
Arachidonic acid 5-LOX
15-LOX
12-LOX
15-HPETE
FLAP 12-HPETE 5-HPETE
15-HETE
12-HETE
5-HETE LTA4 LTA4 hydrolase
LTE4
LTC4 synthase LTC4
LTB4
LTD4
Figure 6. Metabolism of arachidonic acid (AA) through COX and LOX pathways. Prostaglandins and tromboxanes are formed by cyclooxygenase (COX) while leukotriens and 5-HETE are products of 5-lipoxygenase (5-LOX). AA can also be metabolised by 12- or 15-LOX to 12- and 15- hydroxyeicosatetraenoic (12 and 15HETE).
Enzymes that are needed in the AA metabolism must not necessarily be present in the same cell [72]. Neutrophils and platelets have been seen in close proximity and thereby regulate e.g. the metabolism of eicosanoids in both platelets and neutrophils [162-164]. Platelets do not possess 5-LOX and consequently do not generate leukotrienes on their own. In spite of this, platelets are able to release leukotrienes C4, D4 and E4 [83, 85, 86]. This is mediated through transcellular metabolism of eicosanoids, a phenomenon where eicosanoid intermediates diffuse between interacting cells and give rise to eicosanoid metabolites normally not formed in either of the cells alone [72]. It is 22
31
now established that a transcellular eicosanoid metabolism takes place between different cell types in close proximity [165-167]. In addition, PMP have been shown to contain bioactive lipids such as AA that is delivered to different cells, e.g. platelets and endothelial cells, resulting in a metabolisation of AA to other more active lipid metabolites, e.g. TXA2 [168]. It has also been shown that PMP are able to metabolise AA delivered from endothelial cells to TXB2, the stable metabolite of TXA2, indicating that PMP also possess enzymatic activity [169]. Many different cells use AA metabolites in their signal transduction and products from both the COX and LOX pathway are shown to be important in cell spreading, migration and proliferation [170-175]. The type of metabolite and its concentration decide the specific response. LOX metabolites have been shown to display mitogenic activities on endothelial cells, while COX products are considered to be predominantly involved in the stimulation of migration [161]. Some studies have indicated that 12-HETE is a growth-promoting factor and that it facilitates proliferation in fibroblasts [176]. Leukotrienes play a central role in asthma by affecting bronchoconstriction [177-179], inflammation [180, 181] and airway remodeling [182-184]. A number of studies have demonstrated a role for 5-LOX metabolites in cell proliferation, although the effect of the enzyme seems to be cell type specific [185]. Metabolites from the 5-LOX pathway facilitate cell proliferation in glioma cell lines while they have no effect on endothelial cell proliferation [174, 175].
23
32
Reactive oxygen species Reactive oxygen species (ROS) are generated following ligand-receptor interactions, by several sources, including mitochondria and the plasma membrane NADPH oxidase. However, ROS might also be produced by xanthine oxidase and metabolism of AA through COX and LOX [186]. In addition, AA has been shown to affect NADPH-oxidase and thereby induce ROS production [187, 188]. The intracellular redox state is balanced by the ROS production and the antioxidant capacity of the cell based on a variety of antioxidant enzymes, such a superoxide dismutase which reduces superoxide anion to hydrogen peroxide and catalase and gluthatione peroxidase which reduce hydrogen peroxide to water [189]. Phagocytic leukocytes have been considered to play a central role in the innate immunity and one important component is their ability to generate ROS via the membrane-associated NADPH oxidase. Upon activation, this multi-component enzyme uses electrons derived from intracellular NADPH to generate superoxide anion, which is reduced to hydrogen peroxide. These radicals are then used in the host defence against bacterial and fungal pathogens. Furthermore, an extracellular release of ROS may injure the surrounding tissue and cause cell death. However ROS have also been identified as important chemical mediators in the regulation of signal transduction involved in cell growth [190], differentiation [189] and adhesion [191, 192]. External addition of low concentrations of hydrogen peroxide and/or superoxide anion has been shown to stimulate cell proliferation in a variety of cells e.g. smooth muscle cells, fibroblasts and epithelial cells [193-197]. ROS fulfil the role as intracellular second messengers since they are rapidly generated, highly diffusible, easily degraded and present in all cell types [189].
24
33
The leukocytes NADPH oxidase complex consists of four major units: cytochrome b558 (composed of the subunits gp91-phox and p22-phox), p40phox, p47-phox and p67-phox (Figure 7).
O2
Cytochrome b558
gp91
p22
Rap 1A
gp91
Rac 1/2
Rac 1/2
H+
Cytochrome b558
p22
Rap 1A
O2.-
p67 p47
p67 p47
NADPH
p40
NADP+
p40
Resting
Activated
Figure 7. Activation of the leukocyte NADPH-oxidase. In the resting cell, the components p40-phox, p47-phox and p67-phox are located in the cytosol as a complex, while cytochrome b558 is located in membranes. Expose to stimuli results in phosphorylation of p47-phox and movement of the entire cytosolic complex to the membrane, where it associates with cytochrome b558.The activated NADPH oxidase transfers electrons from the substrate NADPH to oxygen. Activation of NADPH oxidase also requires the participation of two small GTP-binding proteins, Rac2 (in some cells Rac1) which is located in the cytoplasm in the resting cell, and Rap1A located in the membrane.
An enzyme with NADPH/NADH activity has also been shown to be present in variety of non-phagocytic cells e.g. smooth muscle cells, epithelial cells, endothelial cells and cancer cells. The oxidase in non-phagocytic cells appears to share some of the components of phagocytic NADPH oxidase, including p2225
34
phox, p47-phox, and p67-phox and in some cases gp-91-phox (or related homologue). But there are some differences, e.g. it takes longer time for the non-phagocytic oxidase to get activated, non-phagocytic oxidases give rise to lower output of ROS and non-phagocytic oxidases prefers NADH rather than NADPH as substrate. The regulation of the non-phagocytic NADPH oxidase appears to be comparable with the signal pathways involved in phagocytic cells [189]. ROS have been shown to regulate growth factor and contractile agent-induced cell proliferation [198, 199]. The downstream effectors are however unknown, but some antioxidants block the activation of the transcription nuclear factor NFκB, suggesting that NFκB is a potential target of ROS generated from NADPH-oxidase [198]. The addition of extracellular ROS has been shown to activate some MAPK pathways, e.g. ERK, c-jun N-terminal kinase (JNK) and p38 MAPK pathways, and PI3K in a variety of celltypes [200]. Although the signal transduction pathways of MAPK are regulated by ROS, the respective tyrosine kinase linked receptors are not necessarily the direct targets of ROS. For example, p21ras is a target of ROS and may be responsible for sensing the intracellular redox status [189]. Furthermore, ROS have also been found to activate several non receptor protein tyrsosine kinases, e.g. Src and FAK, and thereby affect processes such as cell migration and proliferation [200, 201].
26
35
27
36
AIMS Platelets have been suggested to play a part in asthma, an airway disease where a chronic inflammation cause structural changes called airway remodeling. However, mechanisms regarding the role of platelets in airway remodeling are far from understood.
The specific aims of the enclosed studies were therefore to investigate the role of: - 5-lipoxygenase
in
platelet-induced
airway
smooth
muscle
cells
(ASMC)/fibroblast proliferation (paper I-III). - reactive oxygen species (ROS) in platelet-induced ASMC proliferation (paper II). - various platelet fragments in the proliferation of ASMC (paper III). - hyaluronic acid (HA), the HA binding receptor CD44 and focal adhesion kinase (FAK) in the platelet/ASMC interaction and in platelet-induced ASMC proliferation (paper IV).
28
37
29
38
METHODS For further experimental details and source of chemicals and buffers, see material and method sections in Paper I-IV, respectively.
Cell culture Media Starvation medium (DMEM, 1mM sodium pyruvate, 1% non-essential amino acids, 100 U/ml penicillin, and 100 µg/ml streptomycin); Complete medium (starvation medium with 10 % foetal bovine serum). Airway smooth muscle cells and fibroblasts Fibroblasts (Paper I) were bought from NIA Aging cell culture repository (Camden, NJ, USA) and airway smooth muscle cells (ASMC) obtained from guinea pigs (denoted ASMC in paper II and GP-ASMC in Paper III) were isolated using explant technique, approved in advance by the ethical review committee on animal experiments (Linköping, Sweden, Dnr 41-03). ASMC obtained from humans (denoted H-ASMC in paper III and ASMC in paper IV) where bought from Promocell (Heidelberg, Germany). Both ASMC and fibroblasts were cultured in complete medium in a humidified atmosphere at 37°C and 5% CO2. Confluent cells were detached from the cell culture flask using trypsin and subcultivated once a week.
Preparation of platelets, platelet membranes, platelet lysate and supernatant Platelets were isolated according to a modified method of Bengtsson and Grenegård 1994 [202]. Only plastic utensils were used in the preparation 30
39
procedure and all work was performed at room temperature to avoid activation of the platelets. In short, five parts of the blood were mixed with one part of ACD solution and centrifuged for 20 minutes at 220 x g. The platelet-rich plasma obtained in the upper layer were removed and centrifuged for 20 minutes at 480 x g. The platelet pellet was gently washed and resuspended in KRG without calcium, and the platelets were counted in a Bürkner chamber. This platelet suspension was used to prepare lysate, supernatant from platelet lysate and platelet membranes in paper III. Lysate of platelets where prepared using a Branson sonifier cell disrupter B15 (Branson sonic Power company, Danbury, CT, USA) for 3 x 15 seconds. The disrupted platelets where then put in the -70 °C freezer followed by two cycles of thawing, vortexing and freezing. The supernatant was prepared by centrifugation of the cell lysate at 200 000 x g for 30 minutes. Platelet membranes were prepared according to Regan and Matsui 1990 [203] by centrifugation of the platelet suspension for 30 minutes at 2 800 x g at + 4˚C. The pellet was resuspended in lysis buffer and homogenised, and then centrifuged for 45 minutes at 29 500 x g at +4˚C. The lysis-homogenisation procedure was repeated three times, and the resulting platelet membrane fraction was resuspended in calcium-free KRG. The platelet membranes where characterised upon size, density and expression of the platelet specific structures GpIb (CD42b) and GpIIb (CD41) using flow cytometry (Coulter Epics XLMCL, Beckman Coulter, Miami, FL, USA).
31
40
Cell proliferation The CellTiter96®Aqueous One Solution Cell Proliferation Assay Proliferation was measured using the CellTiter96®Aqueous One Solution Cell Proliferation Assay (MTS-assay, Promega, Madison, WI, USA) in Paper I-IV. The MTS-assay contains a compound [3-(4, 5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] (MTS) which is reduced by metabolically active cells into a coloured formazan product. This conversion is presumably accomplished by NADPH or NADH produced by dehydrogenases. The quantity of formazan product measured as 490 nm absorbance is directly proportional to the number of living cells in culture [204]. Briefly, ASMC or fibroblasts seeded into 96-wellplates were incubated with or without platelets and inhibitors for 24 hours. After the incubation new medium and the MTS reagent was added and the amount of viable cells was measured spectrophotometrically at 490 nm using a microplate reader (Spectra MAX, Molecular Devices, Sunnyvale, CA, USA). 3
H-thymidine incorporation
Proliferation was also analysed by measuring 3H-thymidine incorporated into DNA in proliferating cells. Fibroblasts (Paper I) or ASMC (Paper II) seeded into 96-wellplates were incubated with or without platelets and inhibitors together with 2 µCi/ml [3methyl-3H]-thymidine for 24 hours. Thereafter the cells were washed with PBS pH 7.4 and treated with trichloroacetic acid for 30 min at 4 °C. Liquid scintillation cocktail was then added to the 96-wellplate and after 1 hour the amount of thymidine incorporated DNA was counted using a scintillation counter (1450 microbeta Trilux Wallac, Eg & G® Lifescience, Turku, Finland). 32
41
Cell counting and DNA-measurements Proliferation of fibroblasts (Paper I) and ASMC (Paper II) was also analysed by counting the cells. Trypsin detached fibroblasts were counted using a Coulter Channelyser 256 (Coulter Electronics Ltd., England) and ASMC using a Bürkner chamber. Alternatively, proliferation of ASMC was analysed by measuring DNA (Paper II and III) using a NanoDrop ND-1000 UV-visible light spectrophotometer (Saveen Werner AB, Malmö, Sweden).
Microscopic examination of platelet-ASMC/fibroblast interaction The interaction between fibroblasts/platelets and ASMC/platelets was studied morphologically in Paper I-IV by fluorescent staining in combination with fluorescence microscopy. Briefly, ASMC or fibroblasts seeded on coverslips (Paper I-II) and ASMC into 8-well chamber slides (Paper III-IV) were incubated with or without platelets and inhibitors for various periods of times. After stimulation, the cells were fixed with paraformaldehyde (PFA) followed by fluorescent staining and visualisation using a fluorescent microscope (Carl Zeiss, Oberkochen, Germany).
Western blot of focal adhesion kinase and 5-lipoxygenase To study FAK in paper IV ASMC were seeded in 6 well plates and incubated with or without platelets and inhibitors for 1h. To study 5-LOX fibroblasts (Paper I) and ASMC (Paper II) were seeded in petri dishes and stimulated with platelets for 2h. After stimulation, the samples were washed, lysed using a lysis buffer and in paper IV FAK were thereafter immunoprecipetated using a FAK specific antibody. Lysed and denaturated homogenates containing equal amounts of protein (Paper I) or DNA (paper II and IV) were thereafter separated on a 7.5% SDS-PAGE (Paper I-II) or a 3-8% Tris-acetate gel (Paper IV). The 33
42
proteins were transferred to a nitrocellulose membrane (Paper I) or a PVDF membrane (Paper II and IV) and blocked using 3% BSA (Paper I) or 5% (w/v) dry milk (Paper II and IV). Proteins of interest were detected using specific antibodies and horseradish peroxidase-conjugated secondary antibodies and visualised by chemiluminescence. Reverse transcriptase-mediated PCR RNA, isolated from platelets, neutrophils and fibroblasts using Trizol, was in paper I converted to cDNA and amplified using reverse-trancriptase PCR (RTPCR). Briefly, 5 µg RNA were mixed with 1 µl of anchored oligo(dT)20 primer and incubated for 10 min at 70 °C and then kept on ice. Thereafter 1 µl of a reaction cocktail containing 10 mM dNTP mix, buffer, 2 µl 0.1 M DTT and 200 U/ml SuperScript™ II RNase H- was added and the mixture was incubated for another 20 min. 1 µl RNase H was added and the mixture was incubated for another 20 min. The PCR reaction was initiated; denaturation at 94 °C for 5 min, 35 cycles of denaturation at 94 °C for 45 sec, annealing at 62.2 °C for 30 sec, extension at 72 °C for 45 sec followed by extension at 72 °C for 7 min. RNA content was analysed by measuring absorbance at 260 nm and samples containing the same amount of RNA was analysed on a 1.2% agarose gel. Solid phase extraction and HPLC In paper I 5-LOX activity was analysed by measuring 5-HETE formation using isocratic reverse-phase high performance liquid chromatography (HPLC). In short, fibroblasts were incubated with or without platelets and inhibitors for 0.5, 1, 2 or 20h and lysed using water. The samples were thereafter equilibrated prior solid phase extraction using EDTA and isopropyl alcohol followed by centrifugation for 10 min at 2000 x g. The supernatant of the centrifuged samples was added to the extraction columns and solid phase extraction was 34
43
performed using a modified method of Kiss et al. 1998 [205]. Samples were eluted using methanol, dried under a stream of nitrogen and stored in -20 °C before HPLC analysis. Isocratic reverse-phase HPLC was performed on a Nucleosil 100 C18 column at a flow rate of 1 ml/min and the mobile phase consisted of methanol-water-acetic acid (75:25:0.01 by volume). Measurement of intracellular reactive oxygen species In paper II the intracellular redox state was registered using the fluorescent dye 2´,7´-dichlorodihydrofluoroscein diacetate (DCFDA; Molecular probes, Eugene, OR, USA). When applied to intact cells, the nonionic, nonpolar DCFDA crosses cell membranes and is hydrolysed enzymatically by intracellular esterases to nonfluorescent DCFH. In the presence of ROS, DCFH is oxidised to the highly fluorescent dichlorofluorescein (DCF) and the intracellular DCF fluorescence can then be used as an index to quantify the overall ROS-production [206]. Briefly, ASMC seeded into 96-wellplates were incubated with or without platelets and inhibitors for 3 hours followed by incubation for 45 min with 5µM DCFDA. To evaluate the ROS production, the fluorescence was measured using a microplate reader (FL-600 Microplate fluorescence reader, Bio-Tek instruments Inc, Vermont, USA). The excitation filter was set at 485 ± 10 nm and the emission filter was set at 530 ± 12.5 nm. ROS was also generated in a cell free suspension where drugs used in the papers were tested for scavenger effects. ROS generation was analysed in the presence of 0.02 U/ml xanthine oxidase and 0.03 mM hypoxanthine or 1.5M CuSO4 and 0.3% H2O2 using horseradish peroxidase-enhanced chemiluminescence (4 U/ml horseradish peroxidase, 112 uM luminol).
35
44
Statistical analysis Results from paper I-IV are expressed as mean values ± standard deviation (SD) or ± standard error of the mean (S.E.M) as indicated. Statistical difference between groups were calculated using One-way ANOVA followed by Dunnet´s multiple comparison test or Student t-test. A p-value < 0.05 was considered to be significant, and significance is denoted * (p < 0.05),
**
(p < 0.01) and
***
(p
0.05
p < 0.05*
p < 0.05*
AA-861 (5-LOX inhibitor)
n.d
p < 0.05
p < 0.05
ATK (PLA2-inhibitor)
n.d
p < 0.05
p < 0.05
Significant inhibition is denoted p < 0.05 while p > 0.05 indicates no significant inhibition, n.d denotes not determined and * indicates unpublished data. For further details, see results in paper I and II, respectively.
We found that the PLA2 inhibitor DMDA, the combined COX and LOX inhibitor ETYA, the LOX inhibitor ETI and the 5-LOX inhibitor 5,6-dAA all inhibited platelet-induced fibroblast proliferation (Table 3). DMDA, ETI and ETYA also reduced 5-HETE production measured using HPLC (Figure 4 C, Paper I). The results also demonstrated that the PLA2 inhibitor ATK and the 5LOX
inhibitor
AA-861
significantly
reduced
platelet-induced
ASMC
proliferation (Table 3). In addition, platelet membrane-induced ASMC proliferation was found to be 5-LOX dependent due to the antagonising effect of AA-861 (Figure 5A, paper III). ATK also reduced platelet membrane-induced 49
58
ASMC proliferation, although not as effective as compared to its effect on platelet-induced ASMC proliferation (Figure 5B, paper III). These results indicate that other enzymes, e.g. phospholipase C or D might be involved in the proliferative effect of platelet membrane structures. A possible mechanism in the regulation of cell proliferation is that platelet membranes serve as a lipid reservoir for biosynthesis of mitogenic lipids. PMP contain AA that is delivered to cells in close proximity, for example endothelial cells, and through transcellular metabolism generates other more active metabolites e.g. thromboxane A2 [168]. Platelet membranes could also interact with surface structures expressed on ASMC and thereby activate 5-LOX. Since the 5-LOX pathway of AA metabolism appears to be important for platelets ability to induce proliferation, we examined whether 5-LOX metabolites could mimic the mitogenic effect of platelets on fibroblasts (Paper I) or ASMC (Paper II). However, neither 5-HETE (paper I), LTB4 (Paper I-II) nor LTC4 (Paper II) was able to induce cell proliferation indicating that 5-LOX metabolites do not exert mitogenic effects by themselves. This hypothesis is supported by studies indicating that cysteinyl leukotrienes need to act in synergy with other factors to be able to stimulate proliferation [213-215]. We also found in paper I that the 12-LOX inhibitor CDC does not affect platelet-induced fibroblast proliferation, while unpublished data indicates that CDC and Baicalein, another 12-LOX inhibitor, block platelets ability to induce ASMC proliferation. The role of 12-LOX in platelet-induced ASMC proliferation is an interesting area for future research. However, the studies on 5LOX expression and the role of 12-LOX indicate that the platelet-induced fibroblast and ASMC proliferation, dependent on eicosanoids, is not mediated through the same signaling pathways. In summary, results obtained in paper I-III 50
59
show that eicosanoids, especially 5-LOX metabolites, are essential mediators in platelet-induced proliferation of fibroblasts and ASMC. Platelet-induced ASMC proliferation is dependent on reactive oxygen species Reactive oxygen species (ROS) are generated following ligand-receptor interactions by several sources, including mitochondria, xanthine oxidase and NADPH oxidase and has been shown to be involved in cell proliferation [189, 190]. Therefore, we were interested in paper II to investigate the impact of ROS in platelet-induced ASMC proliferation. The ROS production, measured using the fluorescent probe DCFDA, significantly increased in a ratio-dependent manner after coincubating platelets and ASMC for 3h (Figure 5, paper II). Two different NADPH-oxidase inhibitors, diphenyleneiodonium chloride (DPI) and apocynin, were used to investigate the role of NAPH-oxidase dependent ROS production in plateletinduced ASMC proliferation.
51
60
Table 4. The effect of NADPH-oxidase inhibitors on platelet-induced ASMC proliferation. Treatment
ASMC proliferation
ASMC+platelets
p-value
164.0 ± 7.70
ASMC+platelets + DPI 1 µM
108.6 ± 9.30
p < 0.01 (**)
ASMC+platelets +
123.1 ± 11.1
p < 0.01 (**)
Apocynin 1 µM
ASMC were coincubated with platelets (platelets/ASMC ratio 1/1000) for 24h in the presence or absence of DPI (n = 6) or apocynin (n = 4). Proliferation was measured using the MTS-assay. Data are expressed as mean ± SEM and related to unstimulated ASMC (% of control). Paired observations were compared and statistical analysed using One-way ANOVA followed by Dunnet´s multiple comparison test. Different concentrations of the inhibitors have been tested; see results in paper II for further details.
Our results demonstrate that both DPI and apocynin inhibit platelet-induced ASMC proliferation measured using the MTS-assay (Table 3). Thymidine incorporation revealed the same tendency (Figure 6, paper II). Scavengers such as PDTC, NAC and Vitamin D also inhibited platelet-induced ASMC proliferation (unpublished data). However, they also affected basal ASMC proliferation and were therefore not included in paper II. Platelet membraneinduced ASMC proliferation measured using the MTS-assay was also inhibited by DPI (Figure 6, paper III) and these results taken together with the previously described suggest that ROS, produced by NADPH-oxidase expressed either in platelets or ASMC or both cell types, is important for the mitogenic effect of platelets on ASMC proliferation.
52
61
Thymidine incorporation was included as an alternative method since scavengers have been shown to interact with NADH and NADPH, which are involved in the conversion of the MTS substrate in the MTS-assay. However, neither DPI nor apocynin exert scavenging effects measured using horseradish peroxidase-enhanced luminescence, indicating that the effect of DPI and apocynin on platelet-induced ASMC proliferation measured with the MTS-assay is correct. Mechanisms involved in ROS-dependent ASMC proliferation is not fully investigated. Interestingly, low molecular weight HA (LMW-HA) have been shown to be produced through oxidative hydrolysis by ROS of high molecular weight (HMW-HA) [132, 133, 216]. In addition, platelets possess HYAL-2 that, in cooperation with CD44, has the ability to cleave HA into small proproliferative fragments [132, 134, 217, 218]. Interestingly, we found in paper II that platelet/ASMC interaction results in increased ROS-production. The mechanism by which HA affects ASMC proliferation is unknown but one hypothesis based on paper II and IV suggests that platelet binding to HA results in degradation of HA (through HYAL-2 and/or ROS) leading to formation of pro-proliferative fragments. Moreover, studies have shown that there might be a signaling cross-talk between eicosanoids and ROS, e.g. COX and LOX produce superoxide anion during a side reaction that depends on reducing co-substrates [219]. In addition, inhibition of 5-LOX blocks the Rac-1 dependent ROS-generation [220] and a new report suggests that 5-LOX dependent ROS-generation is crucial for fibroblast adhesion [192]. Consequently, we measured the ROS production in coincubating platelets and ASMC in the presence of ATK and AA-861 to investigate a potential relationship between ROS and eicosanoids. Results 53
62
demonstrate that both ATK and AA-861 significantly inhibited the increased ROS production generated after platelet/ASMC interaction (Table 3). In summary, our data suggest that 5-LOX is activated in co-cultures of platelets and ASMC and that this cell-cell interaction generates a NADPH-oxidase dependent ROS production resulting in increased cell proliferation. However, the relationship between ROS and eicosanoid metabolites in platelet-mediated cell proliferation remains to be determined.
54
63
55
64
SUMMARY AND CONCLUSIONS The results demonstrated in paper I-IV lead to the following conclusions:
Unstimulated platelets
1 Activated platelets
2 FAK
Hyaluronic acid
Proliferation ROS
4
5-LOX
Nucleus
5
3 Platelet membranes
1. Platelet binding to HA is CD44 dependent (paper IV). 2. Platelet binding to HA through CD44 induce phosphorylation of FAK and FAK dependent ASMC proliferation (paper IV). 3. Platelet lysate and platelet membranes induce a significant increased ASMC proliferation (paper III). Platelet supernatant, devoid of soluble factors released from platelets, also induces ASMC proliferation, although not to the same extent (paper III). 4. Platelet-induced ASMC/fibroblast and platelet membrane-induced ASMC proliferation is 5-LOX dependent (paper I-II).
56
65
5. The platelet/ASMC interaction results in increased NADPH-oxidase and 5LOX dependent ROS production associated with an increased ASMC proliferation (paper II). In addition, platelet membrane-induced ASMC proliferation is 5-LOX and NADPH-oxidase dependent (paper III). In summary, the results obtained in paper I-IV suggest that platelet/HA interaction mediated through CD44 is of importance for platelets ability to induce cell proliferation. Furthermore, intracellular signaling associated with FAK is also dependent on a CD44-mediated platelet/HA interaction. Moreover, 5-LOX dependent ROS production is essential for the mitogenic effects of platelets. This action of platelets represents a potential important and novel mechanism that may have an impact on the remodeling process and in the development of new pharmacological strategies in the treatment of inflammatory respiratory disease such as asthma.
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TACK Det finns många personer som har betytt mycket för mig under min tid som doktorand och jag vill här passa på att tacka några av dem. Tack alla arbetskamrater, speciellt tack till: Min huvudhandledare Torbjörn Bengtsson, för att du introducerade mig till forskningen och tog dig an mig som doktorand. Tack också för alla goda råd som jag har fått under utbildningen. Min biträdande handledare, Eva Lindström, för ditt engagemang under hela min forskarutbildning. Förutom handledare-doktorand relationen har vi också delat kontor under hela min tid på farmakologen. En plats där vi har delat mycket glädje men även problem och jag vill därför passa på att tacka dig lite extra för all hjälp och alla råd jag har fått. Även ett stort tack för att du fick oss att leta bostad i Vidingsjö, lätt bästa delen av stan =) Ämnesföreträdaren
i
farmakologi,
min
trogna
medförfattare
Magnus
Grenegård, för ditt stora forskningsengagemang och speciellt för din delaktighet i delarbete II-IV där du har varit en ovärderlig inspirations och kunskapskälla. Helena Herbertsson för hjälp med HPLC analyserna i arbete I, Mats Söderström för hjälpen med RT-PCR och Western blot analyserna i arbete I och Sofia Ramström för hjälpen med flödescytometri analyserna i arbete III. Anita Thunberg, farmakologens hjärta. Utan dig vore avdelningen ingenting. 58
67
Mina forna och nuvarande doktorandkollegor: Kristin Engström, Therese Eriksson, Hanna Björk, Andreas Eriksson, Anna Asplund-Persson, Peter Garvin, Louise Levander, Karin Vretenbrandt Öberg, Johanna Lönn och Martina Nylander. Jag vill skänka ett speciellt tack till: Louise Karlsson, tack för allt under alla år vi har känt varandra. Tack för att du får mig att tänka på annat än jobb och familj. Anna Jönsson, min goda vän, jag är så glad att du har flyttat tillbaka till Linköping. Efter nyår behöver jag din hjälp med att komma igång med träningen …… igen =) Caroline Skoglund och Hanna Kälvegren, mina ständiga vapendragare. Tack för allt stöd och all hjälp. Ida Bergström, för att du är en sådan härligt positiv människa. Simon Jönsson, en enastående kost och träningscoach =) Liza Ljungberg, för allt stöd och din positiva inställning. Alla studenter och stipendiater som jag har haft glädjen att undervisa och handleda under doktorandtiden Forum Scintium och speciellt Stefan Klintström. 59
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Ett stort tack till mina goda vänner utöver arbetsplatsen som har fått mig att orka med avhandlingsarbetet, speciellt tack till: Min ”bästis” Nettan Karlsson, mer behöver jag egentligen inte säga =) Även ett stort tack till Niklas, Hanna och Tova. Bea Zechel, tack för alla mysiga promenader och utflykter vi har gjort tillsammans med våra små hulliganer. Tack för ditt positiva tänkande och för din goda vänskap. Tack också Erik, Hedvig och Svea. Jag vill också passa på att tacka min familj: Jag vill först tacka familjen på Andreas sida som består av min svärfar Anders, min svärmor Inga-Lill och sambon Calle, min svägerska Camilla och hennes man Magnus samt deras underbara döttrar Fannie och Filippa och min svåger Christian. Tack också Ann-Margrete, du är saknad. Tack för att ni har tagit in mig som en del i er familj, jag tycker ofantligt mycket om er allihop. Jag vill därefter tacka min familj som består av min mamma Lillemor och mina bröder Niklas och Peter. Tack världens bästa mamma för att du just är världens bästa mamma, utan dig hade jag aldrig orkat hela vägen. Tack för att hjälp med barnpassning, matlagning m.m. under det intensiva skrivarbetet i slutet av forskarutbildningen. Tack Niklas för att du alltid är så positiv. Tack Peter för att du alltid har engagerat och stöttat mig i allt jag har tagit för mig. Tack också Jenny, Lotten, Hedda och Sander. Jag vill också passa på att tacka min pappa Ronny som hastigt avled hösten 1996. Även om han inte finns i livet längre så är minnet av honom ett stort stöd och en viktig inspirationskälla. Tack också 60
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mormor som tyvärr inte heller finns med oss längre; jag hoppas att jag har ärvt din envishet. Slutligen, Andreas och Alva, min fina underbara lilla familj. Andreas, min älskade make och stora trygghet i livet. Tack för all kärlek och allt stöd. Det senaste året har inte varit det lättaste, men vi har snart tagit oss igenom det. Alva, min lilla skrutta, som med kärlek och omtanke får mig prioritera det viktigaste i livet d.v.s. familjen. Jag har tillägnat min avhandling till dig för att du är det absolut finaste och mest värdefulla jag har i mitt liv.
Finansiärerna av denna avhandling är: Strategiområdet Cardiovascular Research Inflammation Centre (CIRC) och Materials in Medicine (MiM) Vetenskapsrådet Hjärt- Lungfonden Hjärt och Lungsjukas Riksförbund Landstinget i Östergötland Fonden för forskning utan djurförsök Lions forskningsfond mot folksjukdomar Stiftelserna Eleonora Demoroutis, Krister Axelssons minnefond, Anna Cederbergs stiftelse för medicinsk forskning, Ssk Siv Olsson forskningsstiftelse, och US stiftelse för medicinsk forskning.
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REFERENCES 1.
2. 3. 4.
5. 6. 7.
8. 9.
10. 11.
12.
13.
Chung KF. Asthma treatments: effects on airway smooth muscle, In: Chung KF, Editor. Airway Smooth Muscle in Asthma and COPD Biology and pharmacology, Chichester: John Wiley & Sons Ltd;2008. pp 277-302. Hui KP, Barnes NC. Lung function improvement in asthma with a cysteinyl-leukotriene receptor antagonist. Lancet 1991 337(8749); 1062-3. Fan Chung K. Phosphodiesterase inhibitors in airways disease. Eur J Pharmacol 2006 533(1-3); 110-7. Tang ML, Wilson JW, Stewart AG, Royce SG. Airway remodelling in asthma: current understanding and implications for future therapies. Pharmacol Ther 2006 112(2); 474-88. Mauad T, Bel EH, Sterk PJ. Asthma therapy and airway remodeling. J Allergy Clin Immunol 2007 120(5); 997-1009; quiz 1010-1. Halwani R, Al-Muhsen S, Hamid Q. Airway remodeling in asthma. Curr Opin Pharmacol 2010 10(3); 236-45. Vignola AM, Mirabella F, Costanzo G, Di Giorgi R, Gjomarkaj M, Bellia V, Bonsignore G. Airway remodeling in asthma. Chest 2003 123(3 Suppl); 417S-22S. Davies DE. The role of the epithelium in airway remodeling in asthma. Proc Am Thorac Soc 2009 6(8); 678-82. Vermeer PD, Harson R, Einwalter LA, Moninger T, Zabner J. Interleukin-9 induces goblet cell hyperplasia during repair of human airway epithelia. Am J Respir Cell Mol Biol 2003 28(3); 286-95. Dunnill MS. The pathology of asthma, with special reference to changes in the bronchial mucosa. J Clin Pathol 1960 13; 27-33. Dunnill MS, Massarella GR, Anderson JA. A comparison of the quantitative anatomy of the bronchi in normal subjects, in status asthmaticus, in chronic bronchitis, and in emphysema. Thorax 1969 24(2); 176-9. Orsida BE, Ward C, Li X, Bish R, Wilson JW, Thien F, Walters EH. Effect of a long-acting beta2-agonist over three months on airway wall vascular remodeling in asthma. Am J Respir Crit Care Med 2001 164(1); 117-21. Li X, Wilson JW. Increased vascularity of the bronchial mucosa in mild asthma. Am J Respir Crit Care Med 1997 156(1); 229-33.
62
71
14.
15.
16.
17.
18.
19.
20. 21.
22.
23.
24.
25.
McDonald DM. Angiogenesis and remodeling of airway vasculature in chronic inflammation. Am J Respir Crit Care Med 2001 164(10 Pt 2); S3945. Makinde T, Murphy RF, Agrawal DK. Immunomodulatory role of vascular endothelial growth factor and angiopoietin-1 in airway remodeling. Curr Mol Med 2006 6(8); 831-41. Lal BK, Varma S, Pappas PJ, Hobson RW, 2nd, Duran WN. VEGF increases permeability of the endothelial cell monolayer by activation of PKB/akt, endothelial nitric-oxide synthase, and MAP kinase pathways. Microvasc Res 2001 62(3); 252-62. Pitchford SC, Yano H, Lever R, Riffo-Vasquez Y, Ciferri S, Rose MJ, Giannini S, Momi S, Spina D, O'Connor B, Gresele P, Page CP. Platelets are essential for leukocyte recruitment in allergic inflammation. J Allergy Clin Immunol 2003 112(1); 109-18. Pitchford SC, Momi S, Giannini S, Casali L, Spina D, Page CP, Gresele P. Platelet P-selectin is required for pulmonary eosinophil and lymphocyte recruitment in a murine model of allergic inflammation. Blood 2005 105(5); 2074-81. Pitchford SC, Momi S, Baglioni S, Casali L, Giannini S, Rossi R, Page CP, Gresele P. Allergen induces the migration of platelets to lung tissue in allergic asthma. Am J Respir Crit Care Med 2008 177(6); 604-12. Roche WR, Beasley R, Williams JH, Holgate ST. Subepithelial fibrosis in the bronchi of asthmatics. Lancet 1989 1(8637); 520-4. Payne DN, Rogers AV, Adelroth E, Bandi V, Guntupalli KK, Bush A, Jeffery PK. Early thickening of the reticular basement membrane in children with difficult asthma. Am J Respir Crit Care Med 2003 167(1); 78-82. Davies DE, Wicks J, Powell RM, Puddicombe SM, Holgate ST. Airway remodeling in asthma: new insights. J Allergy Clin Immunol 2003 111(2); 215-25; quiz 226. Hoshino M, Nakamura Y, Sim JJ. Expression of growth factors and remodelling of the airway wall in bronchial asthma. Thorax 1998 53(1); 21-7. Huang J, Olivenstein R, Taha R, Hamid Q, Ludwig M. Enhanced proteoglycan deposition in the airway wall of atopic asthmatics. Am J Respir Crit Care Med 1999 160(2); 725-9. Aceves SS, Broide DH. Airway fibrosis and angiogenesis due to eosinophil trafficking in chronic asthma. Curr Mol Med 2008 8(5); 350-8.
63
72
26.
27.
28.
29.
30.
31.
32. 33.
34.
35.
36.
Hoshino M, Nakamura Y, Sim J, Shimojo J, Isogai S. Bronchial subepithelial fibrosis and expression of matrix metalloproteinase-9 in asthmatic airway inflammation. J Allergy Clin Immunol 1998 102(5); 783-8. Postlethwaite A, Kang A. Fibroblasts and matrix proteins, In: Gallin J and Snyderman R, Editors. Inflammation: Principles and clinical correlates, Philadelphia: Lippincott Williams & Wilkins;1999. pp 227257. Westergren-Thorsson G, Larsen K, Nihlberg K, Andersson-Sjoland A, Hallgren O, Marko-Varga G, Bjermer L. Pathological airway remodelling in inflammation. Clin Respir J 2010 4 Suppl 1; 1-8. Brewster CE, Howarth PH, Djukanovic R, Wilson J, Holgate ST, Roche WR. Myofibroblasts and subepithelial fibrosis in bronchial asthma. Am J Respir Cell Mol Biol 1990 3(5); 507-11. Hirst SJ. Airway smooth muscle cell culture: application to studies of airway wall remodelling and phenotype plasticity in asthma. Eur Respir J 1996 9(4); 808-20. Black JL, Roth M, Lee J, Carlin S, Johnson PR. Mechanisms of airway remodeling. Airway smooth muscle. Am J Respir Crit Care Med 2001 164(10 Pt 2); S63-6. Hirst SJ. Regulation of airway smooth muscle cell immunomodulatory function: role in asthma. Respir Physiol Neurobiol 2003 137(2-3); 309-26. Walker TR, Moore SM, Lawson MF, Panettieri RA, Jr., Chilvers ER. Platelet-derived growth factor-BB and thrombin activate phosphoinositide 3-kinase and protein kinase B: role in mediating airway smooth muscle proliferation. Mol Pharmacol 1998 54(6); 1007-15. Hirst SJ, Barnes PJ, Twort CH. PDGF isoform-induced proliferation and receptor expression in human cultured airway smooth muscle cells. Am J Physiol 1996 270(3 Pt 1); L415-28. Elias JA, Wu Y, Zheng T, Panettieri R. Cytokine- and virus-stimulated airway smooth muscle cells produce IL-11 and other IL-6-type cytokines. Am J Physiol 1997 273(3 Pt 1); L648-55. Ammit AJ, Hoffman RK, Amrani Y, Lazaar AL, Hay DW, Torphy TJ, Penn RB, Panettieri RA, Jr. Tumor necrosis factor-alpha-induced secretion of RANTES and interleukin-6 from human airway smoothmuscle cells. Modulation by cyclic adenosine monophosphate. Am J Respir Cell Mol Biol 2000 23(6); 794-802.
64
73
37.
38.
39.
40.
41.
42.
43. 44. 45. 46.
47.
48.
Hedges JC, Singer CA, Gerthoffer WT. Mitogen-activated protein kinases regulate cytokine gene expression in human airway myocytes. Am J Respir Cell Mol Biol 2000 23(1); 86-94. Huang CD, Tliba O, Panettieri RA, Jr., Amrani Y. Bradykinin induces interleukin-6 production in human airway smooth muscle cells: modulation by Th2 cytokines and dexamethasone. Am J Respir Cell Mol Biol 2003 28(3); 330-8. Lazaar AL, Albelda SM, Pilewski JM, Brennan B, Pure E, Panettieri RA, Jr. T lymphocytes adhere to airway smooth muscle cells via integrins and CD44 and induce smooth muscle cell DNA synthesis. J Exp Med 1994 180(3); 807-16. Lazaar AL, Amrani Y, Hsu J, Panettieri RA, Jr., Fanslow WC, Albelda SM, Pure E. CD40-mediated signal transduction in human airway smooth muscle. J Immunol 1998 161(6); 3120-7. Johnson PR, Black JL, Carlin S, Ge Q, Underwood PA. The production of extracellular matrix proteins by human passively sensitized airway smooth-muscle cells in culture: the effect of beclomethasone. Am J Respir Crit Care Med 2000 162(6); 2145-51. Halayko AJ, Tran T, Gosens R. Phenotype and functional plasticity of airway smooth muscle: role of caveolae and caveolins. Proc Am Thorac Soc 2008 5(1); 80-8. Halayko AJ, Salari H, Ma X, Stephens NL. Markers of airway smooth muscle cell phenotype. Am J Physiol 1996 270(6 Pt 1); L1040-51. Owens GK. Regulation of differentiation of vascular smooth muscle cells. Physiol Rev 1995 75(3); 487-517. Chamley-Campbell J, Campbell GR, Ross R. The smooth muscle cell in culture. Physiol Rev 1979 59(1); 1-61. Mitchell RW, Halayko AJ, Kahraman S, Solway J, Wylam ME. Selective restoration of calcium coupling to muscarinic M(3) receptors in contractile cultured airway myocytes. Am J Physiol Lung Cell Mol Physiol 2000 278(5); L1091-100. Halayko AJ, Camoretti-Mercado B, Forsythe SM, Vieira JE, Mitchell RW, Wylam ME, Hershenson MB, Solway J. Divergent differentiation paths in airway smooth muscle culture: induction of functionally contractile myocytes. Am J Physiol 1999 276(1 Pt 1); L197-206. Cho SH, Seo JY, Choi DC, Yoon HJ, Cho YJ, Min KU, Lee GK, Seo JW, Kim YY. Pathological changes according to the severity of asthma. Clin Exp Allergy 1996 26(10); 1210-9.
65
74
49. 50.
51.
52.
53.
54. 55. 56.
57.
58.
59.
60.
Beckett PA, Howarth PH. Pharmacotherapy and airway remodelling in asthma? Thorax 2003 58(2); 163-74. Ebina M, Yaegashi H, Takahashi T, Motomiya M, Tanemura M. Distribution of smooth muscles along the bronchial tree. A morphometric study of ordinary autopsy lungs. Am Rev Respir Dis 1990 141(5 Pt 1); 1322-6. Ebina M, Takahashi T, Chiba T, Motomiya M. Cellular hypertrophy and hyperplasia of airway smooth muscles underlying bronchial asthma. A 3-D morphometric study. Am Rev Respir Dis 1993 148(3); 720-6. Woodruff PG, Dolganov GM, Ferrando RE, Donnelly S, Hays SR, Solberg OD, Carter R, Wong HH, Cadbury PS, Fahy JV. Hyperplasia of smooth muscle in mild to moderate asthma without changes in cell size or gene expression. Am J Respir Crit Care Med 2004 169(9); 1001-6. James AL, Bai TR, Mauad T, Abramson MJ, Dolhnikoff M, McKay KO, Maxwell PS, Elliot JG, Green FH. Airway smooth muscle thickness in asthma is related to severity but not duration of asthma. Eur Respir J 2009 34(5); 1040-5. Huber H, Koessler K. The pathology of bronchial asthma. Arch Intern Med 1922 30; 689-760. James A. Remodelling of airway smooth muscle in asthma: what sort do you have? Clin Exp Allergy 2005 35(6); 703-7. Burgess JK, Lee JH, Ge Q, Ramsay EE, Poniris MH, Parmentier J, Roth M, Johnson PR, Hunt NH, Black JL, Ammit AJ. Dual ERK and phosphatidylinositol 3-kinase pathways control airway smooth muscle proliferation: differences in asthma. J Cell Physiol 2008 216(3); 673-9. Johnson PR, Roth M, Tamm M, Hughes M, Ge Q, King G, Burgess JK, Black JL. Airway smooth muscle cell proliferation is increased in asthma. Am J Respir Crit Care Med 2001 164(3); 474-7. Hassan M, Jo T, Risse PA, Tolloczko B, Lemiere C, Olivenstein R, Hamid Q, Martin JG. Airway smooth muscle remodeling is a dynamic process in severe long-standing asthma. J Allergy Clin Immunol 2010 125(5); 1037-1045 e3. Benayoun L, Druilhe A, Dombret MC, Aubier M, Pretolani M. Airway structural alterations selectively associated with severe asthma. Am J Respir Crit Care Med 2003 167(10); 1360-8. Tsurikisawa N, Oshikata C, Tsuburai T, Saito H, Sekiya K, Tanimoto H, Takeichi S, Mitomi H, Akiyama K. Bronchial reactivity to histamine is correlated with airway remodeling in adults with moderate to severe asthma. J Asthma 2010 47(8); 841-8.
66
75
61.
62.
63.
64.
65.
66.
67.
68.
69.
70. 71.
72.
Black JL, Johnson PR. What determines asthma phenotype? Is it the interaction between allergy and the smooth muscle? Am J Respir Crit Care Med 2000 161(3 Pt 2); S207-10. Hakonarson H, Maskeri N, Carter C, Grunstein MM. Regulation of TH1and TH2-type cytokine expression and action in atopic asthmatic sensitized airway smooth muscle. J Clin Invest 1999 103(7); 1077-87. Naureckas ET, Ndukwu IM, Halayko AJ, Maxwell C, Hershenson MB, Solway J. Bronchoalveolar lavage fluid from asthmatic subjects is mitogenic for human airway smooth muscle. Am J Respir Crit Care Med 1999 160(6); 2062-6. Ammit AJ, Kane SA, Panettieri RA, Jr. Activation of K-p21ras and Np21ras, but not H-p21ras, is necessary for mitogen-induced human airway smooth-muscle proliferation. Am J Respir Cell Mol Biol 1999 21(6); 719-27. Gosens R, Roscioni SS, Dekkers BG, Pera T, Schmidt M, Schaafsma D, Zaagsma J, Meurs H. Pharmacology of airway smooth muscle proliferation. Eur J Pharmacol 2008 585(2-3); 385-97. Kelleher MD, Abe MK, Chao TS, Jain M, Green JM, Solway J, Rosner MR, Hershenson MB. Role of MAP kinase activation in bovine tracheal smooth muscle mitogenesis. Am J Physiol 1995 268(6 Pt 1); L894-901. Lee JH, Johnson PR, Roth M, Hunt NH, Black JL. ERK activation and mitogenesis in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 2001 280(5); L1019-29. Orsini MJ, Krymskaya VP, Eszterhas AJ, Benovic JL, Panettieri RA, Jr., Penn RB. MAPK superfamily activation in human airway smooth muscle: mitogenesis requires prolonged p42/p44 activation. Am J Physiol 1999 277(3 Pt 1); L479-88. Krymskaya VP, Penn RB, Orsini MJ, Scott PH, Plevin RJ, Walker TR, Eszterhas AJ, Amrani Y, Chilvers ER, Panettieri RA, Jr. Phosphatidylinositol 3-kinase mediates mitogen-induced human airway smooth muscle cell proliferation. Am J Physiol 1999 277(1 Pt 1); L65-78. White J. Platelet structure, In: Michelson A, Editor. Platelets, San Diego: Elsevier;2007. pp 45-73. Italiano J, Hartwig J. Megakaryocyte development and platelet formation, In: Michelson A, Editor. Platelets, San Diego: Elsevier;2007. pp 23-44. Marcus AJ. Platelets: Their role in Hemostatis, Thrombosis, and Inflammation, In: Snyderman JIGaR, Editor. Inflammation: Basic
67
76
73. 74. 75.
76. 77. 78.
79.
80. 81.
82.
83. 84. 85.
86.
Principles and Clinical Correlates, Philadelphia: Lippincott Williams & Wilkins;1999. pp 77-95. Melamed MR, Cliffton EE, Mercer C, Koss LG. The megakaryocyte blood count. Am J Med Sci 1966 252(3); 301-9. Scheinin TM, Koivuniemi AP. Megakaryocytes in the pulmonary circulation. Blood 1963 22; 82-7. Levine RF, Eldor A, Shoff PK, Kirwin S, Tenza D, Cramer EM. Circulating megakaryocytes: delivery of large numbers of intact, mature megakaryocytes to the lungs. Eur J Haematol 1993 51(4); 233-46. Reed G. Platelet secretion, In: Michelson A, Editor. Platelets, San Diego: Elsevier;2007. pp 309-318. Moritani C, Ishioka S, Haruta Y, Kambe M, Yamakido M. Activation of platelets in bronchial asthma. Chest 1998 113(2); 452-8. Joseph M, Capron A, Ameisen JC, Capron M, Vorng H, Pancre V, Kusnierz JP, Auriault C. The receptor for IgE on blood platelets. Eur J Immunol 1986 16(3); 306-12. Cheng CM, Hawiger J. Affinity isolation and characterization of immunoglobulin G Fc fragment-binding glycoprotein from human blood platelets. J Biol Chem 1979 254(7); 2165-7. Page CP. Platelets and asthma. Ann N Y Acad Sci 1991 629; 38-47. Jeffery PK, Wardlaw AJ, Nelson FC, Collins JV, Kay AB. Bronchial biopsies in asthma. An ultrastructural, quantitative study and correlation with hyperreactivity. Am Rev Respir Dis 1989 140(6); 174553. Metzger WJ, Sjoerdsma K, Richerson HB, Moseley P, Zavala D, Monick M, Hunninghake GW. Platelets in bronchoalveolar lavage from asthmatic patients and allergic rabbits with allergen-induced late phase responses. Agents Actions Suppl 1987 21; 151-9. Hill DJ, Griffiths DH, Rowley AF. Trout thrombocytes contain 12- but not 5-lipoxygenase activity. Biochim Biophys Acta 1999 1437(1); 63-70. Page CP. Platelets and asthma. Agents Actions Suppl 1989 28; 75-84. Edenius C, Heidvall K, Lindgren JA. Novel transcellular interaction: conversion of granulocyte-derived leukotriene A4 to cysteinylcontaining leukotrienes by human platelets. Eur J Biochem 1988 178(1); 81-6. Maclouf JA, Murphy RC. Transcellular metabolism of neutrophilderived leukotriene A4 by human platelets. A potential cellular source of leukotriene C4. J Biol Chem 1988 263(1); 174-81.
68
77
87. 88.
89.
90.
91.
92.
93.
94.
95.
96. 97. 98.
Klinger MH. Platelets and inflammation. Anat Embryol (Berl) 1997 196(1); 1-11. Chihara J, Yasuba H, Tsuda A, Urayama O, Saito N, Honda K, Kayaba H, Yamashita T, Kurimoto F, Yamada H. Elevation of the plasma level of RANTES during asthma attacks. J Allergy Clin Immunol 1997 100(6 Pt 2); S52-5. Cordova C, Musca A, Violi F, Alessandri C, Perrone A, Balsano F. Platelet hyperfunction in patients with chronic airways obstruction. Eur J Respir Dis 1985 66(1); 9-12. Ferroni P, Basili S, Martini F, Vieri M, Labbadia G, Cordova C, Alessandri C, Gazzaniga PP. Soluble P-selectin as a marker of platelet hyperactivity in patients with chronic obstructive pulmonary disease. J Investig Med 2000 48(1); 21-7. Davi G, Basili S, Vieri M, Cipollone F, Santarone S, Alessandri C, Gazzaniga P, Cordova C, Violi F. Enhanced thromboxane biosynthesis in patients with chronic obstructive pulmonary disease. The Chronic Obstructive Bronchitis and Haemostasis Study Group. Am J Respir Crit Care Med 1997 156(6); 1794-9. O'Sullivan BP, Linden MD, Frelinger AL, 3rd, Barnard MR, SpencerManzon M, Morris JE, Salem RO, Laposata M, Michelson AD. Platelet activation in cystic fibrosis. Blood 2005 105(12); 4635-41. Romano M, Collura M, Lapichino L, Pardo F, Falco A, Chiesa PL, Caimi G, Davi G. Endothelial perturbation in cystic fibrosis. Thromb Haemost 2001 86(6); 1363-7. De Sanctis GT, Wolyniec WW, Green FH, Qin S, Jiao A, Finn PW, Noonan T, Joetham AA, Gelfand E, Doerschuk CM, Drazen JM. Reduction of allergic airway responses in P-selectin-deficient mice. J Appl Physiol 1997 83(3); 681-7. Ulfman LH, Joosten DP, van Aalst CW, Lammers JW, van de Graaf EA, Koenderman L, Zwaginga JJ. Platelets promote eosinophil adhesion of patients with asthma to endothelium under flow conditions. Am J Respir Cell Mol Biol 2003 28(4); 512-9. Morley J, Sanjar S, Page CP. The platelet in asthma. Lancet 1984 2(8412); 1142-4. Ross R. Atherosclerosis--an inflammatory disease. N Engl J Med 1999 340(2); 115-26. Massberg S, Brand K, Gruner S, Page S, Muller E, Muller I, Bergmeier W, Richter T, Lorenz M, Konrad I, Nieswandt B, Gawaz M. A critical
69
78
99.
100.
101.
102.
103.
104.
105.
106. 107.
108.
role of platelet adhesion in the initiation of atherosclerotic lesion formation. J Exp Med 2002 196(7); 887-96. Fingerle J, Johnson R, Clowes AW, Majesky MW, Reidy MA. Role of platelets in smooth muscle cell proliferation and migration after vascular injury in rat carotid artery. Proc Natl Acad Sci U S A 1989 86(21); 8412-6. Anderson HV, McNatt J, Clubb FJ, Herman M, Maffrand JP, DeClerck F, Ahn C, Buja LM, Willerson JT. Platelet inhibition reduces cyclic flow variations and neointimal proliferation in normal and hypercholesterolemic-atherosclerotic canine coronary arteries. Circulation 2001 104(19); 2331-7. Ichii T, Koyama H, Tanaka S, Shioi A, Okuno Y, Otani S, Nishizawa Y. Thrombospondin-1 mediates smooth muscle cell proliferation induced by interaction with human platelets. Arterioscler Thromb Vasc Biol 2002 22(8); 1286-92. Fuscaldo AA, Erlick BJ, Brodsky I, Fuscaldo KE. Alteration of platelets and virus-like particles by busulfan in myeloproliferative disorders. Leuk Res 1980 4(1); 105-17. Pitchford SC, Riffo-Vasquez Y, Sousa A, Momi S, Gresele P, Spina D, Page CP. Platelets are necessary for airway wall remodeling in a murine model of chronic allergic inflammation. Blood 2004 103(2); 639-47. Heijnen HF, Schiel AE, Fijnheer R, Geuze HJ, Sixma JJ. Activated platelets release two types of membrane vesicles: microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and alpha-granules. Blood 1999 94(11); 3791-9. Merten M, Pakala R, Thiagarajan P, Benedict CR. Platelet microparticles promote platelet interaction with subendothelial matrix in a glycoprotein IIb/IIIa-dependent mechanism. Circulation 1999 99(19); 2577-82. Nieuwland S. Platelet-derived microparticles, In: Michelson A, Editor. Platelets, San Diego: Elsevier;2007. pp 403-413. Nieuwland R, Berckmans RJ, Rotteveel-Eijkman RC, Maquelin KN, Roozendaal KJ, Jansen PG, ten Have K, Eijsman L, Hack CE, Sturk A. Cell-derived microparticles generated in patients during cardiopulmonary bypass are highly procoagulant. Circulation 1997 96(10); 3534-41. Weber AA, Zucker TP, Schror K. Platelet surface membranes are highly mitogenic for coronary artery smooth muscle cells--A novel mechanism
70
79
109.
110.
111.
112.
113.
114.
115.
116.
117.
118.
119.
for sustained proliferation after vessel injury? Biochem Biophys Res Commun 1999 259(2); 341-3. Weber A, Koppen HO, Schror K. Platelet-derived microparticles stimulate coronary artery smooth muscle cell mitogenesis by a PDGFindependent mechanism. Thromb Res 2000 98(5); 461-6. Pietramaggiori G, Scherer SS, Mathews JC, Gennaoui T, Lancerotto L, Ragno G, Valeri CR, Orgill DP. Quiescent Platelets Stimulate Angiogenesis and Diabetic Wound Repair. J Surg Res 2008. Article in press Sum R, Hager S, Pietramaggiori G, Orgill DP, Dee J, Rudolph A, Orser C, Fitzpatrick GM, Ho D. Wound-healing properties of trehalosestabilized freeze-dried outdated platelets. Transfusion 2007 47(4); 672-9. Pietramaggiori G, Kaipainen A, Czeczuga JM, Wagner CT, Orgill DP. Freeze-dried platelet-rich plasma shows beneficial healing properties in chronic wounds. Wound Repair Regen 2006 14(5); 573-80. Pakala R. Serotonin and thromboxane A2 stimulate platelet-derived microparticle-induced smooth muscle cell proliferation. Cardiovasc Radiat Med 2004 5(1); 20-6. Baj-Krzyworzeka M, Majka M, Pratico D, Ratajczak J, Vilaire G, Kijowski J, Reca R, Janowska-Wieczorek A, Ratajczak MZ. Platelet-derived microparticles stimulate proliferation, survival, adhesion, and chemotaxis of hematopoietic cells. Exp Hematol 2002 30(5); 450-9. Szczeklik A, Krzanowski M, Nizankowska E, Musial J. Bleeding time and PAF-acether-induced platelet aggregation in atopy. Agents Actions Suppl 1987 21; 145-50. Szczeklik A, Milner PC, Birch J, Watkins J, Martin JF. Prolonged bleeding time, reduced platelet aggregation, altered PAF-acether sensitivity and increased platelet mass are a trait of asthma and hay fever. Thromb Haemost 1986 56(3); 283-7. Gresele P, Dottorini M, Selli ML, Iannacci L, Canino S, Todisco T, Romano S, Crook P, Page CP, Nenci GG. Altered platelet function associated with the bronchial hyperresponsiveness accompanying nocturnal asthma. J Allergy Clin Immunol 1993 91(4); 894-902. Beer JH, Wuthrich B, von Felten A. Allergen exposure in acute asthma causes the release of platelet-activating factor (PAF) as demonstrated by the desensitization of platelets to PAF. Int Arch Allergy Immunol 1995 106(3); 291-6. Block LH, Imhof E, Emmons LR, Roth M, Perruchoud AP. PAFdependent phosphatidylinositol turnover in platelets: differences
71
80
120. 121.
122.
123.
124.
125. 126.
127.
128.
129. 130. 131.
132.
between asthmatics and normal individuals. Respiration 1990 57(6); 3728. Yen SS, Morris HG. An imbalance of arachidonic acid metabolism in asthma. Biochem Biophys Res Commun 1981 103(2); 774-9. Audera C, Rocklin R, Vaillancourt R, Jakubowski JA, Deykin D. Altered arachidonic acid metabolism and platelet size in atopic subjects. Clin Immunol Immunopathol 1988 46(3); 352-9. Taytard A, Guenard H, Vuillemin L, Bouvot JL, Vergeret J, Ducassou D, Piquet Y, Freour P. Platelet kinetics in stable atopic asthmatic patients. Am Rev Respir Dis 1986 134(5); 983-5. Ward J, Hirst S. Airway smooth muscle bidirectional interactions with extracellular matrix, In: Chung K, Editor. Airway smooth muscle in asthma and COPD, Chichester: John Wiley and Sons Ltd;2008. pp 105126. Kultti A, Pasonen-Seppanen S, Jauhiainen M, Rilla KJ, Karna R, Pyoria E, Tammi RH, Tammi MI. 4-Methylumbelliferone inhibits hyaluronan synthesis by depletion of cellular UDP-glucuronic acid and downregulation of hyaluronan synthase 2 and 3. Exp Cell Res 2009 315(11); 1914-23. Aruffo A, Stamenkovic I, Melnick M, Underhill CB, Seed B. CD44 is the principal cell surface receptor for hyaluronate. Cell 1990 61(7); 1303-13. Papakonstantinou E, Kouri FM, Karakiulakis G, Klagas I, Eickelberg O. Increased hyaluronic acid content in idiopathic pulmonary arterial hypertension. Eur Respir J 2008 32(6); 1504-12. Klagas I, Goulet S, Karakiulakis G, Zhong J, Baraket M, Black JL, Papakonstantinou E, Roth M. Decreased hyaluronan in airway smooth muscle cells from patients with asthma and COPD. Eur Respir J 2009 34(3); 616-28. Dentener MA, Vernooy JH, Hendriks S, Wouters EF. Enhanced levels of hyaluronan in lungs of patients with COPD: relationship with lung function and local inflammation. Thorax 2005 60(2); 114-9. Fraser JR, Laurent TC, Laurent UB. Hyaluronan: its nature, distribution, functions and turnover. J Intern Med 1997 242(1); 27-33. Laurent TC, Fraser JR. Hyaluronan. Faseb J 1992 6(7); 2397-404. Delpech B, Girard N, Bertrand P, Courel MN, Chauzy C, Delpech A. Hyaluronan: fundamental principles and applications in cancer. J Intern Med 1997 242(1); 41-8. Kothapalli D, Flowers J, Xu T, Pure E, Assoian RK. Differential activation of ERK and Rac mediates the proliferative and anti-
72
81
133.
134.
135.
136.
137.
138.
139.
140.
141.
142.
proliferative effects of hyaluronan and CD44. J Biol Chem 2008 283(46); 31823-9. He Y, Wu GD, Sadahiro T, Noh SI, Wang H, Talavera D, Vierling JM, Klein AS. Interaction of CD44 and hyaluronic acid enhances biliary epithelial proliferation in cholestatic livers. Am J Physiol Gastrointest Liver Physiol 2008 295(2); G305-12. de la Motte C, Nigro J, Vasanji A, Rho H, Kessler S, Bandyopadhyay S, Danese S, Fiocchi C, Stern R. Platelet-derived hyaluronidase 2 cleaves hyaluronan into fragments that trigger monocyte-mediated production of proinflammatory cytokines. Am J Pathol 2009 174(6); 2254-64. Nedvetzki S, Gonen E, Assayag N, Reich R, Williams RO, Thurmond RL, Huang JF, Neudecker BA, Wang FS, Turley EA, Naor D. RHAMM, a receptor for hyaluronan-mediated motility, compensates for CD44 in inflamed CD44-knockout mice: a different interpretation of redundancy. Proc Natl Acad Sci U S A 2004 101(52); 18081-6. Harris EN, Weigel JA, Weigel PH. Endocytic function, glycosaminoglycan specificity, and antibody sensitivity of the recombinant human 190-kDa hyaluronan receptor for endocytosis (HARE). J Biol Chem 2004 279(35); 36201-9. Harris EN, Kyosseva SV, Weigel JA, Weigel PH. Expression, processing, and glycosaminoglycan binding activity of the recombinant human 315kDa hyaluronic acid receptor for endocytosis (HARE). J Biol Chem 2007 282(5); 2785-97. Krettek A, Sjoberg S. CD44 - a new cardiovascular drug target or merely an innocent bystander? Cardiovasc Hematol Disord Drug Targets 2009 9(4); 293-302. Cuff CA, Kothapalli D, Azonobi I, Chun S, Zhang Y, Belkin R, Yeh C, Secreto A, Assoian RK, Rader DJ, Pure E. The adhesion receptor CD44 promotes atherosclerosis by mediating inflammatory cell recruitment and vascular cell activation. J Clin Invest 2001 108(7); 1031-40. Kothapalli D, Zhao L, Hawthorne EA, Cheng Y, Lee E, Pure E, Assoian RK. Hyaluronan and CD44 antagonize mitogen-dependent cyclin D1 expression in mesenchymal cells. J Cell Biol 2007 176(4); 535-44. Bourguignon LY, Zhu H, Shao L, Chen YW. CD44 interaction with c-Src kinase promotes cortactin-mediated cytoskeleton function and hyaluronic acid-dependent ovarian tumor cell migration. J Biol Chem 2001 276(10); 7327-36. Bourguignon LY, Singleton PA, Zhu H, Diedrich F. Hyaluronanmediated CD44 interaction with RhoGEF and Rho kinase promotes
73
82
143.
144. 145. 146.
147.
148.
149. 150.
151.
152.
153.
Grb2-associated binder-1 phosphorylation and phosphatidylinositol 3kinase signaling leading to cytokine (macrophage-colony stimulating factor) production and breast tumor progression. J Biol Chem 2003 278(32); 29420-34. Naor D, Sionov RV, Ish-Shalom D. CD44: structure, function, and association with the malignant process. Adv Cancer Res 1997 71; 241319. Borland G, Ross JA, Guy K. Forms and functions of CD44. Immunology 1998 93(2); 139-48. Lesley J, Hyman R. CD44 structure and function. Front Biosci 1998 3; d616-30. Miyamoto S, Teramoto H, Gutkind JS, Yamada KM. Integrins can collaborate with growth factors for phosphorylation of receptor tyrosine kinases and MAP kinase activation: roles of integrin aggregation and occupancy of receptors. J Cell Biol 1996 135(6 Pt 1); 1633-42. Schneller M, Vuori K, Ruoslahti E. Alphavbeta3 integrin associates with activated insulin and PDGFbeta receptors and potentiates the biological activity of PDGF. Embo J 1997 16(18); 5600-7. Ben Mahdi MH, Andrieu V, Pasquier C. Focal adhesion kinase regulation by oxidative stress in different cell types. IUBMB Life 2000 50(4-5); 291-9. Schlaepfer DD, Hauck CR, Sieg DJ. Signaling through focal adhesion kinase. Prog Biophys Mol Biol 1999 71(3-4); 435-78. Lin CL, Zhang ZX, Xu YJ, Ni W, Chen SX. Focal adhesion kinase antisense oligodeoxynucleotides inhibit human pulmonary artery smooth muscle cells proliferation and promote human pulmonary artery smooth muscle cells apoptosis. Chin Med J (Engl) 2005 118(1); 206. Wu ZM, Yuan XH, Jiang PC, Li ZQ, Wu T. Antisense oligonucleodes targeting the focal adhesion kinase inhibit proliferation, induce apoptosis and cooperate with cytotoxic drugs in human glioma cells. J Neurooncol 2006 77(2); 117-23. Yamamoto D, Sonoda Y, Hasegawa M, Funakoshi-Tago M, Aizu-Yokota E, Kasahara T. FAK overexpression upregulates cyclin D3 and enhances cell proliferation via the PKC and PI3-kinase-Akt pathways. Cell Signal 2003 15(6); 575-83. Mitra SK, Hanson DA, Schlaepfer DD. Focal adhesion kinase: in command and control of cell motility. Nat Rev Mol Cell Biol 2005 6(1); 56-68.
74
83
154.
155.
156.
157.
158.
159.
160. 161.
162.
163. 164. 165.
Huang MM, Lipfert L, Cunningham M, Brugge JS, Ginsberg MH, Shattil SJ. Adhesive ligand binding to integrin alpha IIb beta 3 stimulates tyrosine phosphorylation of novel protein substrates before phosphorylation of pp125FAK. J Cell Biol 1993 122(2); 473-83. Lipfert L, Haimovich B, Schaller MD, Cobb BS, Parsons JT, Brugge JS. Integrin-dependent phosphorylation and activation of the protein tyrosine kinase pp125FAK in platelets. J Cell Biol 1992 119(4); 905-12. Shattil SJ, Haimovich B, Cunningham M, Lipfert L, Parsons JT, Ginsberg MH, Brugge JS. Tyrosine phosphorylation of pp125FAK in platelets requires coordinated signaling through integrin and agonist receptors. J Biol Chem 1994 269(20); 14738-45. Haimovich B, Kaneshiki N, Ji P. Protein kinase C regulates tyrosine phosphorylation of pp125FAK in platelets adherent to fibrinogen. Blood 1996 87(1); 152-61. Hitchcock IS, Fox NE, Prevost N, Sear K, Shattil SJ, Kaushansky K. Roles of focal adhesion kinase (FAK) in megakaryopoiesis and platelet function: studies using a megakaryocyte lineage specific FAK knockout. Blood 2008 111(2); 596-604. Jones ML, Shawe-Taylor AJ, Williams CM, Poole AW. Characterization of a novel focal adhesion kinase inhibitor in human platelets. Biochem Biophys Res Commun 2009 389(1); 198-203. Petr MJ, Origitano TC, Wurster RD. PLA2 activity regulates Ca2+ storage-dependent cellular proliferation. Exp Cell Res 1998 244(1); 310-8. Antoniotti S, Fiorio Pla A, Pregnolato S, Mottola A, Lovisolo D, Munaron L. Control of endothelial cell proliferation by calcium influx and arachidonic acid metabolism: a pharmacological approach. J Cell Physiol 2003 197(3); 370-8. Maclouf J, Murphy RC, Henson PM. Transcellular biosynthesis of sulfidopeptide leukotrienes during receptor-mediated stimulation of human neutrophil/platelet mixtures. Blood 1990 76(9); 1838-44. Weksler BB. Platelets and the inflammatory response. Clin Lab Med 1983 3(4); 667-76. Hamburger SA, McEver RP. GMP-140 mediates adhesion of stimulated platelets to neutrophils. Blood 1990 75(3); 550-4. Marcus AJ, Weksler BB, Jaffe EA, Broekman MJ. Synthesis of prostacyclin from platelet-derived endoperoxides by cultured human endothelial cells. J Clin Invest 1980 66(5); 979-86.
75
84
166.
167.
168.
169.
170.
171.
172.
173.
174.
175.
176.
Fitzpatrick F, Liggett W, McGee J, Bunting S, Morton D, Samuelsson B. Metabolism of leukotriene A4 by human erythrocytes. A novel cellular source of leukotriene B4. J Biol Chem 1984 259(18); 11403-7. Farias SE, Zarini S, Precht T, Murphy RC, Heidenreich KA. Transcellular biosynthesis of cysteinyl leukotrienes in rat neuronal and glial cells. J Neurochem 2007 103(4); 1310-8. Barry OP, Pratico D, Lawson JA, FitzGerald GA. Transcellular activation of platelets and endothelial cells by bioactive lipids in platelet microparticles. J Clin Invest 1997 99(9); 2118-27. Pfister SL. Role of platelet microparticles in the production of thromboxane by rabbit pulmonary artery. Hypertension 2004 43(2); 42833. Roberts W, Riba R, Homer-Vanniasinkam S, Farndale RW, Naseem KM. Nitric oxide specifically inhibits integrin-mediated platelet adhesion and spreading on collagen. J Thromb Haemost 2008 6(12); 2175-85. Mazurov AV, Leytin VL, Repin VS, Taube C, Forster W. Platelet prostanoids in interaction of platelets with collagen substrates. II. Effects of exogenous arachidonic acid, stable PG endoperoxides analogue, and aspirin. Biomed Biochim Acta 1984 43(8-9); S373-6. Kim JI, Lakshmikanthan V, Frilot N, Daaka Y. Prostaglandin E2 promotes lung cancer cell migration via EP4-betaArrestin1-c-Src signalsome. Mol Cancer Res 8(4); 569-77. Potula HS, Wang D, Quyen DV, Singh NK, Kundumani-Sridharan V, Karpurapu M, Park EA, Glasgow WC, Rao GN. Src-dependent STAT-3mediated expression of monocyte chemoattractant protein-1 is required for 15(S)-hydroxyeicosatetraenoic acid-induced vascular smooth muscle cell migration. J Biol Chem 2009 284(45); 31142-55. Yuan YM, Fang SH, Qian XD, Liu LY, Xu LH, Shi WZ, Zhang LH, Lu YB, Zhang WP, Wei EQ. Leukotriene D4 stimulates the migration but not proliferation of endothelial cells mediated by the cysteinyl leukotriene cyslt(1) receptor via the extracellular signal-regulated kinase pathway. J Pharmacol Sci 2009 109(2); 285-92. Ishii K, Zaitsu M, Yonemitsu N, Kan Y, Hamasaki Y, Matsuo M. 5lipoxygenase pathway promotes cell proliferation in human glioma cell lines. Clin Neuropathol 2009 28(6); 445-52. Wen Y, Gu J, Peng X, Zhang G, Nadler J. Overexpression of 12lipoxygenase and cardiac fibroblast hypertrophy. Trends Cardiovasc Med 2003 13(4); 129-36.
76
85
177.
178.
179. 180.
181.
182.
183.
184.
185. 186. 187.
188.
Dahlen SE, Hansson G, Hedqvist P, Bjorck T, Granstrom E, Dahlen B. Allergen challenge of lung tissue from asthmatics elicits bronchial contraction that correlates with the release of leukotrienes C4, D4, and E4. Proc Natl Acad Sci U S A 1983 80(6); 1712-6. Dahlen SE, Dahlen B, Eliasson E, Johansson H, Bjorck T, Kumlin M, Boo K, Whitney J, Binks S, King B, et al. Inhibition of allergic bronchoconstriction in asthmatics by the leukotriene-antagonist ICI204,219. Adv Prostaglandin Thromboxane Leukot Res 1991 21A; 461-4. Dahlen SE, Hedqvist P, Hammarstrom S, Samuelsson B. Leukotrienes are potent constrictors of human bronchi. Nature 1980 288(5790); 484-6. Hua XY, Dahlen SE, Lundberg JM, Hammarstrom S, Hedqvist P. Leukotrienes C4, D4 and E4 cause widespread and extensive plasma extravasation in the guinea pig. Naunyn Schmiedebergs Arch Pharmacol 1985 330(2); 136-41. Laitinen LA, Laitinen A, Haahtela T, Vilkka V, Spur BW, Lee TH. Leukotriene E4 and granulocytic infiltration into asthmatic airways. Lancet 1993 341(8851); 989-90. Wang CG, Du T, Xu LJ, Martin JG. Role of leukotriene D4 in allergeninduced increases in airway smooth muscle in the rat. Am Rev Respir Dis 1993 148(2); 413-7. Palmberg L, Claesson HE, Thyberg J. Leukotrienes stimulate initiation of DNA synthesis in cultured arterial smooth muscle cells. J Cell Sci 1987 88 ( Pt 2); 151-9. Palmberg L, Lindgren JA, Thyberg J, Claesson HE. On the mechanism of induction of DNA synthesis in cultured arterial smooth muscle cells by leukotrienes. Possible role of prostaglandin endoperoxide synthase products and platelet-derived growth factor. J Cell Sci 1991 98 ( Pt 2); 141-9. Ford-Hutchinson AW, Gresser M, Young RN. 5-Lipoxygenase. Annu Rev Biochem 1994 63; 383-417. Kim C, Kim JY, Kim JH. Cytosolic phospholipase A(2), lipoxygenase metabolites, and reactive oxygen species. BMB Rep 2008 41(8); 555-9. Kim C, Dinauer MC. Impaired NADPH oxidase activity in Rac2deficient murine neutrophils does not result from defective translocation of p47phox and p67phox and can be rescued by exogenous arachidonic acid. J Leukoc Biol 2006 79(1); 223-34. Shiose A, Sumimoto H. Arachidonic acid and phosphorylation synergistically induce a conformational change of p47phox to activate the phagocyte NADPH oxidase. J Biol Chem 2000 275(18); 13793-801.
77
86
189.
190.
191.
192.
193. 194. 195. 196.
197.
198. 199.
200.
201.
Sauer H, Wartenberg M, Hescheler J. Reactive oxygen species as intracellular messengers during cell growth and differentiation. Cell Physiol Biochem 2001 11(4); 173-86. Berg C, Trofast C, Bengtsson T. Platelets induce reactive oxygen speciesdependent growth of human skin fibroblasts. Eur J Cell Biol 2003 82(11); 565-71. Chiarugi P, Pani G, Giannoni E, Taddei L, Colavitti R, Raugei G, Symons M, Borrello S, Galeotti T, Ramponi G. Reactive oxygen species as essential mediators of cell adhesion: the oxidative inhibition of a FAK tyrosine phosphatase is required for cell adhesion. J Cell Biol 2003 161(5); 933-44. Taddei ML, Parri M, Mello T, Catalano A, Levine AD, Raugei G, Ramponi G, Chiarugi P. Integrin-mediated cell adhesion and spreading engage different sources of reactive oxygen species. Antioxid Redox Signal 2007 9(4); 469-81. Burdon RH, Rice-Evans C. Free radicals and the regulation of mammalian cell proliferation. Free Radic Res Commun 1989 6(6); 345-58. Burdon RH, Gill V, Rice-Evans C. Cell proliferation and oxidative stress. Free Radic Res Commun 1989 7(3-6); 149-59. Murrell GA, Francis MJ, Bromley L. Modulation of fibroblast proliferation by oxygen free radicals. Biochem J 1990 265(3); 659-65. Craven PA, Pfanstiel J, DeRubertis FR. Role of reactive oxygen in bile salt stimulation of colonic epithelial proliferation. J Clin Invest 1986 77(3); 850-9. Gallagher DL, Betteridge LJ, Patel MK, Schachter M. Effect of oxidants on vascular smooth muscle proliferation. Biochem Soc Trans 1993 21(2); 98S. Zhou L, Hershenson MB. Mitogenic signaling pathways in airway smooth muscle. Respir Physiol Neurobiol 2003 137(2-3); 295-308. Ravasi S, Citro S, Viviani B, Capra V, Rovati GE. CysLT1 receptorinduced human airway smooth muscle cells proliferation requires ROS generation, EGF receptor transactivation and ERK1/2 phosphorylation. Respir Res 2006 7; 42. Mehdi MZ, Azar ZM, Srivastava AK. Role of receptor and nonreceptor protein tyrosine kinases in H2O2-induced PKB and ERK1/2 signaling. Cell Biochem Biophys 2007 47(1); 1-10. Yang S, Yip R, Polena S, Sharma M, Rao S, Griciene P, Gintautas J, Jerome H. Reactive oxygen species increased focal adhesion kinase
78
87
202.
203.
204.
205.
206.
207.
208.
209.
210.
211.
production in pulmonary microvascular endothelial cells. Proc West Pharmacol Soc 2004 47; 54-6. Bengtsson T, Grenegard M. Platelets amplify chemotactic peptideinduced changes in F-actin and calcium in human neutrophils. Eur J Cell Biol 1994 63(2); 345-9. Regan JW, Matsui H. alpha 2-Adrenergic receptor purification, In: Hulme EC, Editor. Receptor Biochemistry: A Practical Approach, Oxford: Oxford University Press;1990. pp 141-161. Cory AH, Owen TC, Barltrop JA, Cory JG. Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture. Cancer Commun 1991 3(7); 207-12. Kiss L, Bieniek E, Weissmann N, Schutte H, Sibelius U, Gunther A, Bier J, Mayer K, Henneking K, Padberg W, Grimm H, Seeger W, Grimminger F. Simultaneous analysis of 4- and 5-series lipoxygenase and cytochrome P450 products from different biological sources by reversed-phase highperformance liquid chromatographic technique. Anal Biochem 1998 261(1); 16-28. Wang H, Joseph JA. Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radic Biol Med 1999 27(5-6); 612-6. Dekkers BG, Bos IS, Gosens R, Halayko AJ, Zaagsma J, Meurs H. The integrin-blocking peptide RGDS inhibits airway smooth muscle remodeling in a guinea pig model of allergic asthma. Am J Respir Crit Care Med 181(6); 556-65. She W, Takeuchi K, Suzuki S, Sakaida H, Ishinaga H, Majima Y. Remodeling of nasal mucosa by allergen exposure in guinea pigs is suppressed by steroid and pranlukast. Rhinology 2009 47(2); 199-206. Kakizaki I, Kojima K, Takagaki K, Endo M, Kannagi R, Ito M, Maruo Y, Sato H, Yasuda T, Mita S, Kimata K, Itano N. A novel mechanism for the inhibition of hyaluronan biosynthesis by 4-methylumbelliferone. J Biol Chem 2004 279(32); 33281-9. McLean GW, Carragher NO, Avizienyte E, Evans J, Brunton VG, Frame MC. The role of focal-adhesion kinase in cancer - a new therapeutic opportunity. Nat Rev Cancer 2005 5(7); 505-15. Fujita Y, Kitagawa M, Nakamura S, Azuma K, Ishii G, Higashi M, Kishi H, Hiwasa T, Koda K, Nakajima N, Harigaya K. CD44 signaling through focal adhesion kinase and its anti-apoptotic effect. FEBS Lett 2002 528(13); 101-8.
79
88
212.
213.
214.
215.
216. 217.
218.
219. 220.
Zhang Y, Thant AA, Hiraiwa Y, Naito Y, Sein TT, Sohara Y, Matsuda S, Hamaguchi M. A role for focal adhesion kinase in hyluronan-dependent MMP-2 secretion in a human small-cell lung carcinoma cell line, QG90. Biochem Biophys Res Commun 2002 290(3); 1123-7. Yoshisue H, Kirkham-Brown J, Healy E, Holgate ST, Sampson AP, Davies DE. Cysteinyl leukotrienes synergize with growth factors to induce proliferation of human bronchial fibroblasts. J Allergy Clin Immunol 2007 119(1); 132-40. Panettieri RA, Tan EM, Ciocca V, Luttmann MA, Leonard TB, Hay DW. Effects of LTD4 on human airway smooth muscle cell proliferation, matrix expression, and contraction In vitro: differential sensitivity to cysteinyl leukotriene receptor antagonists. Am J Respir Cell Mol Biol 1998 19(3); 453-61. Espinosa K, Bosse Y, Stankova J, Rola-Pleszczynski M. CysLT1 receptor upregulation by TGF-beta and IL-13 is associated with bronchial smooth muscle cell proliferation in response to LTD4. J Allergy Clin Immunol 2003 111(5); 1032-40. Stern R, Kogan G, Jedrzejas MJ, Soltes L. The many ways to cleave hyaluronan. Biotechnol Adv 2007 25(6); 537-57. Bourguignon LY, Singleton PA, Diedrich F, Stern R, Gilad E. CD44 interaction with Na+-H+ exchanger (NHE1) creates acidic microenvironments leading to hyaluronidase-2 and cathepsin B activation and breast tumor cell invasion. J Biol Chem 2004 279(26); 26991-7007. Harada H, Takahashi M. CD44-dependent intracellular and extracellular catabolism of hyaluronic acid by hyaluronidase-1 and -2. J Biol Chem 2007 282(8); 5597-607. Thannickal VJ, Fanburg BL. Reactive oxygen species in cell signaling. Am J Physiol Lung Cell Mol Physiol 2000 279(6); L1005-28. Woo CH, Eom YW, Yoo MH, You HJ, Han HJ, Song WK, Yoo YJ, Chun JS, Kim JH. Tumor necrosis factor-alpha generates reactive oxygen species via a cytosolic phospholipase A2-linked cascade. J Biol Chem 2000 275(41); 32357-62.
80
89
