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Platelets and airway remodeling: Mechanisms involved in platelet-induced fibroblast and airway smooth muscle cell proliferation in vitro
Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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 also induce a 5-LOX dependent fibroblast proliferation. Furthermore, morphological analysis demonstrates 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 5-LOX dependent and that platelets induce a HA, CD44, FAK, 5-LOX, and ROSdependent 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.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2010. , 80 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1203
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-61623ISBN: 978-91-7393-324-7 (print)OAI: oai:DiVA.org:liu-61623DiVA: diva2:370621
Public defence
2010-12-03, Berzeliussalen, Universitetssjukhuset, Campus US, Linköpings universitet, Linköping, 13:00 (Swedish)
Opponent
Supervisors
Available from: 2010-11-17 Created: 2010-11-17 Last updated: 2012-01-30Bibliographically approved
List of papers
1. Platelet-induced growth of human fibroblasts is associated with an increased expression of 5-lipoxygenase
Open this publication in new window or tab >>Platelet-induced growth of human fibroblasts is associated with an increased expression of 5-lipoxygenase
Show others...
2006 (English)In: Thrombosis and Haemostasis, ISSN 0340-6245, Vol. 96, no 5, 652-659 p.Article in journal (Refereed) Published
Abstract [en]

Proliferation of fibroblasts is vital for adequate wound healing but is probably also involved in different hyperproliferative disorders such as atherosclerosis and cancer. The regeneration of tissue usually starts with coagulation, involving release of mitogenic and inflammatory factors from activated platelets. This study focuses on the role of eicosanoids in the proliferative effects of platelets on human fibroblasts. We show that the phospholipase A2 inhibitor 7,7-dimethyl-5,8-eicosadienoic acid (DMDA), the combined cyclooxygenase (COX) and lipoxygenase (LOX) inhibitor 5,8,11,14-eicosatetraynoic acid (ETYA) and the LOX inhibitor 5,8,11-eicosatriynoic acid (ETI) block the platelet-induced proliferation of serum starved subconfluent human fibroblasts. Anti-proliferative effects were also obtained by specific inhibition of 5-LOX with 5,6-dehydro arachidonic acid (5,6-dAA), whereas the 12-LOX inhibitor cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC) did not affect the platelet-stimulated growth of fibroblasts. The expression of 5-LOX was analyzed by reverse-transcriptase-mediated PCR (RT-PCR), Western blotting and HPLC. 5-LOX message and protein was detected in fibroblasts but not in platelets. Incubation with platelets markedly increased, already after one hour, the expression of 5-LOX in the fibroblast culture. The increased 5-LOX activity was associated with an elevated level of the 5-LOX metabolite 5-hydroxyeicosatetraenoic acid (5-HETE) reaching its maximum after 1-2 hours of co-incubation of fibroblasts and platelets. The 5-HETE production was reduced by the inhibitors DMDA, ETYA and ETI. In conclusion, this study suggests that platelet-stimulated proliferation of fibroblasts is mediated by an increased 5-LOX activity, which supports recent findings indicating a crucial role for this enzyme in proliferative disorders such as atherosclerosis. © 2006 Schattauer GmbH, Stuttgart.

National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-36111 (URN)10.1160/TH06-02-0069 (DOI)000242168400016 ()29986 (Local ID)29986 (Archive number)29986 (OAI)
Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2017-12-13
2. Platelets stimulate airway smooth muscle cell proliferation through mechanisms involving 5-lipoxygenase and reactive oxygen species
Open this publication in new window or tab >>Platelets stimulate airway smooth muscle cell proliferation through mechanisms involving 5-lipoxygenase and reactive oxygen species
2008 (English)In: Platelets, ISSN 0953-7104, E-ISSN 1369-1635, Vol. 19, no 7, 528-536 p.Article in journal (Refereed) Published
Abstract [en]

Continuous recruitment and inappropriate activity of platelets in the airways may contribute to airway remodeling, a characteristic feature of inflammatory airway diseases that includes increased proliferation of the smooth muscle.

The aim of the present investigation was to examine the effect of platelets on proliferation of airway smooth muscle cells (ASMC) in culture and to determine the possible role of 5-lipoxygenase (5-LOX) and reactive oxygen species (ROS) in this context.

ASMC obtained from guinea pigs were cultured and co-incubated with washed platelets for 24 hours. Thereafter, the proliferation was measured with the MTS-assay, the results were also verified by using thymidine incorporation, DNA measurements and manual counting. The interaction between platelets and ASMC was visualised with fluorescence microscopy.

We found that platelets bind to the ASMC and the presence of platelets caused a significant dose-dependent increase in ASMC proliferation. Co-incubation of ASMC with platelets also increased ROS-production, detected by the fluorescent probe DCFDA. Furthermore, the platelet-induced proliferation was reduced in the presence of the NADPH-oxidase inhibitors DPI and apocynin.

A possible role of 5-LOX in platelet-induced proliferation and ROS-generation was evaluated by using the 5-LOX inhibitor AA-861 and the PLA2-inhibitor ATK. The results showed that inhibition of these enzymes significantly reduced the platelet-induced proliferation. Moreover, Western blot analysis revealed that the ASMC but not the platelets express 5-LOX.

In addition, our experiments revealed that the presence of AA-861 and ATK significantly inhibited the ROS-production generated upon coincubation of platelets and ASMC.

In conclusion, we show that platelets have a marked capacity to induce ASMC proliferation. Furthermore, our study indicates that the interaction between platelets and ASMC leads to activation of 5-LOX in the ASMC followed by an increased ROS-production, events resulting in enhanced ASMC proliferation. The new findings are of importance in understanding possible mechanisms contributing to airway remodeling.

Keyword
platelet-induced proliferation, airway smooth muscle, 5-lipoxygenase, reactive oxygen species, airway remodeling
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-15607 (URN)10.1080/09537100802320300 (DOI)
Note
Original publication: Ann-Charlotte B. Svensson Holm, Torbjörn Bengtsson, Magnus Grenegård and Eva G. Lindström, Platelets stimulate airway smooth muscle cell proliferation through mechanisms involving 5-lipoxygenase and reactive oxygen species, 2008, Platelets, (19), 7, 528-536.http://dx.doi.org/10.1080/09537100802320300. Copyright © Taylor & Francis Group, an informa businessAvailable from: 2008-11-20 Created: 2008-11-20 Last updated: 2017-12-14Bibliographically approved
3. Platelet membranes induce airway smooth muscle cellproliferation
Open this publication in new window or tab >>Platelet membranes induce airway smooth muscle cellproliferation
2011 (English)In: Platelets, ISSN 0953-7104, E-ISSN 1369-1635, Vol. 22, no 1, 45-55 p.Article in journal (Refereed) Published
Abstract [en]

The role of platelets in airway disease is poorly understood although they have been suggested to influence on proliferation of airway smooth muscle cells (ASMC). Platelets have been found localised in the airways in autopsy material from asthmatic patients and have been implicated in airway remodeling. The aim of the present study was to investigate the effects of various platelet fractions on proliferation of ASMC obtained from guinea pigs (GP-ASMC) and humans (H-ASMC). Proliferation of ASMC was measured by the MTS-assay and the results were confirmed by measurements of the DNA content. A key observation was that the platelet membrane preparations induced a significant increase in the proliferation of both GPASMC (129.9 ± 3.0 %) and H-ASMC (144.8 ± 12.2). However, neither supernatants obtained from lysed nor filtrate from thrombin stimulated platelets did induce ASMC proliferation to the same extent as the membrane preparation. We have previously shown the platelet-induced proliferation is dependent on the 5-lipoxygenase (5-LOX) and reactive oxygen species (ROS) pathways. In the present work we established that platelet membrane-induced ASMC proliferation was reduced in the presence of the NADPH oxidase inhibitor DPI and the 5-LOX inhibitor AA-861. In conclusion, our results showed that platelet  membranes significantly induced ASMC proliferation, demonstrating that the mitogenic effect of platelets and platelet membranes on ASMC is mainly due to membrane-associated factors. The effects of platelet membranes were evident on both GP-ASMC and H-ASMC and involved 5-LOX and ROS. These new findings are of importance in understanding the mechanisms contributing to airway remodeling and may contribute to the development of new pharmacological tools in the treatment of inflammatory airway diseases.

Place, publisher, year, edition, pages
Informa, 2011
Keyword
platelets; platelet membranes; airway smooth muscle cell; 5-lipoxygenase; reactive oxygen species; airway remodeling
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-61615 (URN)10.3109/09537104.2010.515696 (DOI)000286937800006 ()
Note
Original Publication: Ann-Charlotte B. Svensson Holm, Torbjörn Bengtsson, Magnus Grenegård and Eva G. Lindström, Platelet membranes induce airway smooth muscle cellproliferation, 2011, Platelets, (22), 1, 45-55. http://dx.doi.org/10.3109/09537104.2010.515696 Copyright: Informa Healthcare http://informahealthcare.com/ Available from: 2010-11-17 Created: 2010-11-17 Last updated: 2017-12-12
4. Platelets bind to hyaluronic acid through CD44 and induce a focal adhesion kinase dependent airway smooth muscle cell proliferation
Open this publication in new window or tab >>Platelets bind to hyaluronic acid through CD44 and induce a focal adhesion kinase dependent airway smooth muscle cell proliferation
2008 (English)Manuscript (preprint) (Other academic)
Abstract [en]

Platelets have been implicated as important players in the remodeling process, e.g. due to their ability to induce airway smooth muscle cell (ASMC) proliferation. The aim of the present study was to investigate the role of the extracellular matrix component hyaluronic acid (HA), the HA-binding surface receptor CD44 and focal adhesion kinase (FAK) in platelet-induced ASMC proliferation. The ability of ASMC to synthesize HA was investigated by fluorescent staining using biotinylated HA-binding protein and streptavidin conjugate. In addition, the interaction between ASMC and platelets was studied by fluorescent staining of the F-actin. We found that ASMC produced HA and that a CD44 blocking antibody and the hyaluronic acid synthase inhibitor 4-Methylumbelliferone (4-MU) inhibited platelet binding to the area surrounding the ASMC. Proliferation of ASMC was measured using the MTS-assay, and we found that the CD44 blocking antibody and 4-MU inhibited platelet-induced ASMC proliferation. We also found that co-culture of ASMC and platelets resulted in increased phosphorylation of FAK as detected by Western blot analysis. Furthermore, the FAKinhibitor PF 573228 inhibited platelet-induced ASMC proliferation. In conclusion, our findings demonstrate that HA, CD44 and FAK contribute to the increased ASMC proliferation caused by platelets. This event is initiated by an interaction between platelets CD44 and HA produced by the ASMC. These new findings may be important in understanding the interplay between ECM, platelets and ASMC in the remodeling process. In conclusion, our results demonstrate that FAK is phosphorylated and on that account activated during the CD44-dependent platelet/ASMC interaction and this contributes to proliferation of the ASMC. These new findings may be important in understanding the interplay between ECM, platelets and ASMC in the remodeling process.

Keyword
airway smooth muscle; airway remodeling, hyaluronic acid, CD44, focal adhesion kinase
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-61622 (URN)
Available from: 2010-11-17 Created: 2010-11-17 Last updated: 2010-11-17Bibliographically approved

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