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					European Respiratory Society
   Annual Congress 2005
Platelets induce proliferation of airway smooth
muscle cells through mechanisms dependent on
          ROS and 5-LOX metabolites



      Ann-Charlotte B Svensson, Torbjörn Bengtsson,
      Magnus Grenegård & Eva G Lindström.
      Cardiovascular Inflammation Research Centre, Division of
      Pharmacology, Department of Medicine and Care,
      Linköping University, Sweden




                       European Respiratory Society
                          Annual Congress 2005
        Introduction – Aim - Material and methods – Results – Conclusion




                        Introduction
   Platelets and asthma?
   Platelets, inflammation and vascular
    smooth muscle cell proliferation
   Remodelling
   5-lipoxygenase (5-LOX) metabolites
    and reactive oxygen species (ROS)



                           European Respiratory Society
                              Annual Congress 2005
      Introduction – Aim - Material and methods – Results – Conclusion




                                Aim
To investigate the effects of platelets
on airway smooth muscle cell (ASMC)
proliferation. Furthermore, the role of
5-LOX metabolites and ROS in these
processes were evaluated




                         European Respiratory Society
                            Annual Congress 2005
        Introduction – Aim - Material and methods – Results – Conclusion


                Material & Methods
                              Cell culture

   Primary culture of
    ASMC from Dunkin
    Hartley guninea pig
    trachea, obtained by
    explant technique
   Primary anti α-actin
    antibody and FITC-
    conjugated
    secondary antibody


                           European Respiratory Society
                              Annual Congress 2005
         Introduction – Aim - Material and methods – Results – Conclusion




                                 Methods

   Washed functional platelets and
    platelet membranes
   Fluorescent staining of F-actin
-   rhodamin phalloidin
   Proliferation
-   MTS-assay, Thymidin incorporation
   Intracellular ROS-production
-   DCFDA


                            European Respiratory Society
                               Annual Congress 2005
         Introduction – Aim - Material and methods – Results – Conclusion




                                 Methods

   Washed functional platelets and
    platelet membranes
   Fluorescent staining of F-actin
-   rhodamin phalloidin
   Proliferation
-   MTS-assay, Thymidin incorporation
   Intracellular ROS-production
-   DCFDA


                            European Respiratory Society
                               Annual Congress 2005
       Introduction – Aim - Material and methods – Results – Conclusion


                             Results
                    Platelets bind to ASMC




ASMC                                     ASMC+platelets 1/1000, 3 hours

                          European Respiratory Society
                             Annual Congress 2005
         Introduction – Aim - Material and methods – Results – Conclusion




                                 Methods

   Washed functional platelets and
    platelet membranes
   Fluorescent staining of F-actin
-   rhodamin phalloidin
   Proliferation
-   MTS-assay, Thymidin incorporation
   Intracellular ROS-production
-   DCFDA


                            European Respiratory Society
                               Annual Congress 2005
         Introduction – Aim - Material and methods – Results – Conclusion




                                 Methods

   Washed functional platelets and
    platelet membranes
   Fluorescent staining of F-actin
-   rhodamin phalloidin
   Proliferation
-   MTS-assay, Thymidin incorporation
   Intracellular ROS-production
-   DCFDA


                            European Respiratory Society
                               Annual Congress 2005
        Introduction – Aim - Material and methods – Results – Conclusion




                           ROS-production

2´,7´-dichlorofluorescein diacetate
 (DCFDA)
        esterases
DCFDA                    ROS (H2O2, OH and ONOO-)

                DCFH               DCF              Measured in a fluorescence
                                                    reader, excitation filer
                                                    485±10 nm and emission filter
                           cell                     530±12.5 nm




                           European Respiratory Society
                              Annual Congress 2005
                                              Introduction – Aim - Material and methods – Results – Conclusion

The presence of platelets and                                                                                              Platelets but not platelet
platelet membranes induce ASMC                                                                                             membranes increase ROS-
proliferation                                                                                                              production




                                                                                           ROS-production (% of control)
 Proliferation (% of control)




                                200                                                                                                                    **
                                                           ***                                                             250
                                                                           ***
                                                                                                                           200

                                                                                                                           150
                                100
                                                                                                                           100

                                                                                                                            50

                                 0                                                                                          0
                                      ASM
                                          C                lets            nes                                                       C                lets            nes
                                                      late              bra                                                      ASM             late              bra
                                                   C+p              me
                                                                       m                                                                      C+p              me
                                                                                                                                                                 m
                                              A SM            C +pl                                                                      A SM            C +pl
                                                        ASM                                                                                        ASM
Proliferation (n=23 resp 9)                                                                ROS-production (n=10 resp 3)


                                                                             European Respiratory Society
                                                                                Annual Congress 2005
         Introduction – Aim - Material and methods – Results – Conclusion


Inhibitors to investigate the role of eicosanoids in platelet-
induced ASMC proliferation

                                                                            CM

                          ATK        PLA2

                                Arachidonic acid
                                       FLA
                                        P
                     AA-861          5- LOX

                                   5-HPETE
                              Leukotriene A4
         LTA4 hydrolase                                LTC4 synthase

   Leukotriene B4                                              Leukotriene C4

                                          Leukotriene D4 & Leukotriene E4


                            European Respiratory Society
                               Annual Congress 2005
        Introduction – Aim - Material and methods – Results – Conclusion


Eicosanoids are involved in platelet-induced ASMC
proliferation


                                            200
             Proliferation (% of control)
                                                                                                       *
                                            150                                                                  ***

                                            100


                                            50


                                             0
                                                                     M



                                                                                   M



                                                                                                                 M



                                                                                                                 M
                                                         l
                                                      +p
                                                  C




                                                                                  1
                                                                  1




                                                                                                               1
                                                                                                               1
                                                  M



                                                                0.




                                                                                                            0.
                                                                             TK




                                                                                                           61
                                              S
                                             A



                                                             TK




                                                                                            61



                                                                                                        -8
                                                                            A
                                                                         l+



                                                                                           -8



                                                                                                       A
                                                            A




                                                                                                      A
                                                                      +p
                                                         l+




                                                                                       A



                                                                                                   l+
                                                                                       A
                                                      +p



                                                                     C




                                                                                                +p
                                                                                    l+
                                                                  M
                                                  C




                                                                                 +p



                                                                                                 C
                                                                 S
                                                  M




                                                                                                              (n=7)
                                                                                                M
                                                                A



                                                                              C
                                              S




                                                                                            S
                                                                             M
                                             A




                                                                                           A
                                                                          S
                                                                         A




                                                          European Respiratory Society
                                                             Annual Congress 2005
         Introduction – Aim - Material and methods – Results – Conclusion


Inhibitors to investigate the role of ROS in platelet-
induced ASMC proliferation

   DPI: NADPH-oxidase inhibitor
   Apocynin: NADPH-oxidase inhibitor
   PDTC: antioxidant
   Vitamin E: antioxidant




                            European Respiratory Society
                               Annual Congress 2005
       Introduction – Aim - Material and methods – Results – Conclusion




ROS are involved in platelet-induced ASMC proliferation


                                            175
             Proliferation (% of control)   150                       *                         **
                                                                                     **
                                            125
                                                                                                            ***
                                            100
                                            75

                                            50
                                            25
                                             0
                                                                    M



                                                                                                            M



                                                                                                            M



                                                                                                            M
                                                      l
                                                   +p
                                                  C



                                                                 I1




                                                                                                          5
                                                  M




                                                                                                          1




                                                                                                         50
                                                                                                       TC
                                              S




                                                                             n
                                                                P




                                                                                                       E
                                                                           ni
                                             A



                                                                D




                                                                                                     D
                                                                         cy
                                                             l+




                                                                                                    in
                                                                                                    P
                                                          +p




                                                                                                  m
                                                                       po




                                                                                                 l+



                                                                                              ita
                                                                                              +p
                                                      C



                                                                      A
                                                      M




                                                                                             V
                                                                   l+



                                                                                 C
                                                  S




                                                                                          l+
                                                                                M
                                                                +p
                                                  A




                                                                                       +p
                                                                            S
                                                               C




                                                                                                         (n=2-5)
                                                                            A



                                                                                      C
                                                              M




                                                                                    M
                                                           S




                                                                                  S
                                                          A




                                                                                A




                                                           European Respiratory Society
                                                              Annual Congress 2005
        Introduction – Aim - Material and methods – Results – Conclusion




                           Conclusion
   Platelets markedly increase the proliferation
    of ASMC through mechanisms dependent on
    5-LOX metabolites and ROS.
   Platelet membranes also increase
    proliferation of ASMC but have no effect on
    ROS-production.
   These findings suggest a role for platelets in
    airway remodelling.



                           European Respiratory Society
                              Annual Congress 2005
Thank you for your attention!




         European Respiratory Society
            Annual Congress 2005
                   [Ca2+]i 
                                                                                         AA
     platelet
                        +

                     PLA2
   phospholipids
                                                  ROS                              5-HETE
                               AA
LTA4→ LTC4→LTD4, LTE4


                                                                                        LTA4


                                                                                        LTB4                   ROS

                                                            H2O2                 .0 -
                                                                                   2

  Proliferation                                             +                                      O2

                                    AA                                            Nox4
                                         H2O2                                +
                                                           PLA2
                                           phospholipids               AA
                               5-HETE
                                                                                  Expression of Nox subunits


                                LTA4                                                      SMC
                                                                   nucleus
              Preparation of human platelets

   Centrifugation process: blood from healthy donors
-   220 x g to obtain platelet rich plasma
-   480 x g to obtain pellet of platelets
-   The collected pellet of platelets were
    carefully resuspended and thereafter
    counted.
                                                    Extracellular

      RTK                             GPCR

                                               Gi
      Shc      GRB         SOS                      Intracellular




ROS
                           p21ras

                                                    PI3K
                Raf-1

                                 p70S6kinase
        MEK1                                   rac1                 Cdc42



  ERK

                     ASMC proliferation
cytochrome b558                         cytochrome b558
                                                                       H+
                                O2

gp91                                     gp91
            p22       Rap1A                            p22           Rap1A


                                      rac1/2
                                                p47
   rac1/2



                                               p67                  NADP+
             p67      p47     NADPH
                                                             O2.-
                                               p40
             p40

            Resting                              Activated
                 Abbreviations
   MTS: [3-(4, 5-dimethylthiazol-2-yl)-5-(3-
    carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-
    tetrazolium, inner salt]
   PTDC: pyrrolidinedithiocarbamate
   DPI: diphenyleneiodonium chloride
   ATK: arachidonyl trifluoromethyl ketone




                   European Respiratory Society
                      Annual Congress 2005
                  IC50-values
   PTDC: in neutrophils 19.3 μM
   DPI: in neutrophils 0.9 μM
   Apocynin: in neutrophils 10 μM
   ATK: in mammalian cells 2 μM
   AA-861: in mammalian cells 0.8 μM
   Vitamin E: ?




                   European Respiratory Society
                      Annual Congress 2005

				
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posted:8/12/2011
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