Platelets stimulate airway smooth muscle cell proliferation through mechanisms involving 5-lipoxygenase and reactive oxygen species

• 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 visualized 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 co-incubation 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. © 2008 Informa UK Ltd.

Nyckelord

5-lipoxygenase

Airway remodeling

Airway smooth muscle

Platelet-induced proliferation

Reactive oxygen species

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