O77	Leukotriene-induced migration and proliferation of vascular smooth muscle cells: implications for atherosclerosis and restenosis.
M.Bäck, Y.Sheikine, G.K.Hansson
Center for Molecular Medicine, Department of Medicine, Karolinska University Hospital, Stockholm, SE.

Leukotriene B4 (LTB4) is a potent inflammatory mediator of the 5-lipoxygenase pathway of arachidonic acid metabolism, recently implicated in the pathogenesis of atherosclerosis. LTB4 exerts its action via BLT1 and BLT2 receptors, representing high and low affinity receptor subtypes, respectively. LTB4 is a potent chemoattractant for human leukocytes, and neutrophils, eosinophils, and T-lymphocytes all migrate in response to LTB4. The aim of the present study was to elucidate the effects of LTB4 on vascular smooth muscle cells. Human coronary artery smooth muscle cells (HCASMC), were cultured using SmGM2 kit medium and cells were used between passage 5 and 8. Migration of HCASMC was studied using 12-well chemotaxis chambers with an 8 μm pore size. Aliquots of 10000 cells were added to the upper chambers and LTB4 (1-1000 nM) was added to the lower chamber after which cells were left to migrate for 8h. Platelet-derived growth factor BB-homodimer (PDGF) was used as a positive control, and results are expressed as percent of the cell migration induced by PDGF (10 ng/mL). For study of proliferation, HCASMC were cultured in 96-well plates (5000 cells/well) and after 48h of serum-starvation, cells were left to proliferate in medium in the absence or presence of LTB4 (0.1-100 nM) for 72 h. Cell proliferation was quantified using WST-1 reagent, and absorbance was measured after 1 hr at 440/620 nm. LTB4 induced migration of HCASMC, with a maximum amount of migrated cells at a concentration of 100 nM. At this concentration, the LTB4-induced migration represented 153±11% (n=4) of the positive control PDGF. Pretreatment of cells in the upper chamber with the BLT1 receptor antagonist, U75302 (1 μM, n=3) significantly inhibited the SMC migration towards an LTB4 concentration of 100 nM (92±20%, P<0.05 vs control). In addition, LTB4 significantly enhanced the proliferation of human coronary artery SMC after 72 h by 17±2.0% (n=5) and 22±3.0% (n=5) compared with control, at concentrations of 10 and 100 nM, respectively (P<0.05). In conclusion, LTB4 induced chemotaxis of vascular smooth muscle cells and stimulated cell proliferation in vitro. These results suggest that LTB4-induced migration and proliferation of vascular smooth muscle cells may represent one possible mechanism linking the 5-lipoxygenase pathway to atherosclerosis and restenosis after angioplasty.

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