P334	Effects of rapamycin on neointimal and medial smooth muscle cells.
1W.Dichtl, 1E-M.Stocker, 2Z-Q.Yan, 1H.F.Alber, 1M.Frick, 3J.Dulak, 1O.Pachinger, 1F.Weidinger
1Clinical Department of Cardiology, Medical University Innsbruck, Innsbruck, AT; 2Center of Molecular Medicine, Department of Medicine, Karolinska Institute, Stockholm, SE; 3Department of Cell Biochemistry, Faculty of Biotechnology, Jagiellonian University, Krakow, PL.

Objective: Local application of rapamycin (sirolimus) by drug-eluting stents prevents lumen loss after angioplasty by inhibition of neointimal hyperplasia. The effects of rapamycin on neointimal smooth muscle cells (niSMCs) which are responsible for the occurence of restenosis have not been investigated so far. Methods and Results: Rat niSMCs were derived from balloon catheter-injured arteries. As compared to medial SMCs (mSMCs), niSMCs elucidated higher basal NF-κB and TNF-α mRNA levels. Incubation with rapamycin (0.1 μg/ml) attenuated basal and TNF-α stimulated nuclear protein binding to NF-IκB-DNA binding in niSMCs but only to a little extend in mSMCs. After 12 hours of incubation, rapamycin (0.001 to 10 μg/ml) led to an induction of IκB-α protein levels in niSMCs, whereas rapamycin had only a weak stimulatory effect on IκB-α levels in mSMCs. Prolonged rapamycin (1 μg/ml) treatment resulted in increased TNF-α mRNA and NF-κB activity in mSMCs, whereas such an effect was not observed in niSMCs. VEGF secretion was higher in mSMCs as compared to niSMCs, and rapamycin decreased VEGF protein levels in both cell types. Conclusions: This study shows that rapamycin influences the inflammatory phenotypes of SMCs by inhibition of enhanced basal NF-κB activity in niSMCs and a delayed up-regulation of NF-κB and TNF-α expression in mSMCs. In addition, rapamycin inhibits VEGF production regardless of the phenotype of SMCs. These findings shed light on molecular mechanisms underlying therapeutic applications of rapamycin in prevention of restenosis, inhibition of chronic transplant arteriosclerosis and reduction of secondary malignoma formation due to immunosuppression.

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