Purpose: Thrombomodulin (TM), an integral endothelial receptor, is known for its anticoagulant functions. Moreover, there is evidence of growth-modulating effects of this cell surface -protein. The aim of our study was to establish by in vitro transfection a stable cell line of vascular smooth muscle cells with overexpression of TM for further investigations concerning the influence of TM on cellular proliferation and its potential role during the formation of restenosis.
Methods: Aortic smooth muscle cells of the rat were transfected with cDNA of mouse TM or one of its three mutants (M1, M2, M4) by a liposome-mediated technique. The expression of mouse TM mRNA in the selected clones was proven with the help of RT-PCR. Changes of cell proliferation were determined by proliferation kinetics over 24 days. The quantification of the total protein TM was made by Western blots.
Results: In 44 of 100 cases the RT-PCR confirmed a successful transfection of mouse-TM. The clones with transfected TM, M1 or M2 showed an inhibited cell growth, whereas M4 demonstrated an increased proliferation compared with controls. The comparison of amounts of total TM with cell growth of individual clones resulted in a negative correlation between proliferation and TM-expression (coefficient of correlation for TM -0.87, for M1 -0.59).
Conclusions: It is possible to reproduce stable cell-lines of vascular smooth muscle cells with overexpression of TM by the presented model of in vitro transfection. Thus, a basis exists for detailed examinations of growth-regulating mechanisms by TM.