siRNA mediated gene silencing has been recently utilized as a powerful molecular tool to study the functional significance of a specific protein. However, due to the transient nature of silencing and insufficient transfection efficiency, this approach can be problematic in primary cell culture. To overcome such weakness of the siRNA based silencing and in order to establish reliable gene silencing in vascular cells, we devised an adenoviral-encoded miRNA based gene silencing system. Here we report the results of silencing ADAM17 in cultured rat vascular smooth muscle cells (VSMCs) and its functional consequences in angiotensin II (AngII) signal transduction. Four distinct miRNA sequences targeting rat ADAM17 were chosen based on recommendations from Invitrogen’s Block-iT RNAi Designer algorithm. The miRNA sequences were inserted into a mammalian expression vector, pcDNA 6.2-GW/EmGFP-miR, and the effective silencing by these vectors was confirmed in HEK cells expressing HA-tagged rat ADAM17. The 4 cassettes carrying the miRNAs were inserted into pAd/CMV/V5-DEST and adenoviral solutions were obtained. Greater than 95% silencing of ADAM17 was achieved when VSMC were infected with 100-200 moi of the ADAM17 miRNA encoding adeonvirus for 72 h with enhancement of infection by fugene6. Relatively linear time and concentration dependencies were observed between 1 to 3 days and 10 to 100 moi of the infection. A miR-ADAM17 (100 moi) but not miR-control (100 moi) completely inhibited 100 nM AngII-induced HB-EGF shedding in VSMCs as assessed by a reporter assay. A miR-ADAM17 but not miR-control also inhibited AngII-induced EGF receptor transactivation and subsequent ERK1/2 activation in VSMCs as assessed by immunoblotting with phospho-selective antibodies. In conclusion, ADAM17 was found to be a major sheddase for HB-EGF contributing to the growth promoting signals induced by AngII in VSMCs. An artificial miRNA-base adenoviral approach appears to be a reliable gene-silencing strategy for signal transduction research in primary cultured vascular cells.
Oxidative stress inhibits insulin signal transduction in cultured human vascular smooth muscle cells: a possible contribution to mechanism of insulin resistance