Abstract. A number of patients with hyperlipidemia are prescribed 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors that are concomitantly used along with the treatment of diabetes mellitus. The effects of atorvastatin and pravastatin on insulin-induced glucose uptake and the related signal transduction in 3T3L1 adipocytes were studied. 3T3L1 fibroblasts were differentiated into adipocytes, pretreated with atorvastatin or pravastatin, and then exposed to insulin. Glucose uptake and the amount of insulin signal proteins were measured. Atorvastatin significantly decreased insulin-stimulated 2-deoxyglucose uptake in 3T3L1 adipocytes associated with the prevention of translocation of GLUT4 into the plasma membrane. The amounts of Rab4 and RhoA that required lipid modification with farnesyl or geranylgeranyl pyrophosphate, in the membrane fraction were decreased by atorvastatin. Insulin-induced tyrosine phosphorylation of IRS-1 and serine / threonine phosphorylation of Akt were reduced by atorvastatin. Pravastatin did not modify these insulin-induced changes in the signal transduction. Inhibitors of the RhoA / Rho kinase system, C3 and Y27632, as well as atorvastatin reduced insulin-induced changes in signal transduction. Atorvastatin and pravastatin did not affect messenger RNA expression, protein level, and tyrosine phosphorylation of insulin receptors. In conclusion, hydrophobic atorvastatin decreases the glucose uptake by 3T3L1 adipocytes since it can enter the cell and prevents lipid modification of some proteins that are involved in the insulin signal transduction process.

A metalloprotease, ADAM17, mediates the generation of mature ligands for the epidermal growth factor receptor (EGFR). This is the key signaling step by which angiotensin II (AngII) induces EGFR transactivation leading to hypertrophy and migration of vascular smooth muscle cells (VSMCs). However, the regulatory mechanism of ADAM17 activity remains largely unclear.Here we hypothesized that caveolin-1 (Cav1), the major structural protein of a caveolae, a membrane microdomain, is involved in the regulation of ADAM17. In cultured VSMCs, infection of adenovirus encoding Cav1 markedly inhibited AngII-induced EGFR ligand shedding, EGFR transactivation, ERK activation, hypertrophy and migration, but not intracellular Ca 2+ elevation. Methyl-β-cyclodextrin and filipin, reagents that disrupt raft structure, both stimulated an EGFR ligand shedding and EGFR transactivation in VSMCs. In addition, non-detergent sucrose gradient membrane fractionations revealed that ADAM17 cofractionated with Cav1 in lipid rafts. These results suggest that lipid rafts and perhaps caveolae provide a negative regulatory environment for EGFR transactivation linked to vascular remodeling induced by AngII. These novel findings may provide important information to target cardiovascular diseases under the enhanced renin angiotensin system.

Abstract. 3-Hydroxy-3-methylglutaryl CoA reductase inhibitors (statins) are safe and welltolerated therapeutic drugs. However, they occasionally induce myotoxicity such as myopathy and rhabdomyolysis. Here, we investigated the mechanism of statin-induced myotoxicity in L6 fibroblasts and in rats in vivo. L6 fibroblasts were differentiated and then treated with pravastatin, simvastatin, or fluvastatin for 72 h. Hydrophobic simvastatin and fluvastatin decreased cell viability in a dose-dependent manner via apoptosis characterized by typical nuclear fragmentation and condensation and caspase-3 activation. Both hydrophobic statins transferred RhoA localization from the cell membrane to the cytosol. These changes induced by both hydrophobic statins were completely abolished by the co-application of geranylgeranylpyrophosphate (GGPP). Y27632, a Rho-kinase inhibitor, mimicked the hydrophobic statin-induced apoptosis. Hydrophilic pravastatin did not affect the viability of the cells. Fluvastatin was continuously infused (2.08 mg / kg at an infusion rate of 0.5 mL / h) into the right internal jugular vein of the rats in vivo for 72 h. Fluvastatin infusion significantly elevated the plasma CPK level and transferred RhoA localization in the skeletal muscle from the cell membrane to the cytosol. In conclusion, RhoA dysfunction due to loss of lipid modification with GGPP is involved in the mechanisms of statin-induced skeletal muscle toxicity.

Small interfering RNA (siRNA) mediated gene silencing has been utilized as a powerful molecular tool to study the functional significance of a specific protein. However, due to transient gene silencing and insufficient transfection efficiency, this approach can be problematic in primary cell culture such as vascular smooth muscle cells. To overcome this weakness, we utilized an adenoviral-encoded microRNA (miRNA)-embedded siRNA “mi/siRNA”-based RNA interference. Here, we report the results of silencing a disintegrin and metalloprotease 17 (ADAM17) in cultured rat vascular smooth muscle cells and its functional mechanism in angiotensin II signal transduction. 3 distinct mi/siRNA sequences targeting rat ADAM17 were inserted into pAd/CMV/V5-DEST and adenoviral solutions were obtained. Nearly 90% silencing of ADAM17 was achieved when vascular smooth muscle cells were infected with 100 multiplicity of infection of each ADAM17 mi/siRNA encoding adenovirus for 3 days. mi/siRNA-ADAM17 but not mi/siRNA-control inhibited angiotensin II-induced epidermal growth factor receptor trans-activation and subsequent extracellular signal-regulated kinase activation and hypertrophic response in the cells. mi/siRNA-ADAM17 also inhibited angiotensin II-induced heparin-binding epidermal growth factor-like factor shedding. This inhibition was rescued with co-infection of adenovirus encoding mouse ADAM17 but not by its cytosolic domain deletion mutant or cytosolic Y702F mutant. As expected, angiotensin II induced tyrosine phosphorylation of ADAM17 in the cells. In conclusion, ADAM17 activation via its tyrosine phosphorylation contributes to heparin-binding epidermal growth factor-like factor shedding and subsequent growth promoting signals induced by angiotensin II in vascular smooth muscle cells. An artificial mi/siRNA-based adenoviral approach appears to be a reliable gene-silencing strategy for signal transduction research in primary cultured vascular cells.

Summary The requirement of a metalloprotease ADAM17 (a disintegrin and metalloprotease 17) for the growth of cultured vascular smooth muscle cells has been demonstrated in vitro. However, whether this metalloprotease is responsible for vascular remodeling in vivo remains unanswered. Rat carotid arteries were analyzed 2 weeks after a balloon angioplasty. The neointimal cells were strongly positive for ADAM17 immunostaining. Marked inhibition of intimal hyperplasia was observed in dominant negative ADAM17 adenovirus treated carotid artery. Proliferating cell nuclear antigen positive cells and phospho-epidermal growth factor receptor positive cells in the neointima were reduced by dominant negative ADAM17 as well. In contrast, the neointima formation, proliferating cell nuclear antigen positive cells, and phospho-epidermal growth factor receptor positive cells were markedly enhanced by wild-type ADAM17 adenovirus. In conclusion, ADAM17 activation is involved in epidermal growth factor receptor activation and subsequent neointimal hyperplasia after vascular injury. ADAM17 could be a novel therapeutic target for pathophysiological vascular remodeling.