PURPOSE: Vascular smooth muscle cell (VSMC) proliferation induced by native low-density lipoprotein (nLDL) stimulation is dependent on superoxide production from activated NADPH oxidase. The present study aimed to investigate whether the novel arginase inhibitor limonin could suppress nLDL-induced VSMC proliferation and to examine related mechanisms. MATERIALS AND METHODS: Isolated VSMCs from rat aortas were treated with nLDL, and cell proliferation was measured by WST-1 and BrdU assays. NADPH oxidase activation was evaluated by lucigenin-induced chemiluminescence, and phosphorylation of protein kinase C (PKC) βII and extracellular signal-regulated kinase (ERK) 1/2 was determined by western blot analysis. Mitochondrial reactive oxygen species (ROS) generation was assessed using MitoSOX-red, and intracellular L-arginine concentrations were determined by high-performance liquid chromatography (HPLC) in the presence or absence of limonin. RESULTS: Limonin inhibited arginase I and II activity in the uncompetitive mode, and prevented nLDL-induced VSMC proliferation in a p21Waf1/Cip1-dependent manner without affecting arginase protein levels. Limonin blocked PKCβII phosphorylation, but not ERK1/2 phosphorylation, and translocation of p47phox to the membrane was decreased, as was superoxide production in nLDL-stimulated VSMCs. Moreover, mitochondrial ROS generation was increased by nLDL stimulation and blocked by preincubation with limonin. Mitochondrial ROS production was responsible for the phosphorylation of PKCβII. HPLC analysis showed that arginase inhibition with limonin increases intracellular L-arginine concentrations, but decreases polyamine concentrations. L-Arginine treatment prevented PKCβII phosphorylation without affecting ERK1/2 phosphorylation. CONCLUSION: Increased L-arginine levels following limonin-dependent arginase inhibition prohibited NADPH oxidase activation in a PKCβII-dependent manner, and blocked nLDL-stimulated VSMC proliferation.
Animals ; Aorta ; Arginase* ; Arginine ; Blotting, Western ; Bromodeoxyuridine ; Cell Proliferation* ; Chromatography, High Pressure Liquid ; Chromatography, Liquid ; Lipoproteins ; Luminescence ; Membranes ; Muscle, Smooth, Vascular* ; NADP* ; NADPH Oxidase* ; Phosphorylation ; Phosphotransferases ; Protein Kinase C ; Rats ; Reactive Oxygen Species ; Superoxides

Arginase inhibition exhibits beneficial effects in vascular endothelial and smooth muscle cells. In human aortic smooth muscle cells (hAoSMCs), native low-density lipoprotein (nLDL) induced the production of interleukin-8 (IL-8) that is involved in the pathogenesis of cardiovascular diseases. Therefore, we examined the effect of arginase inhibition on IL-8 production and the underlying mechanism. In hAoSMCs, reverse transcription–PCR, western blotting and immunocytochemistry with MitoTracker confirmed that arginase II was confined predominantly to mitochondria. The mitochondrial membrane potential (MMP) was assessed using tetramethylrhodamine ethyl ester. The MMP decreased upon nLDL stimulation but was restored upon arginase inhibition. MMP loss caused by nLDL was prevented by treatment with the intracellular Ca(2+) chelator BAPTA-AM. In mitochondrial Ca(2+) measurements using Rhod-2 AM, increased mitochondrial Ca(2+) levels by nLDL were inhibited upon preincubation with an arginase inhibitor. Among the polyamines, spermine, an arginase activity-dependent product, caused mitochondrial Ca(2+) movement. The nLDL-induced MMP change resulted in p38 mitogen-activated protein kinase (MAPK) phosphorylation and IL-8 production and was prevented by the arginase inhibitors BAPTA and ruthenium 360. In isolated AoSMCs from ApoE(−/−) mice fed a high-cholesterol diet, arginase activity, p38 MAPK phosphorylation, spermine and mitochondrial Ca(2+) levels and keratinocyte-derived chemokine (KC) production were increased compared with wild-type (WT) mice. However, in AoSMCs isolated from arginase II-null mice, increases in MMP and decreases in mitochondrial Ca(2+) levels were noted compared with WT and were associated with p38 MAPK activation and IL-8 production. These data suggest that arginase activity regulates the change in MMP through Ca(2+) uptake that is essential for p38 MAPK phosphorylation and IL-8 production.