BackgroundArginase II activity contributes to reciprocal regulation of endothelial nitric oxide synthase (eNOS). We tested the hypotheses that arginase II activity participates in the regulation of Ca2+/Ca2+/calmodulin‐dependent kinase II/eNOS activation, and this process is dependent on mitochondrial p32.Methods and ResultsDownregulation of arginase II increased the concentration of cytosolic Ca2+ ([Ca2+]c) and decreased mitochondrial Ca2+ ([Ca2+]m) in microscopic and fluorescence‐activated cell sorting analyses, resulting in augmented eNOS Ser1177 phosphorylation and decreased eNOS Thr495 phosphorylation through Ca2+/Ca2+/calmodulin‐dependent kinase II. These changes were observed in human umbilical vein endothelial cells treated with small interfering RNA against p32 (sip32). Using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry, fluorescence immunoassay, and ion chromatography, inhibition of arginase II reduced the amount of spermine, a binding molecule, and the release of Ca2+ from p32. In addition, arginase II gene knockdown using small interfering RNA and knockout arginase II‐null mice resulted in reduced p32 protein level. In the aortas of wild‐type mice, small interfering RNA against p32 induced eNOS Ser1177 phosphorylation and enhanced NO‐dependent vasorelaxation. Arginase activity, p32 protein expression, spermine amount, and [Ca2+]m were increased in the aortas from apolipoprotein E (ApoE−/−) mice fed a high‐cholesterol diet, and intravenous administration of small interfering RNA against p32 restored Ca2+/Ca2+/calmodulin‐dependent kinase II‐dependent eNOS Ser1177 phosphorylation and improved endothelial dysfunction. The effects of arginase II downregulation were not associated with elevated NO production when tested in aortic endothelia from eNOS knockout mice.ConclusionsThese data demonstrate a novel function of arginase II in regulation of Ca2+‐dependent eNOS phosphorylation. This novel mechanism drives arginase activation, mitochondrial dysfunction, endothelial dysfunction, and atherogenesis.
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 Ca2+ chelator BAPTA-AM. In mitochondrial Ca2+ measurements using Rhod-2 AM, increased mitochondrial Ca2+ levels by nLDL were inhibited upon preincubation with an arginase inhibitor. Among the polyamines, spermine, an arginase activity-dependent product, caused mitochondrial Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ uptake that is essential for p38 MAPK phosphorylation and IL-8 production.
Present cancer treatments using chemotherapy are limited owing to both significant side effects to normal cells and high recurrence rates. In this study, we demonstrated cancer cell-targeting nanoparticles that load multiple anticancer agents for both specific treatments to cancer and substantial therapeutic effects. For this purpose, hyaluronic acid (HA) was conjugated to mesoporous silica nanoparticles (MSNs) for specifically targeting cancer cells. Moreover, the prepared HA-MSNs exhibited high drug loading potential and sustained drug release. Compared to bare MSNs, the HA-MSNs were internalized at an approximately three times higher rate in squamous cell carcinoma 7 (SCC7) cells. To enhance the anticancer effects of chemotherapy and photodynamic therapy (PDT), doxorubicin (DOX) and chlorin e6 (Ce6) were loaded in HA-MSNs (DOX/Ce6/HA-MSNs); the product exhibited highly effective cytotoxicity on green fluorescent protein-expressing squamous cell carcinoma 7 (SCC7) compared to the corresponding free drugs and HA-MSNs with DOX or Ce6 alone. This study indicates that the application of DOX/Ce6/HA-MSNs in chemotherapy and PDT exerts significant therapeutic effects against SCC7.
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