PURPOSE: Stimulation of human aortic smooth muscle cells (hAoSMCs) with native low-density lipoprotein (nLDL) induced the production of interleukin-8 (IL-8) that is involved in the pathogenesis of cardiovascular diseases. However, the process of signal transduction of nLDL was currently uncharacterized. Therefore, the aim of this study was to investigate the signal transduction pathway of nLDL-dependent IL-8 production and the effect of IL-8 on hAoSMCs migration. MATERIALS AND METHODS: nLDL was prepared by ultracentrifugation with density-adjusted human serum of normocholesterolemia. In hAoSMCs, IL-8 secreted to medium was measured using ELISA assay, and Western blot analysis was performed to detect p38 MAPK activation as a key regulator of IL-8 production. nLDL-dependent H2O2 generation was determined by microscopic analysis using 2',7'-dichlorofluoroscein diacetate (DCF-DA). IL-8-induced migration of hAoSMCs was evaluated by counting the cell numbers moved to lower chamber using Transwell plates. RESULTS: nLDL-induced IL-8 production was completely blocked by preincubation of hAoSMCs with pertussis toxin (PTX), which inhibited nLDL-dependent p38 MAPK phosphorylation. PTX-sensitive G-protein coupled receptor was responsible for nLDL-dependent H2O2 generation that was abrogated with preincubation of the cells with of polyethylene glycol-conjugated catalase (PEG-Cat). Pretreatment of PEG-Cat prevented nLDL-induced p38 MAPK phosphorylation and IL-8 production, which was partly mimicked by treatment with exogenous H2O2. Finally, IL-8 increased hAoSMCs migration that was completely blocked by incubation with IL-8 neutralizing antibody. CONCLUSION: PTX-sensitive G-protein coupled receptor-dependent H2O2 generation by nLDL plays a critical role in IL-8 production in hAoSMC, and IL-8 may contribute to atherogenesis through increased migration of hAoSMCs.
Cell Movement/*physiology ; Cells, Cultured ; Humans ; Hydrogen Peroxide/*metabolism ; Interleukin-8/*biosynthesis ; Lipoproteins, LDL/*pharmacology ; Muscle, Smooth, Vascular/cytology/*metabolism ; Myocytes, Smooth Muscle/cytology/*metabolism ; Pertussis Toxin/pharmacology ; Phosphorylation/drug effects ; Reactive Oxygen Species/metabolism ; Receptors, G-Protein-Coupled/*physiology ; Signal Transduction ; p38 Mitogen-Activated Protein Kinases/metabolism

Atherosclerotic vascular disease is the leading cause of morbidity and mortality in developed countries. While it is a complex condition resulting from numerous genetic and environmental factors, it is well recognized that oxidized low-density lipoprotein produces pro-atherogenic effects in endothelial cells (ECs) by inducing the expression of adhesion molecules, stimulating EC apoptosis, inducing superoxide anion formation and impairing protective endothelial nitric oxide (NO) formation. Emerging evidence suggests that the enzyme arginase reciprocally regulates NO synthase and NO production by competing for the common substrate L-arginine. As oxidized LDL (OxLDL) results in arginase activation/upregulation, it appears to be an important contributor to endothelial dysfunction by a mechanism that involves substrate limitation for endothelial NO synthase (eNOS) and NO synthesis. Additionally, arginase enhances production of reactive oxygen species by eNOS. Arginase inhibition in hypercholesterolemic (ApoE-/-) mice or arginase II deletion (ArgII-/-) mice restores endothelial vasorelaxant function, reduces vascular stiffness and markedly reduces atherosclerotic plaque burden. Furthermore, arginase activation contributes to vascular changes including polyamine-dependent vascular smooth muscle cell proliferation and collagen synthesis. Collectively, arginase may play a key role in the prevention and treatment of atherosclerotic vascular disease.
Animals ; Apoptosis ; Arginase ; Arginine ; Atherosclerosis ; Cell Proliferation ; Collagen ; Developed Countries ; Endothelial Cells ; Lipoproteins ; Lipoproteins, LDL ; Mice ; Muscle, Smooth, Vascular ; Nitric Oxide ; Nitric Oxide Synthase ; Nitric Oxide Synthase Type III ; Plaque, Atherosclerotic ; Reactive Oxygen Species ; Superoxides ; Vascular Diseases ; Vascular Stiffness

Arginase competitively inhibits nitric oxide synthase (NOS) via use of the common substrate L-arginine. Arginase II has recently reported as a novel therapeutic target for the treatment of cardiovascular diseases such as atherosclerosis. Here, we demonstrate that piceatannol-3'-O-beta-D-glucopyranoside (PG), a potent component of stilbenes, inhibits the activity of arginase I and II prepared from mouse liver and kidney lysates, respectively, in a dose-dependent manner. In human umbilical vein endothelial cells, incubation of PG markedly blocked arginase activity and increased NOx production, as measured by Griess assay. The PG effect was associated with increase of eNOS dimer ratio, although the protein levels of arginase II or eNOS were not changed. Furthermore, isolated mice aortic rings treated with PG showed inhibited arginase activity that resulted in increased nitric oxide (NO) production upto 78%, as measured using 4-amino-5-methylamino-2',7'-difluorescein (DAF-FM) and a decreased superoxide anions up to 63%, as measured using dihydroethidine (DHE) in the intact endothelium. PG showed IC50 value of 11.22 microM and 11.06 microM against arginase I and II, respectively. PG as an arginase inhibitor, therefore, represents a novel molecule for the therapy of cardiovascular diseases derived from endothelial dysfunction and may be used for the design of pharmaceutical compounds.
Animals ; Aorta/drug effects/metabolism ; Arginase/*antagonists & inhibitors/metabolism ; Dose-Response Relationship, Drug ; Endothelial Cells/drug effects/enzymology ; Enzyme Activation/drug effects ; Glucosides/chemistry/*pharmacology ; Humans ; Mice ; Mice, Inbred C57BL ; Nitrates/metabolism ; Nitric Oxide/biosynthesis ; Nitric Oxide Synthase Type III/*metabolism ; Nitrites/metabolism ; Reactive Oxygen Species/metabolism ; Rheum/*chemistry ; Stilbenes/chemistry/*pharmacology

Advanced age is one of the risk factors for vascular diseases that are mainly caused by impaired nitric oxide (NO) production. It has been demonstrated that endothelial arginase constrains the activity of endothelial nitric oxide synthase (eNOS) and limits NO generation. Hence, arginase inhibition is suggested to be vasoprotective in aging. In this study, we examined the effects of intravenous injection of Piceatannol, an arginase inhibitor, on aged mice. Our results show that Piceatannol administration reduced the blood pressure in aged mice by inhibiting arginase activity, which was associated with NO production and reactive oxygen species generation. In addition, Piceatannol administration recovered Ca²⁺/calmodulin-dependent protein kinase II phosphorylation, eNOS phosphorylation and eNOS dimer stability in the aged mice. The improved NO signaling was shown to be effective in attenuating the phenylephrine-dependent contractile response and in enhancing the acetylcholine-dependent vasorelaxation response in aortic rings from the aged mice. These data suggest Piceatannol as a potential treatment for vascular disease.
Administration, Intravenous* ; Aging ; Animals ; Arginase* ; Blood Pressure ; Injections, Intravenous ; Mice* ; Nitric Oxide ; Nitric Oxide Synthase Type III ; Phosphorylation ; Protein Kinases ; Reactive Oxygen Species ; Risk Factors ; Vascular Diseases ; Vasodilation

Cardiovascular disease is the prime cause of morbidity and mortality and the population ages that may contribute to increase in the occurrence of cardiovascular disease. Arginase upregulation is associated with impaired endothelial function in aged vascular system and thus may contribute to cardiovascular disease. According to recent research, Korean Red Ginseng water extract (KRGE) may reduce cardiovascular disease risk by improving vascular system health. The purpose of this study was to examine mechanisms contributing to age-related vascular endothelial dysfunction and to determine whether KRGE improves these functions in aged mice. Young (10+/-3 weeks) and aged (55+/-5 weeks) male mice (C57BL/6J) were orally administered 0, 10, or 20 mg/mouse/day of KRGE for 4 weeks. Animals were sacrificed and the aortas were removed. Endothelial arginase activity, nitric oxide (NO) generation and reactive oxygen species (ROS) production, endothelial nitric oxide synthase (eNOS) coupling, vascular tension, and plasma peroxynitrite production were measured. KRGE attenuated arginase activity, restored nitric oxide (NO) generation, reduced ROS production, and enhanced eNOS coupling in aged mice. KRGE also improved vascular tension in aged vessels, as indicated by increased acetylcholine-induced vasorelaxation and improved phenylephrine-stimulated vasoconstriction. Furthermore, KRGE prevented plasma peroxynitrite formation in aged mice, indicating reduced lipid peroxidation. These results suggest KRGE exerts vasoprotective effects by inhibiting arginase activity and augmenting NO signaling and may be a useful treatment for age-dependent vascular diseases.
Aging ; Animals ; Aorta ; Arginase* ; Cardiovascular Diseases ; Humans ; Lipid Peroxidation ; Male ; Mice* ; Mortality ; Nitric Oxide ; Nitric Oxide Synthase Type III ; Panax* ; Peroxynitrous Acid ; Plasma ; Reactive Oxygen Species ; Up-Regulation ; Vascular Diseases ; Vasoconstriction ; Vasodilation ; Water*

The incidence of cardiovascular disease is predicted to increase as the population ages. There is accumulating evidence that arginase upregulation is associated with impaired endothelial function. Here, we demonstrate that arginase II (ArgII) is upregulated in aortic vessels of aged mice and contributes to decreased nitric oxide (NO) generation and increased reactive oxygen species (ROS) production via endothelial nitric oxide synthase (eNOS) uncoupling. Inhibiting ArgII with small interfering RNA technique restored eNOS coupling to that observed in young mice and increased NO generation and decreased ROS production. Furthermore, enhanced vasoconstrictor responses to U46619 and attenuated vasorelaxation responses to acetylcholine in aged vasculature were markedly improved following siRNA treatment against ArgII. These results might be associated with increased L-arginine bioavailability. Collectively, these results suggest that ArgII may be a valuable target in age-dependent vascular diseases.
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid/pharmacology ; Aging ; Animals ; Aorta/enzymology/physiopathology ; Arginase/genetics/*metabolism ; Endothelium, Vascular/*enzymology/physiopathology ; Enzyme Induction ; Gene Knockdown Techniques ; Mice ; Mice, Inbred C57BL ; Nitric Oxide/metabolism ; Nitric Oxide Synthase Type III/*metabolism ; RNA, Small Interfering/genetics ; Reactive Oxygen Species/metabolism ; Up-Regulation ; Vasoconstriction/drug effects

BACKGROUND: Native low-density lipoprotein (nLDL) was one of the modifiable risk factors contributed directly to cardiovascular diseases development. We investigated that nLDL stimulation induced NADPH oxidase activation and superoxide production that was an important factor on human aortic smooth muscle cells (hAoSMC) proliferation. METHODS: Superoxide generation was recorded with fluorescent-staining of dihydroethidine or by measuring lucigenin-induced chemiluminescence for 5 minutes. We examined cell proliferation with 4[-3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) reagent and analyzed the change of gene expression by northern blot analysis. RESULTS: nLDL stimulation increased superoxide anion production in hAoSMC that confirmed through dihydroethidine staining and lucigenin-induced chemiluminescence methods. nLDL-induced proliferation abolished with preincubation of superoxide scavengers or NADPH oxidase inhibitor. NADPH as a substrate of NADPH oxidase increased superoxide generation in both nLDL-stimulated and unstimulated cell homogenate, which was completely blocked at the diphenylene iodinium (DPI)- or apocynin-pretreated hAoSMC homogenates. Furthermore, superoxide generation was only observed at the fraction of cellular precipitate, but not in soluble fraction. Expression of p22phox in mRNA level increased with nLDL treatment as early as 30 minutes and transfection of anti-sense oligonucleotide of p22phox completely abolished nLDL-induced proliferation of hAoSMC. CONCLUSIONS: The above results have shown that nLDL-induced proliferation in hAoSMC depends on superoxide production through NADPH oxidase activation.
Blotting, Northern ; Cardiovascular Diseases ; Cell Proliferation ; Dicarbethoxydihydrocollidine ; Gene Expression ; Humans ; Lipoproteins ; Luminescence ; Muscle, Smooth ; Myocytes, Smooth Muscle ; NADP ; NADPH Oxidase ; Risk Factors ; RNA, Messenger ; Superoxides ; Transfection

Caesalpinia sappan (C. sappan) is a medicinal plant used for promoting blood circulation and removing stasis. During a screening procedure on medicinal plants, the ethylacetate extract of the lignum of C. sappan (CLE) showed inhibitory activity on arginase which has recently been reported as a novel therapeutic target for the treatment of cardiovascular diseases such as atherosclerosis. CLE inhibited arginase II activity prepared from kidney lysate in a dose-dependent manner. In HUVECs, inhibition of arginase activity by CLE reciprocally increased NOx production through enhancement of eNOS dimer stability without any significant changes in the protein levels of eNOS and arginase II expression. Furthermore, CLE-dependent arginase inhibition resulted in increase of NO generation and decrease of superoxide production on endothelium of isolated mice aorta. These results indicate that CLE augments NO production on endothelium through inhibition of arginase activity, and may imply their usefulness for the treatment of cardiovascular diseases associated with endothelial dysfunction.
Animals ; Aorta ; Arginase ; Atherosclerosis ; Blood Circulation ; Caesalpinia ; Cardiovascular Diseases ; Endothelium ; Kidney ; Mass Screening ; Mice ; Nitric Oxide ; Nitric Oxide Synthase Type III ; Plants, Medicinal ; Superoxides

Elevated plasma concentration of native low-density lipoprotein (nLDL) is associated with vascular smooth muscle cell (VSMC) activation and cardiovascular disease. We investigated the mechanisms of superoxide generation and its contribution to pathophysiological cell proliferation in response to nLDL stimulation. Lucigenin-induced chemiluminescence was used to measure nLDL-induced superoxide production in human aortic smooth muscle cells (hAoSMCs). Superoxide production was increased by nicotinamide adenine dinucleotide phosphate (NADPH) and decreased by NADPH oxidase inhibitors in nLDL-stimulated hAoSMC and hAoSMC homogenates, as well as in prepared membrane fractions. Extracellular signal-regulated kinase 1/2 (Erk1/2), protein kinase C-theta (PKCtheta) and protein kinase C-beta (PKCbeta) were phosphorylated and maximally activated within 3 min of nLDL stimulation. Phosphorylated Erk1/2 mitogen-activated protein kinase, PKCtheta and PKCbeta stimulated interactions between p47phox and p22phox; these interactions were prevented by MEK and PKC inhibitors (PD98059 and calphostin C, respectively). These inhibitors decreased nLDL-dependent superoxide production and blocked translocation of p47phox to the membrane, as shown by epifluorescence imaging and cellular fractionation experiments. Proliferation assays showed that a small interfering RNA against p47phox, as well as superoxide scavenger and NADPH oxidase inhibitors, blocked nLDL-induced hAoSMC proliferation. The nLDL stimulation in deendothelialized aortic rings from C57BL/6J mice increased dihydroethidine fluorescence and induced p47phox translocation that was blocked by PD98059 or calphostin C. Isolated aortic SMCs from p47phox-/- mice (mAoSMCs) did not respond to nLDL stimulation. Furthermore, NADPH oxidase 1 (Nox1) was responsible for superoxide generation and cell proliferation in nLDL-stimulated hAoSMCs. These data demonstrated that NADPH oxidase activation contributed to cell proliferation in nLDL-stimulated hAoSMCs.
Animals ; Aorta/*cytology ; Cell Line ; Cell Proliferation ; Cells, Cultured ; Humans ; Lipoproteins, LDL/*metabolism ; Mice, Inbred C57BL ; Mitogen-Activated Protein Kinases/metabolism ; Muscle, Smooth, Vascular/cytology ; Myocytes, Smooth Muscle/*cytology ; NADPH Oxidase/*metabolism ; Phosphorylation ; Protein Kinase C/metabolism ; Signal Transduction ; Superoxides/metabolism

A 73 year-old man was admitted to our hospital because of septic shock. He had undergone drug eluting stent (DES) insertion 40 days before this admission. After about 50 days in the hospital, a coccyx sore required a flap operation. The anticoagulation therapy (aspirin and clopidogrel) was then changed to LMWH. Eleven days after this change, he developed very severe chest pain. The emergency coronary angiography showed occlusion of the left anterior descending artery, left circumflex artery, and right coronary artery due to thrombosis. During the angiography procedure, his heart collapsed. We performed cardio-pulmonary resuscitation (CPR), but were unsuccessful. Patients in very high risk groups need special attention during peri-operative periods. Doctors must have full knowledge about the different anticoagulation strategies, and cooperation among the different clinical departments is needed to properly treat these high risk patients.
Angiography ; Arteries ; Cardiopulmonary Resuscitation ; Chest Pain ; Coccyx ; Coronary Angiography ; Coronary Vessels ; Emergencies ; Heart ; Heparin, Low-Molecular-Weight ; Humans ; Percutaneous Coronary Intervention ; Shock, Septic ; Stents ; Thrombosis