A Water-Soluble Fullerene Vesicle Alleviates Angiotensin II−Induced Oxidative Stress in Human Umbilical Venous Endothelial Cells

141 Hypertens Res Vol.31 (2008) No.1 p.141-151 Original Article A Water-Soluble Fullerene Vesicle Alleviates Angiotensin II–Induced Oxidative Stress in Human Umbilical Venous Endothelial Cells Rui MAEDA1),2), Eisei NOIRI1),3),4), Hiroyuki ISOBE1),2), Tatsuya HOMMA2), Tamami TANAKA4), Kousuke NEGISHI4), Kent DOI4), Toshiro FUJITA3),4), and Eiichi NAKAMURA1),2),5) A water-soluble fullerene vesicle based on the Buckminsterfullerene molecule (Ph5C60K, denoted as PhK) was explored to determine its effects on anti-oxidation of human umbilical endothelial cells (HUVEC) exposed to exogenous and endogenous reactive oxygen species (ROS). Hydrogen peroxide 0.05–0.25 mmol/L remarkably reduced the cellular viability of HUVEC. This reduction in viability was markedly improved when PhK 0.01–1 µmol/L was added simultaneously to the culture medium. The reduction of viability in HUVEC induced by angiotensin II (AII) 10 – 9 to 10 – 7 mol/L was improved by pretreatment with PhK 0.1 or 10 µmol/L 12 h before AII stimulation. The ROS indicator CM-H2DCFDA demonstrated the efficacy of PhK 1 or 10 µmol/L in decreasing AII-induced ROS production to the level induced by the AII receptor blocker RNH-6470 20 µmol/L. The AII-induced peroxynitrite formation, as gauged using hydroxyphenyl fluorescein as a probe, was alleviated significantly by either pretreatment with PhK 0.1 or 1 µmol/L. Electron microscopy revealed intracellular localization of PhK in HUVEC after 12 h incubation. The PhK decreased the AII-induced apoptosis and lipid peroxidation processes as revealed by hexanoyl-lysine adduct formation. These observations show that the PhK water-soluble fullerene vesicle is promising as a compound controlling not only exogenous ROS, but also endogenous AII-mediated pathophysiological conditions. (Hypertens Res 2008; 31: 141–151) Key Words: reactive oxygen species, endothelial cells, nano-compound, apoptosis Introduction It has recently been suggested that angiotensin II (AII) is not simply an autacoid with hemodynamic and renal actions, but rather a biologically active mediator that imparts direct effects on endothelial and vascular smooth muscle cells. Actually, AII plays a key role in the initiation and amplification of pathobiological events leading to vascular disease. In addition, AII is a major mediator of oxidative stress and From the 1)Center for NanoBio Integration, 2)Department of Chemistry, 3)Department of Hemodialysis and Apheresis, 4)Department of Nephrology and Endocrinology, The University of Tokyo, Tokyo, Japan; and 5)Japan Science and Technology Agency, ERATO, Nakamura Functional Carbon Cluster Project, Tokyo, Japan. Part of this study was supported by the NanoBio Integration Program, Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (R.M., E.N., H.I., E.N.), by Health and Labour Sciences Research Grants for Research on Human Genome, Tissue Engineering Food Biotechnology from the Ministry of Health, Labour and Welfare, Japan (057100000661 E.N.), by a Grant-in-Aid for Scientific Research from the MEXT, Japan (19590935 E.N.), by the BioBank Japan Project on the Implementation of Personalized Medicine, MEXT, Japan (3023168 E.N.), by Special Coordination Funds for Promoting Science and Technologies, MEXT, Japan (1200015 E.N.), and by ERATO, JST, Japan (Nakamura Functional Carbon Cluster Project; E.N.). Address for Reprints: Eisei Noiri, M.D., Department of Nephrology 107 Laboratory, University Hospital, The University of Tokyo, 7–3–1 Hongo, Bunkyo-ku, Tokyo 113–8655, Japan. E-mail: Received April 19, 2007; Accepted in revised form August 5, 2007. 142 Hypertens Res Vol. 31, No. 1 (2008) reduces nitric oxide (NO) activity by activating NADH/ NADPH oxidase (1), thereby generating superoxide anion (O2• −) according to the reaction NADPH + 2O2 → 2O2• − + NADP •+ + H •+. Found initially in phagocytic cells, NAD(P)H oxidase has also been identified in non-phagocytic cells such as fibroblasts (2), endothelial cells (3), vascular smooth muscle cells (4), renal mesangial cells (5, 6), and renal tubular cells (7, 8). The upregulation of NAD(P)H oxidase by AII has been demonstrated in aortic vascular smooth muscle (9, 10), afferent arterioles of the glomerulus (11), and endothelial cells. Reaction between the superoxide anion and nitric oxide will generate peroxynitrite, resulting in oxidative stresses that are highly damaging to endothelial cells. Moreover, studies using chronic kidney disease rodent models have suggested that AII receptor blockade has beneficial effects beyond mere blood-pressure lowering (12, 13). It would be of additional interest if AII were to generate superoxide independent of the AII receptor. Therefore, the reduction of AII-mediated oxidative stress presents one approach to control macro-vasculature and micro-vasculature remodeling and endothelial apoptosis (14). Osawa hypothesized in 1970 that fullerenes were the third allotropic carbon form, following graphite and diamond. Because the corannulene molecular structure was a subset of a structure with a soccer ball–like shape, he hypothesized the existence of a full ball (15). In 1985, Osawa’s prediction— which had been made in a local chemistry journal “Kagaku” in Japan and gone mostly unnoticed for over a decade—was rediscovered by Kroto et al. (16), who began the search anew, and the fullerene was finally isolated in 1990 by Krätschmer et al. (17). Among fullerenes, C60, or the Buckminsterfullerene, is the smallest. As predicted, it resembles a soccer ball, comprising hexagons and pentagons with no two hexagons sharing an edge. This fullerene is naturally occurring, and commonly found in candle soot. The anti-oxidative potency of native C60 fullerenes has been reported by Krusic et al. (18). The spheroidal structure of C60 fullerenes, consisting of numerous interconnected double bonds, is highly reactive with oxygen free radicals. Therefore, the chemical capability to receive oxygen free radicals will be superior to that of known scavengers. Water-soluble fullerenes were first reported by Chiang et al. (19), Friedman et al. (20), and Tokuyama et al. (21). The solubility was secured by either allelic hydroxy or carboxylic acid functional groups synthesized on carbon atoms of the fullerene structure, as seen in fullerenol-1 (C60(OH)nOm, n= 18–20, m= 3–7 hemiketal moieties). Subsequently, soluble fullerenes like fullerenol-1 were applied to life sciences. Furthermore, the electric paramagnetic resonance measurement demonstrated that fullerenol-1 virtually abolishes the •OH signal generated by hydrogen peroxide. It is also noteworthy that, by the addition of •OH radicals to the fullerenol-1 molecule, the spheroidal structure of C60 provided more stable adducts of C60. In spite of these theoretical aspects implying an inert nature, accumulating evidence supports the notion that fullerenes have prooxidant characteristics (22–25). Most of these studies were performed using water-insoluble (...truncated)