NADPH Oxidase 1 Mediates Acute Blood Pressure Response to Angiotensin II by Contributing to Calcium Influx in Vascular Smooth Muscle Cells.

BACKGROUND: Reactive oxygen species (ROS) and calcium ions (Ca2+) are among the major effectors of Ang II (angiotensin II) in vascular smooth muscle cells. ROS are related to Ca2+ signaling or contraction induced by Ang II, but little is known about their detailed functions. Here, NOX (NADPH oxidase), a major ROS source responsive to Ang II, was investigated regarding its contribution to Ca2+ signaling. METHODS: Vascular smooth muscle cells were primary cultured from rat aorta. Ca2+ and ROS were monitored mainly using fura-2 and HyPer family probes' respectively. Signals activating NOX were examined with relevant pharmacological inhibitors and genetic manipulation techniques. RESULTS: Ang II-induced ROS generation was found to be biphasic: the first phase of ROS production, which was mainly mediated by NOX1, was small and transient, preceding a rise in Ca2+, and the second phase of ROS generation, mediated by NOX1 and NOX4, was slow but sizeable, continuing over tens of minutes. NOX1-derived superoxide in the first phase is required for Ca2+ influx through nonselective cation channels. AT1R (Ang II type 1 receptor)-Gßγ-PI3Kγ (phosphoinositide 3-kinase γ) signaling pathway was responsible for the rapid activation of NOX1 in the first phase, while in the second phase, NOX1 was further activated by a separate AT1R-Gαq/11-PLC (phospholipase C)-PKCß (protein kinase C ß) signaling axis. Consistent with these observations, aortas from NOX1-knockout mice exhibited reduced contractility in response to Ang II, and thus the acute pressor response to Ang II was also attenuated in NOX1-knockout mice. CONCLUSIONS: NOX1 mediates Ca2+ signal generation and thereby contributes to vascular contraction and blood pressure elevation by Ang II.

A mixture of chloromethylisothiazolinone and methylisothiazolinone impairs rat vascular smooth muscle by depleting thiols and thereby elevating cytosolic Zn2+ and generating reactive oxygen species.

Chloromethylisothiazolinone (CMIT) and methylisothiazolinone (MIT) are biocidal preservatives and the active ingredients in Kathon CG, which contains ca. 1.5% mixture of CMIT and MIT at a ratio of 3:1 (CMIT/MIT). CMIT/MIT was misused as humidifier disinfectant products, which caused serious health problems in Korea. Here, the vascular effects of CMIT/MIT were investigated to evaluate claims of putative cardiovascular toxicity observed in humidifier disinfectant users. CMIT/MIT did not affect the basal tension of the rat thoracic aorta up to 2.5 µg/mL in myograph experiments. Instead, pretreatment with CMIT/MIT impaired phenylephrine- or 5-hydroxytryptamine-induced vasoconstriction in a range of 0.5-2.5 µg/mL, which was largely irreversible and not recovered by washing out the CMIT/MIT. Similarly, the application of CMIT/MIT to pre-contracted aorta caused a gradual loss of tension. In primary cultured vascular smooth muscle cells (VSMCs), CMIT/MIT caused thiol depletion, which in turn led to cytosolic Zn2+ elevation and reactive oxygen species (ROS) formation. CMIT/MIT-induced shrinkage, detachment, and lysis of VSMCs depending on the concentration and the treatment time. All events induced by CMIT/MIT were prevented by a thiol donor N-acetylcysteine (NAC). Cytolysis could be inhibited by a Zn2+ chelator TPEN and a superoxide scavenger TEMPOL, whereas they did not affect shrinkage and detachment. In accordance with these results, CMIT/MIT-exposed aortas exhibited dissociation and collapse of tissue in histology analysis. Taken together, CMIT/MIT causes functional impairment and tissue damage to blood vessels by depleting thiol and thereby elevating cytosolic Zn2+ and generating ROS. Therefore, exposure to CMIT/MIT in consumer products may be a risk factor for cardiovascular disorders.

Cigarette Smoke Extract Produces Superoxide in Aqueous Media by Reacting with Bicarbonate.

The toxicity of cigarette smoke (CS) is largely attributed to its ability to generate reactive oxygen species (ROS). Reportedly, CS generates superoxide in cell culture systems by stimulating the cells to produce superoxide and through direct chemical reactions with components of the culture media. In this study, we investigated CS-induced superoxide formation in biocompatible aqueous media and its characteristics. Cigarette smoke extract (CSE) and total particulate matter (TPM) were prepared from the mainstream smoke of 3R4F reference cigarettes. CSE and TPM generated superoxide in Hank's balanced salt solution (HBSS), Dulbecco's modified Eagle media (DMEM), and blood plasma, but not in distilled water and phosphate-buffered saline. Each constituent of HBSS in solution was tested, and bicarbonate was found to be responsible for the superoxide generation. More than half of the superoxide formation was abolished by pretreating CSE or TPM with peroxidase, indicating that the substrates of peroxidase, presumably peroxides and peroxy acids, mainly contributed to the superoxide production. In conclusion, the presence of bicarbonate in experimental conditions should be considered carefully in studies of the biological activity of CS. Furthermore, the local amount of bicarbonate in exposed tissues may be a determinant of tissue sensitivity to oxidative damage by CS.

Application of Fisetin to the Quantitation of Serum Albumin.

Fisetin (3,3',4',7-tetrahydroxyflavone) is a widely distributed natural flavonol. It interacts with albumin, and thereby generates a fluorescence signal quantitatively. Based on such optical characteristics, we postulated that fisetin was applicable to the quantitation of albumin as an indicator. To establish the fisetin-based albumin assay, we examined the optical properties of fisetin and fisetin-albumin complex. The assay conditions were fine-tuned to fit for the actual concentration of serum albumin and to generate an optimal signal with a high signal-to-background ratio. The reaction between fisetin and albumin was linear in a wide range of concentrations. Non-protein serum components did not interfere with the reaction. The reactivity of fisetin was apparently specific for albumin among serum proteins. Both plasma and serum were compatible with the assay. The samples could be stored in a refrigerator or a freezer without the loss of reactivity toward fisetin. The generation and decay rates of the signal were acceptable for manual handling. The recovery of fortified albumin in serum was confirmed and the assay was validated with human sera. Fisetin-based albumin assay is suitable for clinical laboratory testing, considering the simple and short procedure, high specificity and sensitivity, linearity over a wide range of albumin concentrations, and, presumably, potential automatability.

Inhalation exposure to cigarette smoke induces endothelial nitric oxide synthase uncoupling and enhances vascular collagen deposition in streptozotocin-induced diabetic rats.

Smoking is an acknowledged risk factor for vascular disorders, and vascular complication is a main outcome of diabetes. Hence, we investigated the impact of cigarette smoke on blood vessels in diabetes, postulating that smoking might aggravate diabetic vascular impairment. Sprague-Dawley rats were divided into four groups: control, cigarette smoke-exposed, diabetic, and cigarette smoke-exposed diabetic groups. Streptozotocin-induced diabetic rats were exposed to cigarette smoke by inhalation at total particulate matter concentration of 200 µg/L for 4 h/day, 5 day/week for a total of 4 weeks. Diabetes caused structural change of aorta, but additional cigarette smoke exposure did not induce further alteration. Collagen, a marker for fibrosis, was increased in media of diabetic aorta, and this increase was augmented by cigarette smoke. Cigarette smoke induced endothelial nitric oxide synthase (eNOS) uncoupling in the diabetic group. Malondialdehyde was increased and glutathione was decreased in blood from diabetes, but these effects were not exaggerated by cigarette smoke. Cigarette smoke caused NADPH oxidase (NOX) 2 expression in diabetic aorta and enhanced diabetes-induced NOX4 expression in aorta. Taken together, cigarette smoke exposure can aggravate vascular fibrosis and induce eNOS uncoupling in diabetes under experimental condition, suggesting that smoking might exacerbate diabetic vascular impairments.

Comparative In Vitro Toxicity Study of Docetaxel and Nanoxel, a Docetaxel-Loaded Micellar Formulation Using Cultured and Blood Cells.

Nanoxel-PMTM (Nanoxel) is a docetaxel-loaded methoxy-poly(ethylene glycol)-block-poly(D,L-lactide) (mPEG-PDLLA). This newly developed and marketed nanoformulation exhibits an improved pharmacokinetic profile, efficacy, and safety. Although the safety of Nanoxel to docetaxel as well as its bioequivalence must be clinically confirmed, all biological activities have not been examined in in vitro or in vivo studies. Here, the toxicity in a cultured cell system and the effects on blood cells were tested with Nanoxel and docetaxel. The in vitro cytotoxicity of Nanoxel was found to be comparable to or slightly lower than that of docetaxel depending on the concentrations tested or the cell types. Neither docetaxel nor Nanoxel induced erythrocytes hemolysis and produced reactive oxygen species up to 100 µM. However, Nanoxel was able to enhance the aggregatory response of platelets to collagen, whereas docetaxel attenuated such aggregation in a range of 50-100 µM, while thrombin-induced aggregation was not affected by either of them. Docetaxel or Nanoxel did not alter basal level of Ca2+ and 5-hydroxytryptamine-evoked Ca2+ transient in vascular smooth muscle cells. These results suggest that the mPEG-PDLLA micellar formulation alters the toxicological properties of docetaxel, and that extra cautions are needed when evaluating the safety of nanomedicine.