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J Pharmacol Sci 119, 349 – 358 (2012) Journal of Pharmacological Sciences © The Japanese Pharmacological Society Full Paper Paclitaxel-Induced in Living Rats Is Prevented by via Reduction of

Ken-ichi Serizawa1, Kenji Yogo1, Ken Aizawa1, Yoshihito Tashiro1, Yoko Takahari2, Kaori Sekine3, Toshihiko Suzuki4, Nobuhiko Ishizuka1,*, and Hideyuki Ishida4 1Product Research Department, Chugai Pharmaceutical Co., Ltd., 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan 2Teaching and Research Support Center, 3Department of Pediatrics, 4Department of Physiology, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan

Received March 18, 2012; Accepted June 14, 2012

Abstract. Paclitaxel-eluting stents dramatically reduce rates of in-stent restenosis; however, paclitaxel is known to lead to endothelial dysfunction. Protective effects of nicorandil on paclitaxel- induced endothelial dysfunction by examining flow-mediated dilation (FMD) were investigated in anesthetized rats. After 7-day osmotic infusion of paclitaxel (5 mg/kg per day), FMD was mea- sured by high-resolution ultrasound in the femoral artery of living rats. Paclitaxel significantly re- duced FMD (21.6% ± 3.2% to 7.1% ± 1.7%); this reduction was prevented by co-treatment with nicorandil (15 mg/kg per day), while paclitaxel did not affect -induced . Diazoxide and tempol, but not , had an effect similar to nicorandil in preventing paclitaxel-induced decrease in FMD. Nicorandil significantly prevented paclitaxel-induced reduc- tion in acetylcholine-induced vasodilation. On the underling mechanisms, paclitaxel increased reactive oxygen species (ROS) production (dihydrorhodamine 123, DCF fluorescence intensity) and NADPH oxidase (p47phox, gp91phox mRNA) in arteries and human coronary artery endothelial cells (HCAECs), while paclitaxel reduced (NO) release (DAF-2 fluorescence intensity), but not endothelial NO synthase (eNOS) phosphorylation in HCAECs. Nicorandil prevented the increased ROS production in arteries and HCAECs, which was 5-hydroxydecanoate (5-HD)- sensitive but 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ)-resistant, without significant effect on the reduced NO release. In conclusion, nicorandil prevents paclitaxel-induced endothelial dysfunction, which may be brought by improved NO bioavailability due to the reduction of oxida-

tive stress via KATP channel activation.

Keywords: endothelial dysfunction, flow-mediated dilation, nicorandil, paclitaxel, reactive oxygen species

Introduction (DES) have come to be thought of as another potential cause of endothelial dysfunction, despite their successful It is well known that the endothelium plays an impor- use in the treatment of ischemic heart diseases with the tant role in the regulation of vascular tone via release of accompanying dramatic reduction in in-stent restenosis factors such as nitric oxide (NO), PGI2, EDHF, and en- (2 – 4). Paclitaxel, one of the major drugs used in DES, dothelin. Endothelial dysfunction, a predictor of adverse is known to cause endothelial dysfunction in humans (5) cardiac events in coronary artery disease patients (1), is and swine (6) and to induce apoptosis and cytotoxicity in mainly caused by loss of NO that occurs under several human coronary artery endothelial cells (HCAECs) (7). pathological conditions such as atherosclerosis, hyper- Nicorandil, an anti-anginal drug with ATP-sensitive tension, and . Recently, drug-eluting stents potassium channel (KATP channel)-opening activity and -like activity, was shown in the IONA (Impact Of *Corresponding author. [email protected] Nicorandil in Angina) (8) and JCAD (Japanese Coronary Published online in J-STAGE on July 24, 2012 (in advance) Artery Disease) (9) studies to improve the prognosis of doi: 10.1254/jphs.12067FP patients with ischemic heart disease via a pharmacologi-

349 350 K Serizawa et al cal preconditioning effect (10). It is also reported, both in conform to the Guide for the Care and Use of Laboratory the clinical setting and in animal studies, that the reduc- Animals published by the US National Institutes of tion in cardiac events is due in part to the endothelial Health. All animals were euthanized by exsanguination protective effects of nicorandil: nicorandil improved under anesthesia after experiments. flow-mediated dilation (FMD, an indicator of endothelial function) in patients with ischemic heart disease (11) or Measurements of endothelial function in live rats with cardiovascular risk factors (12) and reduced myo- Male Sprague Dawley rats (250 – 300 g) were used. cardial no-reflow in swine by protecting endothelial All rats were fed ordinary laboratory chow and allowed function (13). However, there is no report of nicorandil free access to water under a constant light and dark cycle having a protective effect on paclitaxel-induced endothe- of 12 h. Rats were anesthetized with an intraperitoneal lial dysfunction. injection of pentobarbital (50 mg/kg) and adequate anes- In the present study, we investigated the protective thetic depth was monitored by pinching the toe. An os- effect of nicorandil on paclitaxel-induced endothelial motic pump (model 2ML1; Durect Corp., Cupertino, dysfunction and also the mechanisms underlying this CA, USA) was implanted subcutaneously into a small protective effect in association with NO bioavailability pouch between the shoulder blades, with topical applica- in vascular endothelial cells. In the clinical setting, en- tion of bupivacaine (5 mg/mL) to the skin wound, for dothelial function is evaluated by measuring FMD, and continuous infusion of paclitaxel (0.5, 1.5, 5 mg/kg per FMD has also been used to evaluate endothelial function day). For the vehicle group, an osmotic pump containing in dogs and pigs (14, 15). Recently, we reported the vehicle for paclitaxel (described above) was subcutane- method for measurement of endothelial function by FMD ously implanted. Nicorandil (15 mg/kg per day) and in rats (16). Therefore, in this study we measured FMD tempol (20 mg/kg per day) were dissolved in drinking in the femoral artery of living rats by using high-resolu- water and administered for 1 week from just after im- tion ultrasound under conditions that maintained blood plantation of the osmotic pump. Diazoxide (15 mg/kg) flow, many humoral factors, and nerve activity. and ISDN (15 mg/kg) were administrated by gastric gavage once a day for 1 week from just after osmotic Materials and Methods pump implantation. FMD was measured in rats 1 week after implantation of the osmotic pump. Chemicals FMD was measured in the rats as previously described Nicorandil [N-(2-hydroxyethyl)nicotinamide nitrate (16). Briefly, rats were anesthetized with thiobutabarbital ester] was synthesized in the Chugai Organic Chemistry (120 mg/kg, i.p.) and adequate anesthetic depth was Laboratory. Paclitaxel and other reagents used were of monitored by pinching the toe. A catheter was inserted analytical grade from Wako Pure Chemicals Co. (Osaka). into the jugular vein for drug administration, while the 4-Hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl body core temperature was kept stable. The femoral arte- (tempol), 5-hydroxydecanoate (5-HD), and 1H-[1,2,4] rial diameter and Doppler flow were measured by using oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), isosorbide a high-resolution ultrasound system with a 30-MHz dinitrate (ISDN), and diazoxide were all purchased from transducer (Vevo770; VisualSonics, Toronto, Canada). Sigma-Aldrich (St. Louis, MO, USA). Nitroglycerin After the femoral artery was identified by its distinctive (NTG; Nippon Kayaku, Tokyo) and acetylcholine flow pattern, the position of the probe was optimized to (Daiichi-Sankyo, Tokyo) were purchased. Paclitaxel was clearly show the vessel wall/lumen interface and was dissolved in 50% dimethylformamide, 25% Cremophor fixed throughout the experiment. Vascular diameter and EL, and 25% distilled water. Diazoxide and ISDN were Doppler flow were obtained from longitudinal sections suspended in 3% gum arabic solution. NTG and acetyl- of the femoral artery before and after 5 min of hind limb choline were dissolved in and diluted with 0.9% saline ischemia. A snare occluder (5–0 nylon surgical suture solution, and the other drugs were freshly dissolved in passed around the artery and through a 4-cm length of distilled water just before the experiment. The concentra- PE-200 tubing) was positioned upstream of the visual- tions of paclitaxel used in this study were chosen on the ized area at the common iliac artery through a transab- basis of previous studies (7, 17). dominal access. After the common iliac artery was oc- cluded, flow arrest was confirmed by abrogation of the Animals Doppler signal. The changes in flow velocity and All animal procedures were conducted in accordance diameter of the femoral artery occurring with reperfusion with Chugai Pharmaceutical’s ethical guidelines for ani- after 5 min of ischemia were monitored until 3 min after mal care, and all experimental protocols were approved reperfusion. After the FMD measurements, NTG (5 μg/ by the Animal Care Committee of the institution and kg, i.v.) was injected via the jugular vein catheter to Endothelial Protection by Nicorandil 351 evaluate endothelium-independent vasodilation. Acetyl- overnight incubation, paclitaxel (10 ng/mL) was added choline (3 ng) was injected into the aorta just proximal to to the culture medium for 24 h. Nicorandil (100 μM), the iliac bifurcation to evaluate endothelium-dependent 5-HD (500 μM), and ODQ (20 μM) were added over the vasodilation. same period. The cellular level of ROS was monitored by using the Real-time PCR analysis in rat femoral arteries fluorescent probe 2′,7′-dichlorodihydrofluorescein diac-

After measurement of FMD, femoral arteries were etate (H2DCFDA) (Invitrogen, Carlsbad, CA, USA). harvested and stored in RNAlater solution (Ambion, Oxidation by ROS converts H2DCFDA to highly fluores- Austin, TX, USA) until RNA isolation was performed. cent 2′,7′-dichlorofluorescein (DCF). Briefly, cultured

Tissues were homogenized using a Micro Smash homog- cells were incubated with 10 μM H2DCFDA for 45 min, enizer (Tomy Digital Biology, Tokyo), and total RNA and then DCF fluorescence from at least 20 cells/dish, 3 was isolated using an RNeasy Fibrous Tissue kit (Qiagen, dishes per experimental condition, was quantified with a Dusseldorf, Germany). TaqMan real-time PCR was confocal microscope (Zeiss Axiovert 200; Carl Zeiss performed using TaqMan Gene Expression Assays for Microscopy, Göttingen, Germany) and quantified with NADPH oxidase components p47phox and gp91phox in an the Image J program (version 1.240, http://rsb.info.nih. ABI PRISM 7500 Sequence Detection System (Applied gov/nih-image/). The data were obtained with three inde- Biosystems, Carlsbad, CA, USA). Gene expression was pendent measurements for each experimental con dition. normalized to the endogenous control, β-actin. Measurements of NO in HCAECs Measurement of reactive oxygen species (ROS) in mouse Intracellular NO was assessed with diaminofluores- testicular arteries cein-2 diacetate (DAF-2DA; Sekisui Medical, Tokyo) In vivo measurement of ROS production monitored by fluorescence probe (10 μM) (19). Briefly, HCAECs were using the fluorescent probe, dihydrorhodamine 123 hy- incubated with PBS containing 10 μM DAF-2 in the dark drochloride (Wako Pure Chemical) in mice was con- for 30 min at 37°C and then washed twice with PBS. ducted as previously described (18). Briefly, male ICR Fluorescence intensity was next measured by spectrofluo- mice (10 – 11-week-old) were anesthetized with pento- rophotometry (SpectraMax Gemini XS; Molecular barbital (50 mg/kg, i.p.). Endothelial cells were observed Devices, Sunnyvale, CA, USA) with excitation and by FITC-labeled isolectin B4, a murine-specific endothe- emission wavelengths of 485 and 545 nm, respectively. lial cell marker (Vector Lab, Burlingame, CA, USA). After the testicular artery (approx. 200 μm in diameter) Statistical analyses was carefully exposed, dihydrorhodamine 123 (2.5 mg/ All data are expressed as the mean ± S.E.M. The n kg) was given intravenously, followed by intravenous values refer to the number of individual animals in which administration of FITC-labeled isolectin B4 (1.25 mg/ experiments were performed. The statistical significance kg). The fluorescence intensity dependent on the increase of differences was determined by the Tukey Multiple in ROS was observed by an ultrafast laser confocal mi- Comparison Test. Probability (P) values of less than 0.05 croscope equipped with a piezoelectric motor control were considered significant. unit and was recorded as arbitrary units (au). Under pentobarbital anesthesia, mice were subcutaneously im- Results planted with the osmotic pump (model 1007D, Durect Corp.), with body temperature kept stable and monitored Measurements of FMD in rats for adequate anesthetic depth by the pinching toe of the In vehicle-treated rats, reperfusion after 5 min of hind animal. Paclitaxel (5 mg/kg per day), was continuously limb ischemia led to an instantaneous increase in femoral infused from the osmotic pump. Nicorandil (15 mg/kg arterial flow velocity (reactive hyperemia) compared per day) was administrated in drinking water from just with baseline (Fig. 1: A, B), followed by rapid decay to after infusion of paclitaxel. baseline values at around 3 min (Fig. 1C). Ischemia also caused an increase in the diameter of the rat femoral ar- Measurements of ROS in HCAECs tery from baseline (Fig. 1D) to 1 min after reperfusion HCAECs were purchased from Lonza (Walkersville, (Fig. 1E). The increase in flow velocity was associated MD, USA) and cultured in EBM-2 supplemented with with the delayed increase in femoral arterial vasodilation 5% fetal bovine serum. For the measurements of ROS that peaked at 1 min (Fig. 1F). This delayed vasodilation production, the cells were seeded onto plastic dishes in the femoral artery is referred to as FMD. (1 × 105 cells / 2 mL per dish) and cultured in monolay- ers in a 5% CO2 humidified incubator at 37°C. After 352 K Serizawa et al

A B C 200 Baseline Just aŌer reperfusion (0 min) 180 20 20 10 10 160 0 0 140 -10 -10 5 min ischemia Velocity (cm/s) Velocity Velocity (cm/s) Velocity 120 -20 -20

Peak velocity (mm/s) velocity Peak 100 Baseline 0 1 2 3 Time aŌer reperfusion (min) D E F Baseline 1 min aŌer reperfusion 30

%) 25 20

i 15 F.A. 10 5 5 min ischemia

Vasodilat on ( Vasodilat 0 -5 Baseline 0 1 2 3 Time aŌer reperfusion (min)

Fig. 1. Measurements of post-ischemic reactive hyperemia and vasodilation of the rat hind limb. Original Doppler flow velocity tracings at baseline (A) and at onset of reperfusion after 5-min occlusion of the common iliac artery (B). C) Time course of peak flow velocity after 5-min ischemia. Representative ultrasound image of femoral artery at baseline (D) and 1 min after reperfusion (E). Longitudinal sections of the rat femoral artery were visualized with a high-resolution ultrasound probe placed on the upper thigh. F) Vasodilation (percentage increase in arterial diameter) after 5 min ischemia (n = 5). F.A. means femoral artery. Scale bar = 1 mm.

Effects of nicorandil on reduced FMD in paclitaxel- that treatment with paclitaxel and nicorandil for 1 week treated rats did not affect bodyweight, blood pressure, heart rate, or Continuous subcutaneous infusion of paclitaxel (0.5, pre-occlusion femoral artery diameter as compared with 1.5, 5 mg/kg per day, 7 days) by the implanted osmotic those parameters in vehicle-treated rats (data not pump reduced FMD in a dose-dependent manner (Fig. shown). 2A), whereas both the increase in peak post-ischemic reactive hyperemia and the NTG-induced vasodilation Effects of nicorandil on acetylcholine-induced vasodila- were similar among all treatment groups (data not tion in paclitaxel-treated rats shown). These results imply that paclitaxel impairs en- In vehicle-treated rats, local administration of acetyl- dothelial function without changing contractility in vas- choline into the iliac artery led to femoral arterial vaso- cular smooth muscle [Fig. 2A; FMD of control group: dilation, which peaked at 10 s and rapidly decayed to 21.6% ± 3.2%; FMD of paclitaxel group (5 mg/kg per baseline value by approximately 1 min. In rats treated day): 7.1% ± 1.7%]. On the other hand, co-treatment with paclitaxel (5 mg/kg per day), acetylcholine-induced with nicorandil prevented the decrease in FMD induced vasodilation was significantly reduced in the femoral by paclitaxel (FMD of paclitaxel + nicorandil group: artery (control: 12.7% ± 2.1%; paclitaxel: 3.9% ± 1.1%; 15.5% ± 2.5%) without changing flow velocity or NTG- P < 0.05 vs. control). On the other hand, nicorandil sig- induced vasodilation (data not shown). We previously nificantly prevented the paclitaxel-mediated decrease in reported that nicorandil did not significantly change acetylcholine-induced vasodilation (Fig. 2B; paclitaxel + FMD in normal rats (16). In the current study we found nicorandil: 12.5% ± 3.5%; P < 0.05 vs. paclitaxel). Endothelial Protection by Nicorandil 353

A 25 # 30 FMD # 25 20 %, 1 min)

%, 1 min) 20 # 15 15 * i i 10 * 10 * * 5 5 Vasodilat on ( Vasodilat Vasodilat on ( Vasodilat 0 0 Control 0.5 1.5 5.0 5.0 Control Paclitaxel (5.0) Paclitaxel Nicorandil Diazoxide ISDN Tempol

Fig. 3. Effects of diazoxide, ISDN, and tempol on the paclitaxel- B dependent impairment of endothelial function. Diazoxide (15 mg/kg Acetylcholine 20 per day) or ISDN (15 mg/kg per day) was suspended in 3% gum ara- # bic solution and administrated by gastric gavage once a day for 1 week. Tempol (20 mg/kg per day, p.o.) was administrated in drinking %, 10 s) 15 water for 1 week. *P < 0.05 vs. control, #P < 0.05 vs. paclitaxel (5 mg/kg per day); n = 5 – 7. 10 i

5 * Vasodilat on ( Vasodilat 0 A Control Paclitaxel (5.0) 1 * Nicorandil 0.8 i

Fig. 2. Effects of nicorandil on paclitaxel-induced impairment of -act n 0.6 # endothelial function. Dose-dependent impairment of FMD by pacli- /

taxel (0.5, 1.5, 5.0 mg/kg per day; 1 week) and the effects of nicorandil phox 0.4 (15 mg/kg per day, 1 week) on paclitaxel-induced impairment of p47 FMD (n = 5 – 9). Columns show mean percentage increase (± S.E.M.) 0.2 in arterial diameter at 1 min after reperfusion. B) Effects of nicorandil on acetylcholine-induced vasodilation (n = 7 – 8). Acetylcholine was 0 Control Paclitaxel (5.0) injected into the common iliac artery via an intra-aortic catheter. Columns show mean percentage increase (± S.E.M.) in arterial Nicorandil diameter at 10 s after acetylcholine injection. *P < 0.05 vs. control, B #P < 0.05 vs. paclitaxel (5 mg/kg per day). 1.2 * 1 i

-act n 0.8 Effects of diazoxide, ISDN, and tempol on reduced FMD / in paclitaxel-treated rats 0.6 phox Diazoxide, a mitochondrial KATP-channel opener, sig- 0.4 gp91 nificantly prevented the decrease in FMD induced by 0.2 paclitaxel, although ISDN, a nitrate, had no effect (Fig. 0 3). Tempol, a ROS scavenger, also significantly pre- Control Paclitaxel (5.0) vented the decrease in FMD induced by paclitaxel (Fig. Nicorandil 3). Neither diazoxide, ISDN, nor tempol affected the flow velocity, NTG-induced vasodilation, bodyweight, Fig. 4. Effects of nicorandil on paclitaxel-induced changes in ex- blood pressure, heart rate, or pre-ischemic femoral artery pression of p47phox and gp91phox mRNA in rat femoral arteries. p47phox phox diameter (data not shown). (A) and gp91 (B) expression levels in femoral arteries were mea- sured using real-time PCR. Data are shown as subunit / β-actin ratio. *P < 0.05 vs. control, #P < 0.05 vs. paclitaxel (5 mg/kg per day); n = 8. 354 K Serizawa et al

Effects of nicorandil on gene expression of NADPH oxi- intensity induced by paclitaxel (control: 33.0 ± 0.5; pa- dase in femoral arteries of paclitaxel-treated rats clitaxel: 52.2 ± 1.1, P < 0.05 vs. control; paclitaxel + In femoral arteries isolated from paclitaxel-treated nicorandil: 44.3 ± 1.0 au, P < 0.05 vs. paclitaxel). rats, the mRNA expressions of p47phox and gp91phox, the major components of NADPH oxidase, were significantly Effects of nicorandil on ROS production, NO release, increased (Fig. 4, P < 0.05 vs. control). On the other and endothelial NO synthase (eNOS) phosphorylation hand, nicorandil significantly prevented the increase in induced by paclitaxel in HCAECs p47phox expression (Fig. 4A, P < 0.05 vs. paclitaxel) and Paclitaxel (10 ng/mL) significantly increased DCF also tended to prevent the increase in gp91phox expression fluorescence compared with the control. Nicorandil sig- (Fig. 4B, P = 0.09 vs. paclitaxel) in the femoral arteries nificantly inhibited the increase in DCF fluorescence of paclitaxel-treated rats. induced by paclitaxel (Fig. 6, P < 0.05 vs. control). This inhibitory effect was abolished by co-treatment with

Effects of nicorandil on ROS production in testicular 5-HD, a mitochondrial KATP-channel inhibitor (Fig. 7A), arteries of paclitaxel-treated mice but not by co-treatment with ODQ, a specific inhibitor of As shown in the Fig. 5, the fluorescence intensity of soluble guanylate cyclase (Fig. 7B). Treatment with dihydrorhodamine 123 hydrochloride in paclitaxel- 5-HD alone showed an increase in DCF fluorescence treated mice was 1.6 times greater than that in vehicle- compared with the control (Fig. 7A), whereas treatment treated mice. On the other hand, co-treatment with nic- with nicorandil or ODQ alone did not affect DCF fluo- orandil significantly inhibited the increase in fluorescence rescence (Fig. 6, 7B). On the other hand, there was no

A Control B Paclitaxel (5.0) C Paclitaxel (5.0) + Nicorandil

D * # (arbitrary units) (arbitrary Fluorescence Fluorescence

Control Paclitaxel (5.0) Nicorandil

Fig. 5. Effect of nicorandil on the increase in oxidative stress by paclitaxel in mice. Paclitaxel-induced production of reactive oxygen species in mouse testicular artery was measured using dihydrorhodamine 123 hydrochloride. Representative images showing fluorescence in control (A), after 7-day administration of paclitaxel (B), and after 7-day administration of nicorandil co- administered with paclitaxel (C). Scale bar = 25 μm. D) Each column denotes mean fluorescence intensity from 3 mice. Paclitaxel increased the fluorescence intensity of especially the edge of the spindle-shaped vascular endothelial cells (B). *P < 0.05 vs. control, #P < 0.05 vs. paclitaxel (5 mg/kg per day). Endothelial Protection by Nicorandil 355

ABCD A 2 * † * 1.5 # * E 1

0.5

2 in changes Fold

* intensity Fluorescence 0 1.5 Control Paclitaxel Nicorandil # 5-HD 1 B

Fold changes in 0.5 2 * Fluorescence intensity (normalized to control) 1.5 0 Control Paclitaxel 1 # #

Nicorandil in changes Fold 0.5 Fluorescence intensity Fluorescence 0 Fig. 6. Effects of nicorandil on the increase in oxidative stress in- Control Paclitaxel duced by paclitaxel in HCAECs. Representative images showing fluo- Nicorandil rescence in control (A), after 24 h treatment with paclitaxel (10 ng/ ODQ mL) (B), paclitaxel + nicorandil (100 μM) (C), and nicorandil alone (100 μM) (D). Fold changes in DCF fluorescence intensity is provided as the normalized ratio to the control (E). Intracellular production of Fig. 7. Effects of 5-HD and ODQ on the inhibitory effects of nic- reactive oxygen species was measured by confocal microscopy after orandil against oxidative stress induced by paclitaxel in HCAECs. 45 min DCF (10 μM) labeling as described in the Materials and Co-treatment with 5-HD (500 μM, 24 h), a mitochondrial KATP chan- Methods section. Nicorandil inhibited paclitaxel-induced fluorescence nel inhibitor (A), but not co-treatment with ODQ (20 μM, 24 h), a intensity in HCAECs. *P < 0.05 vs. control, #P < 0.05 vs. paclitaxel. specific inhibitor of soluble guanylate cyclase (B), abolished the in- Each column denotes mean fluorescence intensity of 20 cells/dish hibitory effects of nicorandil (100 μM) on the increase in oxidative from 3 dishes. Scale bar = 100 μm. stress induced by paclitaxel (10 ng/mL). Fold changes in DCF fluo- rescence intensity is reported as the normalized ratio to the vehicle control. Intracellular production of reactive oxygen species was mea- sured by confocal microscopy after 45 min DCF (10 μM) labeling as difference in phosphorylation of eNOS protein among described in the Materials and Methods sections. *P < 0.05 vs. con- trol; #P < 0.05 vs. paclitaxel, †P < 0.05 vs. paclitaxel with nicorandil. the 3 groups. Each column denotes mean fluorescence intensity of 20 cells/dish Paclitaxel (10 ng/mL) significantly decreased DAF-2 from 3 dishes. fluorescence intensity compared with the control (Fig. 8, P < 0.05 vs. control), while nicorandil tended to inhibit the decrease in DAF-2 fluorescence intensity induced by 1.2 paclitaxel. Nicorandil alone had no effect on the fluores- cence intensity. On the other hand, there was no signifi- 1 cant difference in the phosphorylation of eNOS protein 0.8 (phosphorylated eNOS / total eNOS ratio) among the 0.6 vehicle control, paclitaxel (10 ng/mL), and paclitaxel * with nicorandil (100 μM) in cultured HCAECs (data not 0.4 Fold changes in changes Fold shown). 0.2

Fluorescence intensity of DAF-2 of DAF-2 intensity Fluorescence 0 Discussion Control Paclitaxel Nicorandil Although paclitaxel-eluting stents (PES) provide dra- matic reductions in in-stent restenosis, paclitaxel is Fig. 8. Effects of nicorandil (100 μM, 24 h) on the paclitaxel (10 ng/ known to lead to endothelial dysfunction. We investi- mL, 24 h)-induced decreases in fluorescent intensity of DAF-2 in cultured HCAECs. The data are reported as the mean ± S.E.M. Each gated the protective effect of nicorandil on paclitaxel- column denotes mean fluorescence intensity from 6 dishes. *P < 0.05 induced endothelial dysfunction and its underlying vs. control. 356 K Serizawa et al mechanisms by using FMD in anesthetized rats. The that ROS has the potential to impair not only the NO main finding of the present study was that paclitaxel in- bioavailability but also the vascular smooth muscle func- duced endothelial dysfunction in anesthetized rats via tion. However, in this study, NTG-induced relaxation both an increase in oxidative stress and a decrease in NO was unimpaired by paclitaxel, implying that paclitaxel- production. Furthermore, based on the results of func- induced ROS production did not impair smooth muscle tional FMD, ROS production, mRNA expression, NO function in this study. The reasons for this discrepancy synthesis, and eNOS phosphorylation, the present study may lie in differences in degree of ROS production or in also demonstrated that nicorandil would protect against the methods of measurement of vascular relaxation. paclitaxel-induced endothelial dysfunction mainly Earlier studies suggested that nicorandil had a protec- through the inhibition of ROS production. tive effect on the endothelium through its antioxidative With respect to the mechanism of paclitaxel-induced effect. On the basis of experimental results showing that endothelial dysfunction, it has been postulated that pacli- nicorandil reinforced the anti-aggregatory activity of taxel could reduce NO availability. A critical determinant endothelial cells through the reduction of ROS produc- of endothelial function is the balance between NO and tion under hypoxia–reoxygenation conditions, Tajima et ROS, and it has been judged that the availability of en- al. suggested that the protective effect of nicorandil on dothelium-derived NO can be limited by enhanced for- the endothelium was mediated by inhibition of NADPH mation of ROS (20, 21). NO can be trapped with ROS in oxidase activity via mitochondrial KATP channel opening a very rapid reaction, forming and resulting (28). Eguchi et al. suggested that nicorandil prevented in the reduction of the amount of available NO (22). An thrombus formation via activation of mitochondrial KATP earlier report has indicated that a significant source of the channels and inhibition of ROS-induced ROS release in produced following treatment with paclitaxel the mitochondria of endothelial cells (18). The present is NADPH oxidase and that an NADPH oxidase inhibitor study suggests that nicorandil prevents paclitaxel-induced or suppression of gp91phox by siRNA significantly attenu- endothelial dysfunction by reduction of oxidative stress. ated paclitaxel-induced cell death (23). Furthermore, re- The action of nicorandil in this study seems to be depen- duction of NO availability by paclitaxel may also enhance dent on its mitochondrial KATP channel opening proper- thrombotic processes because reduced NO availability ties because diazoxide, another mitochondrial KATP- not only attenuates vasodilation but also adversely affects channel opener, also prevented the paclitaxel-induced anti-thrombotic processes such as reduced leukocyte reduction of FMD (Fig. 3) and because the reduction of adhesion, platelet adhesion, aggregation, and expression ROS by nicorandil was inhibited by 5-HD, a mitochon- of plasminogen activator inhibitor (PAI-1) (21), and in- drial KATP-channel blocker, in HCAECs (Fig. 7A). Inter- deed, paclitaxel increased PAI-1 mRNA expression in estingly, treatment with 5-HD alone showed an increase HCAECs (24). Our findings are consistent with those in oxidative stress (Fig. 7A), suggesting that the mito- earlier reports. In this study, paclitaxel increased ROS chondrial KATP channel is important in the regulation of production both in vivo (Fig. 5) and in HCAECs (Fig. 6), oxidative stress induced in the endothelium by paclitaxel. with the concurrent increases in mRNA expression of Ozcan et al. also demonstrated that nicorandil prevented phox phox NADPH oxidase (p47 and gp91 , Fig. 4) in the rat. oxidative stress via opening of the mitochondrial KATP Moreover, tempol prevented the decrease in FMD by channel (29). Although it cannot be ruled out completely, paclitaxel in rat femoral arteries (Fig. 3). On the other the action of nicorandil in this study does not seem to be hand, paclitaxel decreased NO release in HCAECs (Fig. dependent on its nitrate-like activity. In fact, ISDN did 8), but did not change eNOS phosphorylation. These data not prevent the paclitaxel-induced decrease in FMD (Fig. clearly suggest that paclitaxel induces endothelial dys- 3), and ODQ did not inhibit the decrease in ROS by function by limiting the availability of endothelium- nicorandil in HCAECs (Fig. 7B). Thus, nicorandil might derived NO via both increased ROS production and de- attenuate ROS-induced ROS release elicited by paclitaxel creased NO release. Earlier reports indicated that through mitochondrial stabilization. paclitaxel induced endothelial dysfunction in humans We chose in vivo measurements of FMD for evalua- (5), animals (6, 25), and cultured cells (7, 26) through tion of endothelial function instead of in vitro evaluation apoptosis by increasing the level of pro-apoptotic mRNA for the following reasons: Firstly, in most reports en- transcripts, although the relationship between reduction dothelial function was evaluated experimentally as ace- of NO availability induced by paclitaxel and endothelial tylcholine-induced vasodilation in isolated arteries; clini- dysfunction was not fully revealed. Tawa et al. reported cally, however, endothelial function is determined at the that hypoxia-induced ROS production in smooth muscle brachial artery under the influence of nervous innervation cells impaired NO donor-mediated relaxation in endothe- and humoral factors within the blood stream. Secondly, lium-denuded monkey coronary arteries (27). This means endothelial function can be evaluated either by NO avail- Endothelial Protection by Nicorandil 357 ability or by sensing of shear stress (30). NO availability plasma concentration of paclitaxel would be lower in the is determined by NO production depending on the ex- clinical setting than that in these experiments, and it re- pression level and the activity of eNOS, both of mains unclear whether systemically administered pacli- which are modulated by various humoral factors such as taxel has other points of action. However, in arteries increased ROS (21), increased asymmetric dimethylargi- isolated from PES-implanted swine, the PES enhanced nine (ADMA) (21, 31), depletion of tetrahydrobiopterin local oxidative stress, which may contribute to endothe- (32, 33), and increased advanced glycation end products lium-dependent vasomotor dysfunction (6); therefore, it (34). Moreover, blood flow–induced shear stress stimu- is clear that the PES itself causes endothelial dysfunction lates NO production from the concerted action of multiple in the artery downstream of the implantation point. mechanotransducer molecules including the glycocalyx, In conclusion, we demonstrate in rat femoral artery integrins, and caveolae (30), which can be reduced under that nicorandil protects against paclitaxel-induced en- certain genetic or pathological conditions. dothelial dysfunction as evaluated by FMD, which may Measurement of FMD in rats is considered a useful be brought about by improved NO bioavailability due to experimental tool for estimating endothelial dysfunction the reduction of oxidative stress via KATP channel in serious vascular diseases. We previously reported the activation. impairment of FMD in streptozotocin-induced diabetic rats (16) according to the FMD method described in the References report by Heiss et al. (35). Endothelial function could be successfully assessed by in vivo FMD measurement in 1 Kitta Y, Obata JE, Nakamura T, Hirano M, Kodama Y, Fujioka D, et al. Persistent impairment of endothelial vasomotor function our current experiment because 5-min ischemia/reperfu- has a negative impact on outcome in patients with coronary artery sion or intra-arterial acetylcholine induced femoral vaso- disease. J Am Coll Cardiol. 2009;53:323–330. dilation without affecting the reactive hyperemia or 2 Dawkins KD, Grube E, Guagliumi G, Banning AP, Zmudka K, NTG-induced endothelium-independent vasodilation in Colombo A, et al. Clinical efficacy of polymer-based paclitaxel- anesthetized rats. The present study suggests that mea- eluting stents in the treatment of complex, long coronary artery surement of FMD in rats provides a useful and reproduc- lesions from a multicenter, randomized trial: support for the use ible method not only for investigating the side effects of of drug-eluting stents in contemporary clinical practice. Circula- pharmacological treatments, such as endothelial dysfunc- tion. 2005;112:3306–3313. 3 Stone GW, Ellis SG, Cox DA, Hermiller J, O’Shaughnessy C, tion induced by paclitaxel used in DES or in Mann JT, et al. A polymer-based, paclitaxel-eluting stent in pa- therapy (36), but also for evaluating the therapeutic ef- tients with coronary artery disease. N Engl J Med. 2004;350: fects on endothelial dysfunction in several models of 221–231. vascular disease such as , heart failure, dia- 4 Stone GW, Ellis SG, Cannon L, Mann JT, Greenberg JD, Spriggs betes, and . D, et al. Comparison of a polymer-based paclitaxel-eluting stent In the IONA trial, nicorandil significantly improved with a bare metal stent in patients with complex coronary artery the prognosis by reducing the incident rates of major disease: a randomized controlled trial. JAMA. 2005;294: 1215–1223. coronary events (8). One of the underlying mechanisms 5 Shin DI, Kim PJ, Seung KB, Kim DB, Kim MJ, Chang K, et al. for prognosis improvement was explained by pharmaco- Drug-eluting stent implantation could be associated with long- logical preconditioning (37). Recent clinical studies have term coronary endothelial dysfunction. Int Heart J. 2007;48: suggested that nicorandil’s protective effects against 553–567. endothelial dysfunction would be another possible 6 Pendyala LK, Li J, Shinke T, Geva S, Yin X, Chen JP, et al. En- mechanism because endothelial dysfunction is a risk dothelium-dependent vasomotor dysfunction in pig coronary ar- factor for cardiovascular disease. It has been reported teries with Paclitaxel-eluting stents is associated with inflamma- that in patients with cardiovascular risk factors, long- tion and oxidative stress. JACC Cardiovasc Interv. 2009;2: 253–262. term administration of nicorandil improves FMD (11, 12, 7 Wessely R, Blaich B, Belaiba RS, Merl S, Gorlach A, Kastrati A, 38) and that this improvement is due to the reduction of et al. Comparative characterization of cellular and molecular anti- oxidative stress (12, 38). In the current study, nicorandil restenotic profiles of paclitaxel and sirolimus. Implications for prevented the paclitaxel-induced decrease of FMD due to local drug delivery. Thromb Haemost. 2007;97:1003–1012. the reduction of oxidative stress. Therefore, these results 8 IONA Study Group. Effect of nicorandil on coronary events in suggest that nicorandil might have favorable prognostic patients with stable angina: the Impact Of Nicorandil in Angina effects against endothelial dysfunction in PES-implanted (IONA) randomised trial. Lancet. 2002;359:1269–1275. patients as well as in patients with cardiovascular risk 9 Horinaka S, Yabe A, Yagi H, Ishimitsu T, Yamazaki T, Suzuki S, et al. Effects of nicorandil on cardiovascular events in patients factors. To assess the clinical implications of our find- with coronary artery disease in the Japanese Coronary Artery ings, further clinical studies are needed. Disease (JCAD) study. Circ J. 2010;74:503–509. There is a limitation in the present study. Systemic 10 Horinaka S. Use of nicorandil in cardiovascular disease and its 358 K Serizawa et al

optimization. Drugs. 2011;71:1105–1119. mus and paclitaxel provoke different vascular pathological re- 11 Sekiya M, Sato M, Funada J, Ohtani T, Akutsu H, Watanabe K. sponses after local delivery in a murine model for restenosis on Effects of the long-term administration of nicorandil on vascular underlying atherosclerotic arteries. Heart. 2007;93:922–927. endothelial function and the progression of arteriosclerosis. J 26 Pasquier E, Carre M, Pourroy B, Camoin L, Rebai O, Briand C, Cardiovasc Pharmacol. 2005;46:63–67. et al. Antiangiogenic activity of paclitaxel is associated with its 12 Ishibashi Y, Takahashi N, Tokumaru A, Karino K, Sugamori T, cytostatic effect, mediated by the initiation but not completion of Sakane T, et al. Effects of long-term nicorandil administration on a mitochondrial apoptotic signaling pathway. Mol Cancer Ther. endothelial function, inflammation, and oxidative stress in pa- 2004;3:1301–1310. tients without coronary artery disease. J Cardiovasc Pharmacol. 27 Tawa M, Yamamizu K, Geddawy A, Shimosato T, Imamura T, 2008;51:311–316. Ayajiki K, et al. Impairment by hypoxia or hypoxia/reoxygen- 13 Zhao JL, Yang YJ, Chen JL, Kang LM, Wu Y, Gao RL. Nicorandil ation of nitric oxide-mediated relaxation in isolated monkey coro- reduces myocardial no-reflow by protection of endothelial func- nary artery: the role of intracellular superoxide. J Pharmacol Sci.

tion via the activation of KATP channel. Clin Chim Acta. 2006; 2011;116:188–196. 374:100–105. 28 Tajima M, Ishizuka N, Saitoh K, Sakagami H. Nicorandil en- 14 Jones ID, Fuentes VL, Wrigglesworth D, Mort E, Elliott J. Com- hances the effect of endothelial nitric oxide under hypoxia-reox-

parison of flow-mediated vasodilation in femoral and brachial ygenation: role of the KATP channel. Eur J Pharmacol. 2008; arteries in healthy dogs. Am J Vet Res. 2011;72:1029–1037. 579:86–92. 15 Weber LP, Al-Dissi A, Marit JS, German TN, Terletski SD. Role 29 Ozcan C, Bienengraeber M, Dzeja PP, Terzic A. Potassium chan- of carbon monoxide in impaired endothelial function mediated nel openers protect cardiac mitochondria by attenuating oxidant by acute second-hand tobacco, incense, and candle smoke expo- stress at reoxygenation. Am J Physiol Heart Circ Physiol. 2002; sures. Environ Toxicol Pharmacol. 2011;31:453–459. 282:H531–H539. 16 Serizawa K, Yogo K, Aizawa K, Tashiro Y, Ishizuka N. Nic- 30 Yamamoto K, Ando J. New molecular mechanisms for cardio- orandil prevents endothelial dysfunction due to antioxidative ef- vascular disease:blood flow sensing mechanism in vascular en- fects via normalisation of NADPH oxidase and nitric oxide syn- dothelial cells. J Pharmacol Sci. 2011;116:323–331. thase in streptozotocin diabetic rats. Cardiovasc Diabetol. 2011; 31 Cooke JP. Does ADMA cause endothelial dysfunction? Arterio- 10:105. scler Thromb Vasc Biol. 2000;20:2032–2037. 17 Kim DW, Kwon JS, Kim YG, Kim MS, Lee GS, Youn TJ, et al. 32 Landmesser U, Dikalov S, Price SR, McCann L, Fukai T, Holland Novel oral formulation of paclitaxel inhibits neointimal hyper- SM, et al. Oxidation of tetrahydrobiopterin leads to uncoupling plasia in a rat carotid artery injury model. Circulation. 2004; of endothelial cell in hypertension. J Clin 109:1558–1563. Invest. 2003;111:1201–1209. 18 Eguchi Y, Takahari Y, Higashijima N, Ishizuka N, Tamura N, 33 Crabtree MJ, Hale AB, Channon KM.

Kawamura Y, et al. Nicorandil attenuates FeCl3-induced throm- protects endothelial nitric oxide synthase from uncoupling in bus formation through the inhibition of reactive oxygen species tetrahydrobiopterin deficiency. Free Radic Biol Med. 2011;50: production. Circ J. 2009;73:554–561. 1639–1646. 19 Wang S, Peng Q, Zhang J, Liu L. Na+/H+ exchanger is required 34 Rojas A, Romay S, Gonzalez D, Herrera B, Delgado R, Otero K. for hyperglycaemia-induced endothelial dysfunction via calcium- Regulation of endothelial nitric oxide synthase expression by dependent calpain. Cardiovasc Res. 2008;80:255–262. albumin-derived advanced glycosylation end products. Circ Res. 20 Muller G, Morawietz H. NAD(P)H oxidase and endothelial dys- 2000;86:E50–E54. function. Horm Metab Res. 2009;41:152–158. 35 Heiss C, Sievers RE, Amabile N, Momma TY, Chen Q, Natarajan 21 Landmesser U, Hornig B, Drexler H. Endothelial function: a criti- S, et al. In vivo measurement of flow-mediated vasodilation in cal determinant in atherosclerosis? Circulation. 2004;109: living rats using high-resolution ultrasound. Am J Physiol Heart II27–II33. Circ Physiol. 2008;294:H1086–H1093. 22 Gryglewski RJ, Palmer RM, Moncada S. Superoxide anion is 36 Mailloux A, Grenet K, Bruneel A, Beneteau-Burnat B, involved in the breakdown of endothelium-derived vascular re- Vaubourdolle M, Baudin B. Anticancer drugs induce necrosis of laxing factor. Nature. 1986;320:454–456. human endothelial cells involving both oncosis and apoptosis. 23 Jang HJ, Hwang S, Cho KY, Kim do K, Chay KO, Kim JK. Taxol Eur J Cell Biol. 2001;80:442–449. induces oxidative neuronal cell death by enhancing the activity 37 Tsuchida A, Miura T, Tanno M, Sakamoto J, Miki T, Kuno A, of NADPH oxidase in mouse cortical cultures. Neurosci Lett. et al. Infarct size limitation by nicorandil: roles of mitochondrial

2008;443:17–22. KATP channels, sarcolemmal KATP channels, and protein kinase C. 24 Muldowney JA 3rd, Stringham JR, Levy SE, Gleaves LA, Eren J Am Coll Cardiol. 2002;40:1523–1530. M, Piana RN, et al. Antiproliferative agents alter vascular plas- 38 Ono H, Osanai T, Ishizaka H, Hanada H, Kamada T, Onodera H, minogen activator inhibitor-1 expression: a potential prothrom- et al. Nicorandil improves cardiac function and clinical outcome botic mechanism of drug-eluting stents. Arterioscler Thromb in patients with acute myocardial infarction undergoing primary Vasc Biol. 2007;27:400–406. percutaneous coronary intervention: role of inhibitory effect on 25 Pires NM, Eefting D, de Vries MR, Quax PH, Jukema JW. Siroli- reactive oxygen species formation. Am Heart J. 2004;148:E15.