Mechanistic Studies of AVE3085 Against Homocysteine

Yang et al, 2013

CDTY-D-13-00086 R2

Mechanistic Studies of AVE3085 Against Homocysteine

in Endothelial Protection

Qin Yang, MD, PhDa#*; Hong-Mei Xue,PhDb#; Malcolm John Underwood,MDc;

Cheuk-Man Yu, MDd

aDivision of Cardiology, Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Hong Kong & TEDA International Cardiovascular Hospital, Medical College, Nankai University, Tianjin, China

bDepartment of Physiology, Hebei Medical University, Hebei, China

cDivision of Cardiothoracic Surgery, Department of Surgery, dDivision of Cardiology, Department of Medicine and Therapeutics, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong

#Contribute equally

*Correspondence to

Professor Qin Yang, MD, PhD

Division of Cardiology

Department of Medicine and Therapeutics

The ChineseUniversity of Hong Kong

Tel: (852)37636069

E-mail:

CDTY-D-13-00086 R2

Supplementary Data

Methods

Direct measurement of NO

Calibration of NO microsenser

The NO microsensor (ISO-NOP30L, World Precision Instruments, Sarasota, FL, USA) was calibrated prior to daily experiment. The calibration was performed by decomposition of S-Nitroso-N-acetyl-D,L-penicillamine (SNAP) using copper sulfate as a catalyst. SNAP was used in combination with copper sulfate to generate a known quantity of NO in solution. In details, a known amount of standard SNAP (WPI) was injected into the vial containing copper sulfate solution. The current output rapidly increased upon addition of the aliquot and reached a plateau within a few seconds. Each subsequent aliquot was injected as soon as the previous signal reached its plateau. A standard curve was constructed upon the completion of the calibration procedure. After calibration, the NO microsensor was slowly introduced into the vessel lumen by means of a micromanipulator (WR-6, Narishige International).

Western blot analysis

Porcine coronary small arteries were snapped frozen in liquid nitrogen and subsequently homogenized in an ice-cold RIPA lysing buffer, containing 100 µg/ml phenylmethylsulfonyl fluoride (PMSF), 1 mmol/L sodium orthovanadate, 1 mmol/L EGTA, protease inhibitor cocktail with 2 mmol/L 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), 1 mmol/L EDTA, 130 µmol/L Bestain, 14 µmol/L E-64, 1 µmol/L Leupeptin, and 0.3 µmol/L Aprotinin. The lysates were sonicated on ice and then centrifuged at 20,000 g for 20 minutes. The protein concentration was determined by the Lowry method (Bio-Rad). The protein samples were electrophoresed through 8% SDS-poly-acrylamide gel and transferred onto an immobilon-P polyvinylidene difluoride (PVDF) membrane (Amersham Pharmacia) using wet transfer at 100V for 120 minutes at 4ºC. Non-specific bindings were blocked with 5% non-fat milk in 0.05% Tween-20 Tris-buffered saline (TBST) for 60 minutes at room temperature and probed overnight at 4ºC with antibodies against eNOS(BD Transduction Laboratories, BD, Franklin Lakes, NJ, USA),phosphorylated eNOS at Ser1177 (p-eNOSSer1177)(Upstate BiotechnologyInc., Lake Placid, NY, USA),and iNOS(Abcam, Cambridge, UK)in TBST with 5% non-fat milk. After washes in TBST, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG (Santa Cruz Technologies, Santa Cruz, CA, USA)or anti-mouse IgG (Dako, Glostrup, Denmark)for 60 minutes at room temperature. Blots were developed with an enhanced chemiluminescence detection system (ECL reagents, Amersham Pharmacia) and exposed to X-ray films. Densitometry was performed using Quantity One imaging system (Bio-Rad).

Lucigenin-enhanced chemiluminescence

Levels of O2.- produced by endothelial cells were detected by using the lucigenin-enhanced chemiluminescence method with a Sirius Luminometer and FB12 software from Berthold Detection System GmbH (Pforzheim, Germany) as described elsewhere20,25. Porcine coronary small arterial rings were cut open longitudinally with endothelium side facing down in the tissue chamber. The segments wererinsed briefly in PBS and maintained at 37ºC for 30 minutes. Lucigenin-enhanced chemiluminescence was measured in each segment with using low-concentration lucigenin (5 µmol/L)20,25. It was reported that lucigenin at higher concentration may lead to enhanced rates of production of O2.- and accumulation of the dismutation product, H2O2 in vascular cells25. Time-based reading of the luminometer was recorded by FB12 software. The illuminescence in relative light units (RLU) per second for each sample were averaged between 5 and 10 minutes. Values of blank chambers that contain the same reagents were recorded as background and the background values were subtracted from that of the corresponding vessel samples. Data were normalized to vessel dry weight and represented as RLU per second per milligram dry weight.

Arginase activity analysis

Arginase activity was measured by determining levels of urea production as described elsewhere26. Briefly, porcine coronary small arteries were sonicated for 10 minutes in lysis buffer (50 mmol/L Tris.HCl, pH 7.5, 0.1 mmol/L EDTA, 0.1% Triton X-100, and protease inhibitor) and centrifuged at 14,000 g for 30 minutes at 4ºC. Supernatant (50 µl) was then added to 75 µl of Tris.HCl (50 mmol/L, pH 7.5) containing 10 mmol/L MnCl2, and the mixture was activated by heating for 10 minutes at 55-60ºC. The mixture was incubated with l-arginine (50 µl, 0.5 mol/L, pH 9.7) at 37ºC for 1h, and the reaction was stopped by adding 400 µl of acid solution (H2SO4-H3PO4-H2O=1:3:7). For colorimetric determination of urea, α-isonitrosopropiophenone (25 µl, 9% in ethanol) was added, and the mixture was heated at 100 ºC for 45 minutes. After the sample was placed in the dark for 10 minutes at room temperature, the urea concentration was determined spectrophotometrically by measuring absorbance at 550 nm. Urea production was calculated from a standard curve (urea) and was expressed as nanomoles of urea per minute per gram of tissue.

Results

Table 1.Resting force and U46619-precontraction ofporcine coronary small arteries.

Data are shown as mean±SEM. n=8 in each group. Hcy: homocysteine; AVE: AVE3085; 1400W: iNOS inhibitor; nor-NOHA: arginase inhibitor; wortmannin and LY294002: PI3 kinase inhibitors.

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