Olmesartan

Chondrocytes were isolated from articular cartilage of MTP joints of a 10‐month‐old cow by digestion with 0.08% collagenase (Wako Pure Chemical Industries, Osaka, Japan) for 6 h at 37 °C. After filtration, cells were seeded at a density of 2 × 10⁴ cells·mL⁻¹ in 100 mm plates and cultured until confluent as previously described 4. The cells were then embedded in agarose hydrogels with a cell density of 2 × 10⁶ cells·mL⁻¹, as described previously 16, 17 (Fig. 1A,B). The cell–agarose constructs were maintained in culture for 8 days in DMEM at 37 °C in a humidified hypoxic atmosphere (5% O₂ and 5% CO₂).
The cell–agarose constructs were allocated into four groups (groups A–D). The constructs in groups A and B were submitted to cyclic compressive loads. The cell–agarose constructs in group B were treated with 10 μm Olmesartan, which is an Ang II receptor blocker (ARB), for 12 h before the load. The constructs in group C were treated with 1 μm Ang II for 30 min without load. The constructs in group D were cultured without the addition of any agents or cyclic compressive loads. Olmesartan was obtained from Daiichi‐Sankyo Co., Ltd. (Tokyo, Japan). Ang II was purchased from Sigma‐Aldrich Co., Ltd. (St. Louis, MO, USA).

Olmesartan was kindly provided by Daiichi Sankyo Pharmaceutical Co. (Tokyo, Japan).

C57BL/6 male mice (8–10 weeks old, weighing 22–25 g, provided by the Animal Center of Southern Medical University) were anesthetized with a mixture of xylazine (5 mg/kg, intraperitoneally) and ketamine (100 mg/kg, intraperitoneally), and the adequacy of anesthesia was monitored from the disappearance of pedal withdrawal reflex. Pressure overload model was created by transverse aortic constriction (TAC) as described elsewhere [18] (link), [19] (link). Some of the TAC mice were randomized to treatment with olmesartan (Daiichi Sankyo, Tokyo, 10 mg/kg/d added in the food) or vehicle alone for 4 weeks. The mice were sacrificed by overdose anesthesia (pentobarbital sodium 150 mg/kg intraperitoneally) and cervical dislocation.

hANG and hRN mice were previously generated in our laboratory (Fukamizu et al. 1989 (link); Takahashi et al. 1991 (link)). hANG females were mated with hRN males to create PAH mice. The day of mating was defined as day 0 of pregnancy (P0). PAH mice were treated with Olmesartan or hydralazine from afternoon of P13 to P19. Olmesartan (15 mg/L, kindly gifted from Daiichi Sankyo, Tokyo, Japan) was administrated as described previously (Sakairi et al. 2008 (link)). Hydralazine hydrochloride (151278, MP Biomedicals, Santa Ana, CA) was administrated in drinking water. The dose of hydralazine was gradually increased to sufficiently suppress hypertension (62.5, 250, 375 mg/L). Hydralazine solution was changed at P15 and P17, respectively. C57BL/6J mice were used as control (wild-type [WT] mice, purchased from CLEA Japan, Tokyo, Japan). All animal experiments were carried out in a humane manner and approved by the Institutional Animal Experiment Committee of the University of Tsukuba. Experiments were conducted in accordance with the Regulation of Animal Experiments of the University of Tsukuba and the Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Chloramine-T was purchased from Nacalai Tesque Inc. (Kyoto, Japan). Methylcellulose 400, benzbromarone, dihydroethidium (DHE), and ANG II were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Olmesartan was a kind gift from Daiichi Sankyo Pharmaceutical Co. Ltd. (Tokyo, Japan). All other chemicals were of the highest grade and obtained from commercial sources.