Cell experiment:

Unless otherwise stated, for each BMEC experiment cells are randomly divided into 4 groups: (1) normoxia vehicle control (Nx-CTRL); (2) normoxia-treated; (3) hypoxia (24 h) control (Hx-CTRL) and (4) hypoxia (24 h) treated. As previously described, Nrf2 activators are added 24 h prior to any hypoxic exposures. Cell treatments are; Protandim (100 μg/mL), methazolamide (125 μg/mL, nifedipine (7 μg/mL) or Ambrisentan (40 μg/mL). In addition, some cells are treated with Nrf2 siRNA. In these experiments, siRNA is added 24 h prior to drug treatments. The rationale for 24 h hypoxia exposure for BMEC is to ensure that cells remained transfected with siRNA for the pre-treatment of drugs (24 h in normoxia) and during the 24 h hypoxia exposure. Data is collected from at least three separate cell culture preparations on three separate days (n=9)[2].

Animal experiment:

Mice[1] A total of 13 male FLS-ob/ob mice (age, 8 wk; body weight, 42.88 g±1.74 g) are used. At the age of 12 wk, male FLS-ob/ob mice are randomly assigned to the Ambrisentan (n=8) or control (n= 5) group. Intragastric gavage administration is carried out in conscious animals with an appropriately sized gastric tube. Ambrisentan (2.5 mg/kg per day) is orally administered every afternoon for 4 wk as a bolus through a gastric tube. Water is administered to the control group. At week 4, animals are fasted for 4 h and tail vein blood is drawn and subjected to blood glucose determination. Animals are killed by pentobarbital anesthesia injection after 4 wk and blood is collected from the right ventricle. Plasma samples are frozen and stored at -80℃ Liver and visceral fat are then weighed, snap-frozen in liquid nitrogen, and stored at -80℃. Additional liver specimens are fixed in 10% buffered formalin and embedded in paraffin for histological analysis.

Ambrisentan is a selective antagonist of ETA-receptor with Ki value of 1nM [1].

Ambrisentan is an orally active diphenyl propionic acid derivative. It is usually used to treat for pulmonary arterial hypertension. In the in vitro assay, ambrisentan shows selective affinity with ETA-receptor over ETB-receptor expressed in CHO cells. The Ki values of ETA- and ETB-receptor are 1nM and 195nM, respectively. This selectivity is much more higher for the recombinant human ET-receptors in intact cells. The Ki values of ETA- and ETB-receptor are 0.63nM and 48.7nM, respectively. As a selective antagonist of ETA-receptor, ambrisentan is preferential to non-selective receptor antagonism as it permitting maintenance of vasodilator and clearance functions specific to ETB- receptors on the endothelial cells. Moreover, ambrisentan also has possible use in the prevention of reperfusion injury and is appropriate to treat for cerebrovascular disorders [1, 2].

References:
[1] Vatter H, Seifert V. Ambrisentan, a Non-peptide Endothelin Receptor Antagonist. Cardiovascular drug reviews, 2006, 24(1): 63-76.
[2] Barst R J. A review of pulmonary arterial hypertension: role of ambrisentan. Vascular health and risk management, 2007, 3(1): 11.