2-Methoxyestradiol Ameliorates Angiotensin II–Induced Hypertension by Inhibiting Cytosolic Phospholipase A ₂ α Activity in Female Mice

Figure 1.. Ang II (angiotensin II) increased blood pressure (BP) and renal collagen deposition and 2-hydroxyethdium (2-OHE) fluorescence in intact and ovariectomized (OVX)-cPLA2α+/+/Cyp1b1+/+ but not intact cPLA2α−/−/Cyp1b1+/+ mice. Mean arterial pressure (MAP; A and B) was measured by radiotelemetry. Saline was used as a vehicle (Veh) for Ang II. Collagen deposition was detected by Masson trichrome Staining (C), and reactive oxygen species were measured by DHE staining (E), and the quantitative values were expressed as arbitrary units (AU; D and F), respectively. Saline was used as a vehicle (Veh) for Ang II. Data are mean±SEM (n=5 per group) and analyzed by repeated measures 2-way ANOVA followed by Tukey multiple comparisons test. *P<0.05 and §<0.05 vs day 0 value before osmotic pump with Ang II. †P<0.05 vs intact cPLA2α–/–/Cyp1b1+/+; ‡<0.05, OVX-cPLA2α–/–/Cyp1b1+/+. Data in (D) and (F) are mean±SEM (n=4–6 per group) and analyzed by 1-way ANOVA and Tukey multiple comparisons test. A indicates Ang II; cPLA2α, cytosolic phospholipase A2α; and V, Veh. Scale bars 50 μm.

Figure 2.. Ang II (angiotensin II) increased renal cPLA2α (cytosolic phospholipase A2α) activation in intact and ovariectomized (OVX)-cPLA2α+/+/Cyp1b1+/+ and intact cPLA2α+/+/Cyp1b1−/− mice. OVX-cPLA2α+/+/Cyp1b1+/+ mice expressed Ang II-induced phospho (p)-cPLA2α, activation form of cPLA2α, more than intact-cPLA2α+/+/Cyp1b1+/+ mice (A). 17β-estradiol (E2) diminished Ang II–induced p-cPLA2α expression in OVX-cPLA2α+/+/Cyp1b1+/+ (B) but not in OVX-cPLA2α+/+/Cyp1b1−/− mice (C). 2-methoxyestradiol (2-ME) abolished Ang II–induced cPLA2α activation in OVX-cPLA2α+/+/Cyp1b1+/+ (D) and in intact cPLA2α+/+/Cyp1b1−/− mice (E). cPLA2α proteins were not detected in OVX-cPLA2α−/−/Cyp1b1+/+ mice (F). Saline was used as a vehicle (Veh) of Ang II (A and F) and dimethyl sulfoxide (B–D) for 2-ME. p-cPLA2α and cPLA2α expressions were analyzed by Western blot. Quantitative data were presented as the relative ratio, p-cPLA2α, and cPLA2α, and on the top of each panel is the representative blot for each group of experiments, respectively. β-actin was used for loading control in intact and OVX-cPLA2α−/−/Cyp1b1+/+ mice (F). Data are mean±SEM (n=3–6 per group) and analyzed by 1-way ANOVA and Tukey multiple…

Figure 3.. 2-Methoxyestradiol (2-ME) inhibited Ang II (angiotensin II)–induced increase in systolic blood pressure (SBP), renal collagen deposition, and 2-hydroxyethidium (2-OHE) fluorescence in ovariectomized (OVX)-cPLA2α+/+/Cyp1b1+/+ and intact cPLA2α+/+/Cyp1b1−/− mice. Systolic blood pressure (SBP) was measured by the tail-cuff method (A and B). Collagen deposition was detected by Masson trichrome staining (C), and reactive oxygen species were measured by DHE staining (E), and the quantitative values were expressed as arbitrary units (AU; D) and (F), respectively. Data are mean±SEM (n=4–6 per group). Data for SBP were analyzed using repeated measures 2-way ANOVA followed by Tukey multiple comparison test (A and B) and, data for collagen deposition and 2-OHE fluorescence by 1-way ANOVA followed by Tukey multiple comparison test (D and F). *P<0.05 vs day 0 value before osmotic pump with Ang II. †P<0.05 vs Ang II alone. A indicates Ang II; and V, vehicle. Saline was used as a vehicle of Ang II and DMSO for 2-ME. Scale bars 50 μm.

Figure 4.. ETYA (5,8,11,14-eicosatetraynoic acid) inhibited Ang II (angiotensin II), increased systolic blood pressure (SBP), renal collagen deposition, and 2-hydroxyethidium (2-OHE) fluorescence in ovariectomized (OVX)-cPLA2α+/+/Cyp1b1+/+ and intact cPLA2α+/+/Cyp1b1−/− mice. SBP was measured by the tail-cuff method in OVX-cPLA2α+/+/Cyp1b1+/+ (A) and intact cPLA2α+/+/Cyp1b1−/− mice (B), and collagen deposition was measured by Masson trichrome staining (C), and reactive oxygen species were measured by 2-OHE fluorescence (E), and the quantitative values were expressed as arbitrary units (AU; D and F), respectively. Saline was used as a vehicle (Veh) of Ang II and dimethyl sulfoxide for ETYA. Data are mean±SEM (n=5–6 per group). Statistical analysis was performed using repeated measures 2-way ANOVA and Tukey multiple comparisons test (A and B) and 1-way ANOVA and Tukey multiple comparisons test (D and F). *P<0.05 vs day 0 value before osmotic pump with Ang II; †P<0.05 vs Ang II alone. A indicates Ang II; and V, Veh. Scale bars 50 μm.

Figure 5.. Prostaglandin E2 receptor antagonist (RA) EP1, EP3, and thromboxane A2 (TP) RA diminished Ang II (angiotensin II) –induced increased systolic blood pressure (SBP) and collagen deposition in ovariectomized (OVX)-cPLA2α+/+/Cyp1b1+/+ and intact cPLA2α+/+/Cyp1b1−/− mice. Blood pressure was measured by the tail-cuff method in OVX-cPLA2α+/+/Cyp1b1+/+ (A), and intact cPLA2α+/+/Cyp1b1−/− mice (D), and the collagen deposition was detected by Masson trichrome staining (B and E), and the quantitative values expressed as an arbitrary unit (AU; C and F), respectively. Saline was used as a vehicle (Veh) of Ang II and dimethyl sulfoxide (DMSO) for EP1, EP3, and TP receptor antagonists. Data are mean±SEM (n=5–6 per group). Statistical analyses were performed using repeated-measures 2-way ANOVA followed by Tukey multiple comparison test (A and D) and 1-way ANOVA followed by Tukey multiple comparison test (C and F). *P<0.05 vs day 0 value before osmotic pump with Ang II; †P<0.05 vs Ang II alone. A indicates Ang II; and V, Veh. Scale bars 50 μm.

Figure 6.. EP1, EP3, and TP receptor antagonists prevented Ang II (angiotensin II)–induced increase 2-hydroxyethidium (2-OHE) fluorescence in ovariectomized (OVX)-cPLA2α+/+/Cyp1b1+/+ and intact cPLA2α+/+/Cyp1b1−/− mice. Reactive oxygen species were measured by 2-OHE fluorescence in the kidney section of OVX-cPLA2α+/+/Cyp1b1+/+ (A), and intact cPLA2α+/+/Cyp1b1−/− mice (C), and quantitative values expressed as arbitrary units (AU), respectively (B and D). Saline was used as a vehicle (Veh) of Ang II and dimethyl sulfoxide for EP1, EP3, and TP receptor antagonists. Data are mean±SEM (n=5–6 per group) and analyzed using 1-way ANOVA and Tukey multiple comparisons test. A indicates Ang II; V, Veh; EP1RA, EP1 receptor antagonist; EP3RA, EP3 receptor antagonist; and TPRA, TP receptor antagonist. Scale bars 50 μm.