key: cord-0022133-okmikvec
authors: Song, Chi Young; Singh, Purnima; Motiwala, Mustafa; Shin, Ji Soo; Lew, Jessica; Dutta, Shubha R.; Gonzalez, Frank J.; Bonventre, Joseph V.; Malik, Kafait U.
title: 2-Methoxyestradiol Ameliorates Angiotensin II–Induced Hypertension by Inhibiting Cytosolic Phospholipase A(2)α Activity in Female Mice
date: 2021-10-11
journal: Hypertension
DOI: 10.1161/hypertensionaha.121.18181
sha: f24ea4137133da369407cc2d24dbfcc495712e93
doc_id: 22133
cord_uid: okmikvec

We tested the hypothesis that CYP1B1 (cytochrome P450 1B1)-17β-estradiol metabolite 2-methoxyestradiol protects against Ang II (angiotensin II)–induced hypertension by inhibiting group IV cPLA(2)α (cytosolic phospholipase A(2)α) activity and production of prohypertensive eicosanoids in female mice. Ang II (700 ng/kg per minute, SC) increased mean arterial blood pressure (BP), systolic and diastolic BP measured by radiotelemetry, renal fibrosis, and reactive oxygen species production in wild-type mice (cPLA(2)α(+/+)/Cyp1b1(+/+)) that were enhanced by ovariectomy and abolished in intact and ovariectomized-cPLA(2)α(−/−)/Cyp1b1(+/+) mice. Ang II–induced increase in SBP measured by tail-cuff, renal fibrosis, reactive oxygen species production, and cPLA(2)α activity measured by its phosphorylation in the kidney, and urinary excretion of prostaglandin E(2) and thromboxane A(2) metabolites were enhanced in ovariectomized-cPLA(2)α(+/+)/Cyp1b1(+/+) and intact cPLA(2)α(+/+)/Cyp1b1(−/−) mice. 2-Methoxyestradiol and arachidonic acid metabolism inhibitor 5,8,11,14-eicosatetraynoic acid attenuated the Ang II–induced increase in SBP, renal fibrosis, reactive oxygen species production, and urinary excretion of prostaglandin E(2), and thromboxane A(2) metabolites in ovariectomized-cPLA(2)α(+/+)/Cyp1b1(+/+) and intact cPLA(2)α(+/+)/Cyp1b1(−/−) mice. Antagonists of prostaglandin E(2) and thromboxane A(2) receptors EP1 and EP3 and TP, respectively, inhibited Ang II–induced increases in SBP and reactive oxygen species production and renal fibrosis in ovariectomized-cPLA(2)α(+/+)/Cyp1b1(+/+) and intact cPLA(2)α(+/+)/Cyp1b1(−/−) mice. These data suggest that CYP1B1-generated metabolite 2-methoxyestradiol mitigates Ang II–induced hypertension and renal fibrosis by inhibiting cPLA(2)α activity, reducing prostaglandin E(2), and thromboxane A(2) production and stimulating EP1 and EP3 and TP receptors, respectively. Thus, 2-methoxyestradiol and the drugs that selectively block EP1 and EP3 and TP receptors could be useful in treating hypertension and its pathogenesis in females.

W e reported previously that Ang II (angiotensin II), the main product of the renin-angiotensin system, produces hypertension by activating group IV cPLA 2 α (cytosolic phospholipase A 2 α), resulting in arachidonic acid (AA) release and the generation predominantly of eicosanoids with prohypertensive effects in male mice. 1, 2 The eicosanoids produced by COX (cyclooxygenase), PGE 2 (prostaglandin E 2 ) by acting on EP1 and EP3, and TXA 2 (thromboxane A 2 ) by acting on its TP (prostanoid receptor), contribute to the hypertensive effect of Ang II. [3] [4] [5] [6] Also, AA metabolites generated via 12/15-lipoxygenase and cytochrome P450 A1, 12Sand 20-hydroxyeicosatetraenoic acids, respectively, participate in the vasoconstrictor effect of Ang II and contribute to its prohypertensive effect. 7, 8 There is sexual dimorphism in blood pressure (BP) levels in humans and various animal models of hypertension which is attributed to gonadal hormones and sex chromosomes affecting renin-angiotensin and immune cell system activity. [9] [10] [11] Ang II produces a greater pressor effect in men than women. 12 In addition, Ang II infusion produces a higher increase in BP in males than in female animals. [9] [10] [11] Ovariectomy enhances in female mice, and castration reduces the effect of Ang II in male mice to increase BP. 13 However, whether the protection against Ang II-induced hypertension in the females depends on alteration in the activity of the cPLA 2 α/AA system is unknown.

Previously, we showed that E2 (17β-estradiol)-CYP1B1 generated metabolite 2-hydroxyestradiol by its subsequent metabolism by catechol-O-methyltransferase to 2-ME (2-methoxyestradiol) protected against Ang II-induced hypertension in female mice. [14] [15] [16] [17] However, the mechanism by which 2-ME protects against Ang II-induced hypertension is not known. Our preliminary observation that cPLA 2 α gene disruption in females, as in male mice, 1 also prevents Ang II-induced hypertension led to the following hypothesis: CYP1B1-E2 generated metabolite 2-ME acts upstream by inhibiting cPLA 2 α activity and reducing the generation of prohypertensive eicosanoids, protects against Ang II-induced hypertension and its pathogenesis.

The authors declare that a detailed Methods Section and all supporting data are available within the article and in the Data Supplement. Other details of analytic methods, study materials, and the data will be made available from the corresponding author upon reasonable request.

All experiments were performed in female mice according to protocols approved by the University of Tennessee Health Science center Institutional Animal Care and Use Committee according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. cPLA 2 α +/+ /Cyp1b1 +/+ , cPLA 2 α −/− /Cyp1b1 +/+ , and cPLA 2 α +/+ /Cyp1b1 −/− female mice on the C57BL/6J background were used in this study. cPLA 2 α −/− /Cyp1b1 +/+ and cPLA 2 α +/+ /Cyp1b1 −/− female mice, respectively, were bred and genotyped as described previously 18, 19 and also as detailed in the Data Supplement. The animals were randomly divided into various treatment groups and infused Nonstandard Abbreviations and Acronyms 

What Is New?

• 17β-estradiol (E2)-CYP1B1 (cytochrome P450 1B1)derived metabolite 2-ME (2-methoxyestradiol) protects against Ang II (angiotensin II)-induced hypertension, reactive oxygen species production, and renal fibrosis by inhibiting the activity of group IV cPLA 2 α (cytosolic phospholipase A 2 α) and generation of arachidonic acid metabolites prostaglandins (PG) E 2 and thromboxane (TX) A 2 in female mice. • The decrease in PGE 2 and TXA 2 levels by reducing stimulation of the EP1/EP3 and TP receptors, respectively, ameliorates Ang II-induced hypertension, reactive oxygen species production, and renal fibrosis.

What Is Relevant?

• 2-ME and EP1, EP3, and TP receptor antagonists could be useful in treating hypertension and its pathogenesis in postmenopausal and hypoestrogenemic premenopausal women and women with menstrual irregularities ovarian failure as well as in males.

We have previously shown that E2 protects against Ang II-induced hypertension and associated cardiovascular and renal pathogenesis via CYP1B-generated metabolite 2-ME in female mice. The present study demonstrates that 2-ME exerts its protective effect against Ang II-induced hypertension and associated production of reactive oxygen species and renal fibrosis by inhibiting group IV cPLA 2 α and generation of arachidonic acid metabolites PGE 2 and TXA 2 in female mice. The decrease in PGE 2 and TXA 2 production leads to decreased stimulation of the EP1/ EP3 and TP receptors, respectively, extenuate Ang II-induced hypertension, reactive oxygen species production, and renal fibrosis. Therefore, 2-ME and EP1, EP3, and TP receptor antagonists could be beneficial in treating hypertension and associated pathogenesis in postmenopausal and hypoestrogenemic premenopausal women and women with menstrual irregularities caused by ovarian failure.

subcutaneously with Ang II (700 ng/kg per minute) or saline for 14 days through implanted micro-osmotic pumps (Alzet, Cupertino, CA; model 1002). Mean arterial BP, systolic BP (SBP), and diastolic BP, and heart rate was measured by radiotelemetry. In some experiments, as described in the Results section, SBP was measured by the noninvasive tail-cuff method (Kent Scientific; model XBP 1000). To test our hypothesis, we investigated (1) the effect of cPLA 2 α gene disruption on Ang IIinduced hypertension and associated pathogenesis in intact and ovariectomized-cPLA 2 α +/+ /Cyp1b1 +/+ mice; (2) the effect of Ang II on renal cPLA 2 α expression and activity in ovariectomized-cPLA 2 α +/+ /Cyp1b1 +/+ and intact and ovariectomized-cPLA 2 α +/+ / Cyp1b1 −/− mice treated with E2 or 2-ME (1.5 mg/kg, IP, every third day), respectively, and their vehicle, dimethyl sulfoxide (DMSO); (3) the effect of Ang II on plasma levels of E2 and 2-ME in intact cPLA 2 α +/+ /Cyp1b1 +/+ and cPLA 2 α −/− /Cyp1b1 +/+ mice, and in ovariectomized-cPLA 2 α +/+ /Cyp1b1 +/+ and intact cPLA 2 α +/+ /Cyp1b1 −/− mice treated with exogenous E2 or 2-ME and their vehicle (DMSO); (4) the effect of AA metabolism inhibitor ETYA (5, 8, 11,14-eicosatetraeynoic acid, 50 mg/kg, IP, every third day), 20 2-ME, and antagonists of EP1 (SC19220, 10 μg/g, SC, second day), 21 as well as EP3 (L-798106, 10 μg/g, SC, every second day), 4 and (terutroban, 10 μg/g, SC, every second day) 22 receptors and their vehicle (DMSO) on Ang II-induced increase in SBP in ovariectomized-cPLA 2 α +/+ /Cy p1b1 +/+ and intact cPLA 2 α +/+ /Cyp1b1 −/− mice. Administration of E2, 2-ME, ETYA, EP1, EP3, and TP receptor antagonists or their vehicle DMSO was initiated with the implantation of microosmotic pumps for infusion of Ang II or its vehicle (saline).

Kidney sections from various treatment groups were stained with Masson trichrome for collagen detection. The sections were viewed blinded with an Olympus inverted system microscope (Olympus America Inc, model BX41) and photographed using a SPOT Insight digital camera (Diagnostic Instruments Inc, model Insight 2MP Firewire). Images were quantified by ImageJ software version 1.53a (National Institutes of Health, Bethesda, MD).

Kidneys were lysed in TissureLyser II (Qiagen) and centrifuged; an equal amount of protein from each lysate was subjected to SDS-PAGE and transferred onto a nitrocellulose membrane. The blots were probed with anti-phospho (p)-cPLA 2 α and cPLA 2 α and corresponding secondary antibodies.

Urine samples were collected by placing the mice in metabolic cages for 24 hours. PGE 2 is rapidly converted to its 13,14-dihydro-15-keto metabolite in vivo and further undergoes degradation to PGA products. We measured the concentration of its metabolites using a Prostaglandin E Metabolite ELISA kit (Cayman Catalog No. 514531) following the manufacturer's instructions. This kit enables the conversion of 13,14-dihydro-15-keto PGA 2 and 13,14-dihydro-15-keto PGE 2 to a single, stable derivative that can be quantified. TXA 2 is rapidly hydrolyzed nonenzymatically to form TXB 2 and excreted to urine. TXB 2 levels were measured using an ELISA kit (Cayman Catalog No. 501020).

Five micrometer frozen sections of the kidney were exposed to dihydroethidium, and the yellow fluorescence selective for 2-hydoxyethidium as an index of ROS was visualized on a fluorescence microscope (model IX50, Olympus America) with dual-wavelength filter, excitation at 375 nm and emission at 585 nm as described previously. 23 Images were quantified by ImageJ software version 1.53a.

The data were analyzed by one-way ANOVA and Tukey test for multiple comparisons. BP data were analyzed by repeated measures 2-way ANOVA followed by Tukey multiple comparison test. The values of different experiments are expressed as mean±SEM, and P<0.05 was considered statistically significant.

In most of our experiments, the main comparisons exceeded a power of 0.8 with the number of animals used (Data Supplement).

Ang II Increased Mean Arterial BP, SBP, and Diastolic BP in cPLA 2 α +/+ /Cyp1b1 +/+ But Not in Intact or Ovariectomized-cPLA 2 α -/-/Cyp1b1 +/+ Mice Ang II activates cPLA 2 α and releases AA from tissue phospholipids, 24 and AA metabolites with prohypertensive effects contribute to the development of Ang II-induced hypertension in male mice. Ang II produced a smaller increase in BP in females than in male animals, [9] [10] [11] which is enhanced by ovariectomized. 13 To determine if Ang IIinduced hypertension in both intact and ovariectomized female mice is also dependent on cPLA 2 α, we examined the effect of Ang II on BP in intact and ovariectomized-cPL A 2 α +/+ /Cyp1b1 +/+ and cPLA 2 α -/-/Cyp1b1 +/+ mice. Ang II infusion for 2 weeks increased the mean arterial BP, and SBP and diastolic BP, as measured by radiotelemetry in intact ( Figure 1A , Figure S1A and S1B in the Data Supplement) and to a much higher level, in ovariectomized-cPL A 2 α +/+ /Cyp1b1 +/+ ( Figure 1B , and Figure S1D and S1E), but not in intact and ovariectomized-cPLA 2 α -/-/Cyp1b1 +/+ mice ( Figure 1A and 1B, and Figure S1A , S1B, S1D, and S1E); heart rate was not altered in these groups of mice ( Figure S1C and S1F). The increase in SBP produced by Ang II, measured by tail-cuff and radiotelemetry at the same time of the day, are comparable. Therefore, we measured SBP in the rest of the experiments by tail-cuff.

Ovariectomized-cPLA 2 α +/+ /Cyp1b1 +/+ But Not Intact and Ovariectomized-cPLA 2 α -/-/Cyp1b1 +/+ Mice Infusion of Ang II-induced collagen deposition as detected by Masson trichrome staining ( Figure 1C and 1D), and ROS production, as determined by 2-hydroxyethidium fluorescence ( Figure 1E and 1F) in the kidney of intact and ovariectomized-cPLA 2 α +/+ /Cyp1b1 +/+ but not intact and ovariectomized-cPLA 2 α -/-/Cyp1b1 +/+ mice.

Ang II-Induced Increase in Renal cPLA 2 α Activity Was Enhanced in Ovariectomized-cPLA 2 α +/+ /Cyp1b1 +/+ and Intact cPLA 2 α +/+ / Cyp1b1 −/− Mice and Attenuated by E2 Only in Ovariectomized-cPLA 2 α +/+ /Cyp1b1 +/+ , and by 2-ME in Both the Groups Several vasoactive agents, including Ang II, increase cPLA 2 α activity and release AA in the vascular smooth muscle cells. [24] [25] [26] Infusion of Ang II did not alter cPLA 2 α expression but increased cPLA 2 α activity, as determined by a higher ratio of p-cPLA 2 α to cPLA 2 α in the kidneys of cPLA 2 α +/+ /Cyp1b1 +/+ mice. To determine if E2-CYP1B1 generated metabolite 2-ME acts upstream or downstream of cPLA 2 α, we examined the effect of Ang II on cPLA 2 α activity and expression in the kidneys of intact and ovariectomized-cPLA 2 α +/+ /Cyp1b1 +/+ mice and intact cPLA 2 α +/+ /Cyp1b1 -/mice treated with E2 and 2-ME. Ang II-induced increase in the ratio of p-cPLA 2 α was enhanced to a much greater degree in the kidneys of ovariectomized-cPLA 2 α +/+ /Cyp1b1 +/+ compared with the intact cPLA 2 α +/+ /Cyp1b1 +/+ mice ( Figure 2A ). E2 inhibited the ability of Ang II to increase p-cPLA 2 α expression in ovariectomized-cPLA 2 α +/+ / Cyp1b1 +/+ ( Figure 2B ) but not ovariectomized-cPLA 2 α +/+ /Cyp1b1 +/+ mice ( Figure 2C ). Treatment with 2-ME attenuated the Ang II-induced increases in the ratio of p-cPLA 2 α to cPLA 2 α in both ovariectomized-cPLA 2 α +/+ /Cyp1b1 +/+ ( Figure 2D ) and intact cPLA 2 α +/+ / Cyp1b1 -/mice ( Figure 2E ). As expected, cPLA 2 α expression was not detected in the kidneys of cPLA 2 α -/-/ Cyp1b1 +/+ mice with the cPLA 2 α antibody used in our experiment, indicating that this antibody detects only cPLA 2 α ( Figure 2F ). cPLA 2 α Gene Disruption Did not Reduce Plasma Levels of E2 or 2-ME, and Cyp1b1 Gene Disruption Inhibited Plasma Levels of 2-ME but not E2

To determine if cPLA 2 α and Cyp1b1 gene disruption affected the production of E2 and 2-ME, we examined the effect of Ang II infusion on plasma levels of E2 and 2-ME in intact cPLA 2 α +/+ /Cyp1b1 +/+ and cPLA 2 α -/-/ Cyp1b1 +/+ mice and ovariectomized-cPLA 2 α +/+ / Cyp1b1 +/+ and intact and ovariectomized-cPLA 2 α +/+ / Cyp1b1 -/mice with exogenous E2 and 2-ME treatment or their vehicle DMSO. The plasma levels of E2 in intact cPLA 2 α -/-/Cyp1b1 +/+ mice were not different from that observed in cPLA 2 α +/+ /Cyp1b1 +/+ mice and were not altered by Ang II ( Figure S2A ). Plasma levels of E2 and 2-ME were reduced in ovariectomized-cPLA 2 α +/+ /Cyp1b1 +/+ and ovariectomized-cPLA 2 α +/+ / Cyp1b1 -/mice, and treatment with exogenous E2 but not 2-ME increased levels of E2 ( Figure S2A and S2B). Ang II increased plasma levels of 2-ME in both intact cPLA 2 α +/+ /Cyp1b1 +/+ and cPLA 2 α -/-/Cyp1b1 +/+ mice ( Figure S2C ). In ovariectomized-cPLA 2 α +/+ / Cyp1b1 +/+ and intact and ovariectomized-cPLA 2 α +/+ / Cyp1b1 -/mice, 2-ME levels were reduced, and treatment with exogenous E2 raised the basal level of 2-ME that was increased by Ang II in the former but not in the latter groups of mice ( Figure S2C and S2D). Administration of exogenous 2-ME increased its plasma levels in both ovariectomized-cPLA 2 α +/+ / Cyp1b1 +/+ and ovariectomized-cPLA 2 α +/+ /Cyp1b1 -/mice that were not altered by Ang II (Figure S2C and S2D).

Increase in SBP, Renal Collagen Deposition, and ROS Production in Ovariectomized-

We showed that 2-ME treatment inhibits Ang II-induced increase in SBP in ovariectomized-cPLA 2 α +/+ /Cyp1b1 +/+ and intact cPLA 2 α +/+ /Cyp1b1 -/mice. 17 We confirmed this observation ( Figure 3A and 3B) and show here that 2-ME also prevents the ability of Ang II to cause renal collagen deposition ( Figure 3C and 3D) end ROS production in these mice ( Figure 3E and 3F).

Ang II-Induced Increase in SBP, Renal Collagen Deposition, and ROS Production in Ovariectomized-cPLA 2 α +/+ /Cyp1b1 +/+ and Intact

Ang II failed to increase SBP ( Figure 4A and 4B), produce renal collagen deposition ( Figure 4C and 4D), and increase ROS production ( Figure 4E and 4F) in ovariectomized-cPLA 2 α +/+ /Cyp1b1 +/+ and intact cPLA 2 α +/+ /Cyp1b1 -/mice treated with ETYA, an AA metabolism inhibitor. 20 cPLA 2 α Gene Disruption, ETYA, and 2-ME Inhibited Ang II-Induced Increase in Urinary Excretion of PGE 2 and TXA 2 Metabolites

Ang II increased urinary excretion of stable metabolites of PGE 2 (13,14-dihydro-15-keto PGE 2 /PGA 2 ) and TXA 2 (TXB 2 ), respectively, in intact cPLA 2 α +/+ / Cyp1b1 +/+ but not cPLA 2 α -/-/Cyp1b1 +/+ mice ( Figure  S3A and S3B). The AA metabolism inhibitor ETYA, E2, and 2-ME attenuated the ability of Ang II to increase the urinary excretion of metabolites of PGE 2 and TXB 2 in ovariectomized-cPLA 2 α +/+ /Cyp1b1 +/+ mice ( Figure S3A and S3B) , as well as ETYA and 2-ME in intact cPLA 2 α +/+ /Cyp1b1 -/mice ( Figure S3C and S3D). E2 failed to inhibit Ang II-induced increased urinary excretion of PGE 2 metabolites and TXB 2 in ovariectomized-cPLA 2 α +/+ /Cyp1b1 -/mice ( Figure  S3C and S3D) . 4 and TXA 2 (terutroban) 22 reduced Ang II-induced increases in SBP, renal collagen deposition, and ROS production in ovariectomized-cPLA 2 α +/+ /Cyp1b1 +/+ ( Figure 5A through 5C and Figure 6A and 6B) and in intact cPLA 2 α +/+ /Cyp1b1 -/mice ( Figure 5D through 5F and Figure 6C and 6D).

The main findings of the present study are that (1) Ang II-induced hypertension and associated renal ROS production and fibrosis are dependent on cPLA 2 α in female mice; (2) the E2-CYP1B1 derived metabolite 2-ME acts upstream of cPLA 2 α and by inhibiting its activation by Ang II, prevented the production of the AA-generated COX metabolites PGE 2 and TXA 2 ; (3) ovariectomy and Cyp1b1 gene disruption promote the Ang II-induced increases in cPLA 2 α activation and production of PGE 2 and TXA 2 , BP, renal fibrosis, and ROS production, all of which were mitigated by 2-ME; and (4) the AA metabolism inhibitor ETYA and antagonists of PGE 2 and TXA 2 receptors EP1, EP3, and TP, respectively, inhibited Ang II-induced hypertension and associated renal fibrosis and ROS production in ovariectomized-cPLA 2 α +/+ / Cyp1b1 +/+ and intact cPLA 2 α +/+ /Cyp1b1 -/mice. Previously, we showed that the lower increase in BP by Ang II in females rather than in male mice is maintained by the E2 metabolite 2-ME, generated from its sequential metabolism by CYP1B1 and 2-hydroxyestradiol by catechol-O-methyl transferase. 15 Moreover, by activating group IV cPLA 2 α, Ang II releases AA, and generates prohypertensive eicosanoids that contribute to the development of hypertension and its associated pathogenesis in male mice. 1, 2 In the present study, cPLA 2 α gene disruption also attenuated Ang II-induced increases in mean arterial BP, SBP, and diastolic BP, as measured by radiotelemetry, and increased associated renal fibrosis, which was determined by collagen deposition and ROS production as detected by 2-hydroxyethidium fluorescence in female mice. Our demonstration that (1) Ang II-induced increase in cPLA 2 α activity measured by its phosphorylation was enhanced in ovariectomized-cPLA 2 α +/+ /Cyp1b1 +/+ and intact and ovariectomized-cPLA 2 α +/+ /Cyp1b1 -/mice; (2) treatment with E2 and 2-ME inhibited cPLA 2 α activity in ovariectomized-cPLA 2 α +/+ Cyp1b1 +/+ mice, and 2-ME but not E2 attenuated cPLA 2 α activity in intact and ovariectomized-cPLA 2 α +/+ /Cyp1b1 -/mice, which suggests that the E2-CYP1B1-generated metabolite 2-ME acts upstream of cPLA 2 α to inhibit its activity. Supporting this view was our demonstration that Ang II increased plasma levels of 2-ME but not E2 in intact mice, demonstrating that Ang II promotes the conversion of E2 to 2-ME. Moreover, this effect was absent in Cyp1b1 but not cPLA 2 α gene disrupted mice suggesting that this occurs by increased CYP1B1 activity. Supporting this notion were our findings that treatment with exogenous E2 increased both basal and Ang II-induced plasma levels of 2-ME in ovariectomized-cPLA 2 α +/+ /Cyp1b1 +/+ but not in intact and ovariectomized-cPLA 2 α +/+ /Cyp1b1 -/mice. However, the administration of exogenous 2-ME increased its plasma levels in all these groups. Previously, we reported that Ang II increased renal CYP1B1 activity without altering its expression that was associated with increased plasma levels of 2-ME. 15 The mechanism by which 2-ME inhibits Ang IIinduced activation of cPLA 2 α is not known. Since 2-ME treatment alone did not alter the activity of cPLA 2 α, it appears that 2-ME acts as an inhibitory modulator of Ang II-induced activation of cPLA 2 α. 2-ME, which could attenuate cPLA 2 α activity via genomic E2 receptor and its nongenomic G-protein coupled estrogen receptor 1 27 by interfering with one or more signaling molecules including the influx of extracellular calcium, calcium-calmodulin-dependent kinase, and extracellular signal-regulated kinase 1/2 activities required for cPLA 2 α stimulation by Ang II, 24, 25 remains to be determined. 2-ME was reported to inhibit an Ang II-induced rise in cytosolic calcium while also downregulating AT1 receptor in rat vascular smooth muscle cells via G-protein coupled estrogen receptor 1. 28 In our study, the ability of 2-ME to inhibit cPLA 2 α activity is unlikely due to downregulation of the AT1a receptor because treatment with 2-ME did not alter AT1 receptor expression in the kidney. 17 Ang II increased the urinary excretion of PGE 2 metabolites and TXB 2 in intact cPLA 2 α +/+ /Cyp1b1 +/+ but not in cPLA 2 α −/− /Cyp1b1 +/+ mice, and these effects of Ang II were enhanced in ovariectomized-cPLA 2 α +/+ /Cyp1b1 +/+ and intact and ovariectomized-cPLA 2 α +/+ /Cyp1b1 -/mice. Treatment with E2 and 2-ME in ovariectomized-cPLA 2 α +/+ /Cyp1b1 +/+ and 2-ME but not E2 in intact and ovariectomized-cPLA 2 α +/+ /Cyp1b1 -/mice inhibited Ang II-induced increases in urinary excretion of PGE 2 metabolites and TXB 2 . These data suggest that 2-ME, by inhibiting cPLA 2 α activity and AA-COX-generated metabolites of PGE 2 and TXA 2, ameliorates Ang IIinduced hypertension, renal fibrosis, and ROS production. Supporting these conclusions are our findings that 2-ME and the AA metabolism inhibitor ETYA 20 attenuated the Ang II-induced increases in SBP, renal fibrosis, ROS production, and urinary excretion of PGE 2 metabolites and TXB 2 in ovariectomized-cPLA 2 α +/+ /Cyp1b1 +/+ and intact cPLA 2 α +/+ /Cyp1b1 -/mice.

Ang II-induced increases in BP were reported to be responsible for cardiac hypertrophy and fibrosis, along with renal injury. [29] [30] [31] [32] Cyclic stretch via stress-induced calcium influx activates cPLA 2 α and releases AA in rabbit proximal tubule cells 33 and increases ROS production in striated muscle. 34 Therefore, the mechanical stretch caused by Ang II-induced increases in BP might also, by activating cPLA 2 α, produce PGE 2 and TXA 2 , and 2-ME, by inhibiting cPLA 2 α activity, prevents the production of these eicosanoids, ROS production, and renal fibrosis in female mice. PGE 2 via EP1 and EP3, 3, 4 and TXA 2 through TP receptors, 5,35 promote vasoconstrictor and hypertensive effects 

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.

of Ang II. 3,4,36 PGE 2 /EP3 also contributes to L-NAME (Nω-nitro-L-arginine methyl ester hydrochloride) 37 and high salt diet-fed S-P467L mice with decreased PPARγ (peroxisome proliferator-activated receptor γ) activity-induced hypertension. 38 In our study, antagonists of EP1 (SC19220), 21 EP3 (L-798106), 4 and TXA 2 -TP (terutroban) 22 receptors reduced Ang II-induced increases in SBP, renal fibrosis, and ROS production in ovariectomized-cPLA 2 α +/+ /Cyp1b1 +/+ and intact cPLA 2 α +/+ /Cyp1b1 −/− mice. From these observations, it follows that the E2-CYP1B1 generated metabolite 2-ME by inhibiting Ang II-induced cPLA 2 α activation and production of PGE 2 and TXA 2 , reduced stimulation of EP1 and EP3 and TP receptors, respectively, which ameliorates hypertension and associated renal fibrosis and ROS production most likely consequent to decreased BP. Although EP3 and TP receptor antagonists attenuated Ang II-induced increase in BP in ovariectomized and intact Cyp1b1 gene disrupted mice, EP1 receptor antagonist SC19220 abolished the effect of Ang II in Figure 5 . Prostaglandin E 2 receptor antagonist (RA) EP1, EP3, and thromboxane A 2 (TP) RA diminished Ang II (angiotensin II)induced increased systolic blood pressure (SBP) and collagen deposition in ovariectomized (OVX)-cPLA 2 α +/+ /Cyp1b1 +/+ and intact cPLA 2 α +/+ /Cyp1b1 -/mice. Blood pressure was measured by the tail-cuff method in OVX-cPLA 2 α +/+ /Cyp1b1 +/+ (A), and intact cPLA 2 α +/+ /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.

ovariectomized and significantly reduced it in cPLA 2 α + /+ /Cyp1b1 −/− mice. EP1 receptor antagonist SC19220 used in our study has been reported not to alter the binding of [ 3 H]PGE 2 to mouse cloned EP1 receptor expressed in Chinese hamster ovary cells, 39 but it attenuated the effect of PGE 2 to upregulate renin expression in mouse cortical CD-M1 cells expressing EP1 receptors 21 and inhibited binding of [ 3 H]PGE 2 to human cloned EP1 receptor expressed in COS cells. 40 One of the limitations of our study is that we did not confirm the selectivity of EP1, EP3, and TP receptor antagonists used in our study. Therefore, we cannot rule out their nonselective effects. Further studies using EP1, EP3, and TP receptor knockout mice are required to confirm our findings.

In the present study, EP1, EP3, and TP receptor antagonists did not reduce basal SBP in ovariectomized-cPL A 2 α +/+ /Cyp1b1 +/+ and intact cPLA 2 α +/+ /Cyp1b1 −/− mice probably because the amount of PGE 2 and TXA 2 produced due to low basal cPLA 2 α activity in the absence of Ang II, was insufficient to alter BP. Therefore, PGE 2 and TXA 2 via EP1 and EP3 and TP receptors, respectively, appear to promote Ang II-induced increased BP, renal ROS production, and fibrosis by acting as permissive factors in ovariectomized-and Cyp1b1 gene disrupted mice. Ang II promotes PGE 2 -EP3 receptor-mediated constriction of the femoral artery by increasing cell calcium and activating proline-rich tyrosine kinase and Rho-kinase. 41 However, as stated above, antagonists of EP1 and EP3 and TP receptors did not alter the basal BP. Therefore, activation of EP1, EP3, and TP receptors by PGE 2 and TXA 2 , respectively, in our study could promote the effect of Ang II on vascular tone by facilitating the increase in cell calcium and sensitization to calcium by Rho-kinase activation. 4, 25 Ang II also increases ROS generation and isolevuglandin protein adducts in dendritic cells, T cells activation, and cytokine production that contribute to its hypertensive effect and renal fibrosis. 43 In the L-NAME/ high salt-induced model of hypertension, ROS production, dendritic cell activation, accumulation of isolevuglandin protein adducts in spleen cells, and production of proinflammatory cytokines are dependent on PGE 2 activated EP3 receptors. 37 PGE 2 , also via the EP1 receptor, acts directly on dendritic cells to increase isolevuglandin adducted proteins. 37 The protection against Ang IIinduced hypertension in females has been attributed to decreased proinflammatory T cells and increased Treg cell activation, along with increased AT2 and Mas receptor expression and ACE2 in the kidneys of females compared with males. 44 Moreover, L-NAME, an inhibitor of the NOS system, increases BP and reduces the compensatory increase in Treg cells in female rats. 45 2-ME stimulates NO production and inhibits L-NAME-induced increases in BP and cardiac and renal injury, collagen deposition, and infiltration of ED1 + macrophages in the hearts and kidneys. 46,47 2-ME also inhibits activation and proliferation of T and B cells and production of cytokines in vitro and in a model of autoimmune encephalomyelitis. 48 Since cPLA 2 α is also required for L-NAME-induced hypertension and its associated endothelial dysfunction, 49 and deoxycorticosterone salt-induced hypertension (Song and Malik, unpublished work), isolevuglandins are formed from AA, 50 2-ME, by inhibiting Ang II-induced cPLA 2 α activation, AA release, and generation of PGE 2 and TXA 2 would reduce stimulation of EP1 and EP3 receptors. This, in turn, would decrease ROS production and isolevuglandin protein adducts in dendritic cells, activation of T cells and generation of cytokines, and increased BP and associated renal fibrosis. However, further studies are required to determine the effect of 2-ME on PGE 2 /EP1 and EP3 and TXA 2 /TP receptormediated Ang II-and L-NAME-induced isolevuglandin protein adducts formation in dendritic cells, and T lymphocytes and Treg cells activation. Furthermore, protection by 2-ME against eicosanoids with prohypertensive effects generated from AA via lipoxygenase and cytochrome P450 4A1 including 12S-HETE and 20-HETE, respectively, that contribute to Ang II-induced hypertension 1 are currently under investigation. Moreover, the contribution of renal sodium transport and renal vascular function to the eicosanoid-mediated effect of 2-ME in lowering Ang II-induced increased BP in ovariectomized-and Cyp1b1 gene disrupted mice remains to be determined. Finally, the protective effects of 2-ME on pressure-dependent and independent cardiac hypertrophy and fibrosis and renal injury, lowering cholesterol levels, reducing neointimal growth, angiogenesis, pulmonary hypertension, and preeclampsia, magnesium insufficiency-induced salt-sensitive hypertension with reduced catechol-O-methyltransferase activity, and anticarcinogenic actions 47,51-55 all of which could be due to its inhibitory effect on cPLA 2 α activity and generation of eicosanoids that have been implicated in these disorders remains to be explored.

In conclusion, the E2-CYP1B1-generated metabolite 2-ME, by acting upstream of cPLA 2 α and inhibiting its activation by Ang II, attenuates the COX-AA metabolites PGE 2 and TXA 2 and stimulation of EP1 and EP3, and TP receptors, respectively. This, in turn, reduces BP, and renal fibrosis, and ROS production (Graphical Abstract, Figure S4 ). Therefore, selective inhibitors of EP1, EP3, and TP receptors and 2-ME could be useful for treating hypertension and its pathogenesis in postmenopausal females; hypoestrogenemic premenopausal women; women with menstrual irregularities due to ovarian failure; as well as in males.

The protection against Ang II-induced hypertension and associated cardiovascular and renal pathogenesis is mediated by the CYP1B1-E2 derived metabolite 2-ME in female mice. 14-17 Moreover, Ang II-induced hypertension and its associated pathogenesis are mediated by prohypertensive eicosanoids produced by activation of cPLA 2 α in male mice. 1 This study furthers our understanding of the mechanism by which the E2-CYP1B1 metabolite 2-ME protects against Ang II-induced hypertension and its pathogenesis by acting upstream of cPLA 2 α and by inhibiting cPLA 2 α activation, attenuates the generation of AA metabolites PGE 2 and TXA 2 and their prohypertensive effects mediated via stimulation of EP1 and EP3, and TP receptors, respectively. However, the site of interaction of 2-ME and cPLA 2 α is unknown. We recently demonstrated that E2-CYP1B1 generated 2-ME in the paraventricular nucleus, which, by decreasing sympathetic activity, ameliorates Ang II-induced hypertension. 27 Hence, further studies are needed to determine the interaction of the CYP1B1generated E2 metabolite 2-ME and cPLA 2 α and AA metabolites in the paraventricular nucleus to elucidate the mechanism that contributes to the protective effect of E2 against Ang II-induced hypertension and its associated pathogenesis. Moreover, it was recently reported that there is an association of the CYP1B1 Leu432Val gene polymorphism with hypertension in a small group of Slovak midlife women. 56 Therefore, it would be important to explore CYP1B1 gene polymorphism further in a larger population of females and males to determine its impact on the contribution of cPLA 2 α and AA-derived eicosanoids in hypertension and its pathogenesis. Thus, the regulation of CYP1B1-generated E2 metabolite 2-ME and cPLA 2 α and AA metabolites in the kidney and the paraventricular nucleus is important to explore further how E2 protects against Ang II-induced hypertension and its associated pathogenesis. 

Cytosolic phospholipase A2α is critical for angiotensin II-induced hypertension and associated cardiovascular pathophysiology

Cytosolic phospholipase A2α is essential for renal dysfunction and endorgan damage associated with angiotensin ii-induced hypertension

Antihypertensive effects of selective prostaglandin E2 receptor subtype 1 targeting

Inactivation of the E-prostanoid 3 receptor attenuates the angiotensin II pressor response via decreasing arterial contractility

Thromboxane mediates renal hemodynamic response to infused angiotensin II

Role for thromboxane receptors in angiotensin-II-induced hypertension

12-Hydroxyeicosatetraenoic acid participates in angiotensin II afferent arteriolar vasoconstriction by activating L-type calcium channels

Role of 20-hydroxyeicosatetraenoic acid in the renal and vasoconstrictor actions of angiotensin II

Gender differences in the regulation of blood pressure

Sex differences in primary hypertension

Sex differences in hypertension: recent advances

Gender differences in response to acute and chronic angiotensin II infusion: a translational approach

Sex differences in the development of angiotensin II-induced hypertension in conscious mice

Sex hormones and hypertension

Estrogen metabolism by cytochrome P450 1B1 modulates the hypertensive effect of angiotensin II in female mice. Hypertension

Disruption of the cytochrome P-450 1B1 gene exacerbates renal dysfunction and damage associated with angiotensin II-induced hypertension in female mice

2-Methoxyestradiol reduces angiotensin II-induced hypertension and renal dysfunction in ovariectomized female and intact male mice

Reduced fertility and postischaemic brain injury in mice deficient in cytosolic phospholipase A2

Fetal mouse Cyp1b1 and transplacental carcinogenesis from maternal exposure to dibenzo(a,l) pyrene

Inhibition of platelet arachidonic acid 12-lipoxygenase by acetylenic acid compounds

PGE2 upregulates renin through E-prostanoid receptor 1 via PKC/cAMP/CREB pathway in M-1 cells

Renal effects of S18886 (Terutroban), a TP receptor antagonist, in an experimental model of type 2 diabetes

Brain testosterone-CYP1B1 (Cytochrome P450 1B1) generated metabolite 6β-hydroxytestosterone promotes neurogenic hypertension and inflammation

Signal transduction mechanisms involved in angiotensin-(1-7)-stimulated arachidonic acid release and prostanoid synthesis in rabbit aortic smooth muscle cells

Calcium/calmodulin-dependent protein kinase IIalpha mediates activation of mitogen-activated protein kinase and cytosolic phospholipase A2 in norepinephrine-induced arachidonic acid release in rabbit aortic smooth muscle cells

Endothelin-1-induced arachidonic acid release by cytosolic phospholipase A2 activation in rat vascular smooth muscle via extracellular signal-regulated kinases pathway

Central CYP1B1 (Cytochrome P450 1B1)-estradiol metabolite 2-methoxyestradiol protects from hypertension and neuroinflammation in female mice

2-Methoxyestradiol causes matrix metalloproteinase 9-mediated transactivation of epidermal growth factor receptor and angiotensin type 1 receptor downregulation in rat aortic smooth muscle cells

Pressure overload induces cardiac hypertrophy in angiotensin II type 1A receptor knockout mice. Circulation

Angiotensin II stimulates left ventricular hypertrophy in hypertensive patients independently of blood pressure

Direct actions of AT1 (Type 1 Angiotensin) receptors in cardiomyocytes do not contribute to cardiac hypertrophy

Role of pressure in angiotensin II-induced renal injury: chronic servo-control of renal perfusion pressure in rats

Cyclic stretch-induced cPLA2 mediates ERK ½ signaling in rabbit proximal tubule cells

Mechanical stretch-induced activation of ROS/RNS signaling in striated muscle

Role of TXA2 in the pathogenesis of severe angiotensin II-salt hypertension

The deleterious role of the prostaglandin E2 EP3 receptor in angiotensin II hypertension

Central EP3 (E Prostanoid 3) receptors mediate salt-sensitive hypertension and immune activation

EP3 (E-Prostanoid 3) receptor mediates impaired vasodilation in a mouse model of salt-sensitive hypertension

Ligand binding specificities of the eight types and subtypes of the mouse prostanoid receptors expressed in Chinese hamster ovary cells

Cloning and expression of a cDNA for the human prostaglandin E receptor EP1 subtype

Regulation of arterial reactivity by concurrent signaling through the E-prostanoid receptor 3 and angiotensin receptor 1

Thromboxane A2-induced contraction of rat caudal arterial smooth muscle involves activation of Ca2+ entry and Ca2+ sensitization: Rho-associated kinase-mediated phosphorylation of MYPT1 at Thr-855, but not Thr-697

DC isoketal-modified proteins activate T cells and promote hypertension

Renal NOS activity, expression, and localization in male and female spontaneously hypertensive rats

Hypertensive female Sprague-Dawley rats require an intact nitric oxide synthase system for compensatory increases in renal regulatory T cells

Effects of estradiol and its metabolites on glomerular endothelial nitric oxide synthesis and mesangial cell growth

Estradiol metabolites attenuate renal and cardiovascular injury induced by chronic nitric oxide synthase inhibition

2-Methoxyestradiol inhibits experimental autoimmune encephalomyelitis through suppression of immune cell activation

Cytosolic phospholipase A2alpha contributes to blood pressure increases and endothelial dysfunction under chronic NO inhibition

Roberts LJ 2 nd . Identification of extremely reactive gamma-ketoaldehydes (isolevuglandins) as products of the isoprostane pathway and characterization of their lysyl protein adducts

2-Methoxyestradiol protects against pressure overload-induced left ventricular hypertrophy

2-Methoxyestradiol attenuates angiotensin II-induced hypertension, cardiovascular remodeling, and renal injury

Potential vascular actions of 2-methoxyestradiol

2-Methoxyestradiol in pulmonary arterial hypertension: a new disease modifier

Dietary magnesium insufficiency induces salt-sensitive hypertension in mice associated with reduced kidney catechol-o-methyl transferase activity

Association of cytochrome P450 1B1 gene polymorphisms and environmental biomarkers with hypertension in Slovak midlife women

We thank Nathan G. Tipton, PhD, Executive Assistant, Department of Physiology, University of Tennessee Health Science Center for his editorial assistance.

This work was supported by the National Institutes of Health (NIH) National Heart, Lung, and Blood Institute grants R01HL-19134-45 and University of Tennessee Health Science Center CORNET Award (K.U. Malik). J.V. Bonventre was supported by the NIH, National Institutes of Diabetes, Digestive and Kidney Diseases (NIDDK) Grants R37DK039773 and R01DK072381. The contents of this article are solely the authors' responsibility and do not necessarily represent the official views of the National Heart, Lung, and Blood Institute.