Androgen-Sensitive Hypertension Associates with Upregulated Vascular CYP4A12–20-HETE Synthase

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† Cheng-Chia Wu,* Shaojun Mei, Jennifer Cheng,* Yan Ding,* Adam Weidenhammer,* ‡ ‡ Victor Garcia,* Fan Zhang,* Katherine Gotlinger,* Vijaya L. Manthati, John R. Falck, † Jorge H. Capdevila, and Michal L. Schwartzman*

*Department of Pharmacology, New York Medical College, Valhalla, New York; †Departments of Medicine and Biochemistry, Vanderbilt University, Nashville, Tennessee; and ‡Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas

ABSTRACT Although the mechanism underlying the effect of androgen on BP and cardiovascular disease is not well understood, recent studies suggest that 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE), a primary cytochrome P450 4 (Cyp4)–derived eicosanoid, may mediate androgen-induced hypertension. Here, treatment of normotensive mice with 5a-dihydrotestosterone increased BP and induced both Cyp4a12 expression and 20-HETE levels in preglomerular microvessels. Administration of a 20-HETE antagonist prevented and reversed the effects of dihydrotestosterone on BP. Cyp4a14(2/2) mice, which exhibit androgen-sensitive hypertension in the male mice, produced increased levels of vascular 20-HETE; fur- thermore, administration of a 20-HETE antagonist normalized BP. To examine whether androgen-independent increases in 20-HETE are sufﬁcient to cause hypertension, we studied Cyp4a12-transgenic mice, which express the CYP4A12–20-HETE synthase under the control of a doxycycline-sensitive promoter. Administration of doxycycline increased BP by 40%, and administration of a 20-HETE antagonist prevented this increase. Levels of CYP4A12 and 20-HETE in preglomerular microvessels of doxycycline-treated transgenic mice approximately doubled, correlating with increased 20-HETE–dependent sensitivity to phenylephrine-mediated vasoconstriction and with decreased acetylcholine-mediated vasodilation in the renal microvasculature. We observed a similar contribution of 20-HETE to myogenic tone in the mesenteric microvasculature. Taken together, these results suggest that 20-HETE both mediates androgen- induced hypertension and can cause hypertension independent of androgen.

J Am Soc Nephrol 24: 1288–1296, 2013. doi: 10.1681/ASN.2012070714

The v-hydroxylation of arachidonic acid (AA) to spontaneously hypertensive rats (SHRs),13,14 (2) in- 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20- hibition of vascular 20-HETE synthesis by CYP4A2 HETE) is catalyzed by members of the cytochrome antisense oligonucleotides decreases BP in SHRs,15,16 P450 4 (CYP4) gene family and regulated by factors such as age, sex hormones, and dietary lipids.1,2 CYP4 expression and 20-HETE synthesis have Received July 20, 2012. Accepted February 20, 2013. been implicated in the regulation of vascular and tu- Published online ahead of print. Publication date available at bular function and the development of hypertension www.jasn.org. 3–5 in experimental models. Studies demonstrating Present address: Dr. Jennifer Cheng, National Institute of 6–8 that 20-HETE is a vasoconstrictor suggest that in- Environmental Health Sciences, Research Triangle Park, North creased 20-HETE synthesis and/or effects in the renal Carolina. 9–12 vasculature underlies its prohypertensive property. Correspondence: Dr. Michal L. Schwartzman, Department of This notion has been substantiated by several reports Pharmacology, New York Medical College, 15 Dana Road, showing the following: (1) the synthesis of and vas- Valhalla, NY, 10595. Email: [email protected] cular reactivity to 20-HETE are signiﬁcantly higher in Copyright © 2013 by the American Society of Nephrology

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most prominently within the vasculature.27,29 Importantly, the androgen-induced increases in BP in rats are abrogated by administration of an inhibitor of 20-HETE synthesis or a 20-HETE antagonist,27,30 suggesting that 20- HETE mediates, at least in part, the hypertensive effect of androgen. However, the relative contribution of upregulating the expression of CYP4A12 and the production of 20- HETE in renal vascular and tubular structures to androgen-induced hypertension as op- posed to other androgen-dependent actions such as activation of the renin-angiotensin system31 and upregulation of sodium trans- porters32 is unclear. This study aimed to address this issue by developing transgenic mice in which the expression of the CYP4A12– 20-HETE synthase is under the control of an androgen-independent, tetracycline (doxycycline [DOX])–sensitive promoter. Here we show that androgen and/or DOX- mediated induction of CYP4A12 expression leads to 20-HETE–dependent vascular dysfunction and hypertension.

RESULTS

20-HETE Mediates Androgen-Driven Figure 1. Androgen-driven 20-HETE–dependent hypertension in mice. Mice are Hypertension treated with either vehicle (placebo pellet) or DHT for 18 days. A subgroup of DHT- Androgen has been shown to induce treated mice is administered 20-HEDE (10 mg/kg per day) starting at day 12 of CYP4A12 in mice but the relationship treatment. (A) CYP4A12 protein levels in renal PGMVs. (B) 20-HETE levels in renal between androgen-mediated CYP4A12 in- PGMV. (C) Systolic BP (n=4–6). *P,0.05 versus vehicle. duction, 20-HETE synthesis, and hypertension has not been established. As seen in and (3) endothelial-specific transduction of CYP4A2 cDNA in- Figure 1, A and B, after 17 days of treatment with androgen (5a- creases vascular 20-HETE synthesis and causes hypertension.17–19 dihydrotestosterone [DHT]), mouse renal preglomerular mi- The aforementioned studies, along with clinical reports in- crovessels (PGMVs) showed a 40% increase in CYP4A12 levels dicating an association between 20-HETE and hypertension in and a 3- to 4-fold increase in endogenous 20-HETE production. humans,20,21 prompted studies to identify the putative 20-HETE Moreover, within the first 9 days of androgen treatment, systolic synthase hypertensive gene. The murine Cyp4a genes include BP of DHT-treated mice increased significantly (137.660.7 Cyp4a10, Cyp4a12,andCyp4a14. There are two Cyp4a12 genes, mmHg versus 109.960.5 mmHg for DHT and vehicle-treated Cyp4a12a and Cyp4a12b, the product of a tandem 100-kb dupli- mice, respectively) and reached 141.360.6 mmHg by day 15. cation within the Cyp4abx cluster.22 Cyp4a10 is expressed in both Importantly, addition of 20-hydroxyeicosa-6(Z),15(Z)-dienoic male and female mice, whereas Cyp4a12a is male specificand acid (20-HEDE), a 20-HETE antagonist, at day 13 of the above androgen regulated and Cyp4a14 is highly expressed in female regimen, rapidly normalized the BP of DHT-treated mice, in- mice.23–25 Among these CYP4A proteins, only CYP4A12 exhibits dicating that the pressure effects of DHTare 20-HETE mediated significant 20-HETE synthase activity.23,24,26 Interestingly, (Figure 1C). Control experiments showed that 20-HEDE did not Cyp4a14(2/2) mice display male-specifichypertensionthatis affect CYP4A12 expression nor did it change 20-HETE levels in associated with androgen-driven increases in CYP4A12 expres- the renal PGMVs (Figure 1, A and B). sion and 20-HETE biosynthesis.23 Indeed, androgen administration to male or female rats or mice causes hypertension.23,27–29 Cyp4a14(2/2) Mice Display 20-HETE–Dependent Moreover, the hypertensive response to androgen has been asso- Male-Specific Hypertension ciated with increases in CYP4A827,29 and CYP4A1223,24 expres- Previous studies by Holla et al.23 showed that Cyp4a14(2/2) sion in rats and mice, respectively, as well as 20-HETE production, mice displayed male-specific hypertension that was associated

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was attenuated (P,0.05) by treatment with 20-HEDGE (EC50=0.2860.05 mM), suggesting that 20-HETE contributed to increased vascular reactivity in these mice.

Characterization of Cyp4a12tg Mice To examine the roles played by the CYP4A12–20-HETE synthase in androgen-sensitive hypertension, transgenic mice overexpressing CYP4A12 under the control of a tetracycline-sensitive promoter (Tet-on) were developed. Inasmuch as transgene copy number and insertion site (s) influence their expression level and/or could alter the expression, integrity, or functional properties of alternate unknown genes, several founder lines were character- ized for their transgene relative copy numbers (Supplemental Figure 1) and BP responses to DOX administration. As seen in Figure 3, lines 22, 89, and 96, which express the rtTA-M2 and Cyp4a12 transgenes, showed BP increases in response to DOX. In contrast, the BP of the DOX- treated line 13 mice remained more or less normal (Figure 3). RT-PCR analysis of liver and kidney RNAs from mice of lines 13 and 89 indicated that DOX increased Cyp4a12 mRNA levels only in mice from line 89 (Figure 4, A and B). Like- wise, DOX increased the levels of CYP4A12 Figure 2. BP elevation in Cyp4a14(2/2) male mice is 20-HETE dependent. (A) Sys- protein (Figure 4C) and the microsomal tolic BP in male and female Cyp4a14(2/2) mice (n=4–6). *P,0.05 versus female mice. CYP4A12 activity as measured by conver- (B) 20-HETE levels in renal PGMV from male and female Cyp4a14(2/2) mice (n=4). sion of AA to 20-HETE (Figure 4D), only in *P,0.05 versus female mice. (C) Systolic BP in wild-type (WT) and Cyp4a14(2/2) mice kidneys of line 89 mice. Immunofluores- treated with and without 20-HEDGE (10 mg/kg per day) for 12 days (n=4–6). (D) 2 2 cence of kidney sections from DOX-treated Cumulative concentration-response curve to phenylephrine (10 9 to 10 4 M) in 2 2 Cyp4a12tg mice showed CYP4A12 expres- PGMVs from WT and Cyp4a14( / ) mice treated with and without 20-HEDGE for 12 sion in all kidney structures with intense days. *P,0.05 versus WT; #P,0.05 versus Cyp4a14(2/2). signals in the vasculature (Figure 4E, panel a). Additional costaining with CD31 anti- with increases in plasma androgen levels and renal CYP4A12 bodies indicated substantial expression of CYP4A12 in kidney expression, and suggested that an increase in 20-HETE bio- vessels that appeared to be localized to the smooth muscle layer synthetic capacity contributed to the hypertension in these (Figure 4E, panels d–f). Lastly, as seen in Figure 5A, DOX treat- mice. As seen in Figure 2, A and B, BP as well as renal vascular ment increased CYP4A12 levels in renal PGMV by 2-fold. This production of 20-HETE were sex dependent, and importantly, was associated with a 2-fold increase in endogenous produc- administration of the 20-HETE antagonist, N-(20-hydroxyei- tion of 20-HETE (Figure 5B). Mice from line 89 were used for cosa-6(Z),15(Z)-dienoyl)glycine (20-HEDGE), lowered the all subsequent experiments as the Cyp4a12tg mice. Both male BP of male Cyp4a14(2/2) mice (Figure 2C), suggesting that and female CYP4a12tg mice displayed DOX-sensitive hyper- the hypertension in these mice is mediated by 20-HETE. In tension that was associated with CYP4A12 induction and in- addition, as seen in Figure 2D, renal interlobar arteries from creased 20-HETE production. Cyp4a14(2/2) mice displayed increased sensitivity to phenylephrine-induced constriction as evidenced by a significant Blockade of 20-HETE Action Prevents DOX-Induced decrease in the phenylephrine EC50 (0.7260.14 and Hypertension in Cyp4a12tg Mice 0.1160.01 mM in wild-type mice and Cyp4a14(2/2), respec- To determine whether the DOX-induced increase in 20- tively; P,0.05). This increased sensitivity to phenylephrine HETE contributes to the hypertension, 20-HEDE was either

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shown in Figure 7, C and D, the myogenic tone of the third branch of the mesenteric arteries (approximately 60–80 mm) significantly increased in DOX-treated as com- pared with nontreated Cyp4a12tg mice. Moreover, DOX-induced increase in myogenic response was abrogated by cotreatment with 20-HEDE, suggesting that 20-HETE contributed to increased vascular tone in the mesenteric microcirculation of the Cyp4a12tg mice.

DISCUSSION

Epidemiologic, clinical, and experimental studies have shown that androgen is an important determinant of sex-specificdif- Figure 3. BP measurements in four different lines of Cyp4a12tg mice after adminis- ferences in arterial BP; men aged ,60 years tration of DOX. Systolic BP is measured by tail-cuff after placing mice on DOX (1 mg/ml in drinking water). Line 13 mice express the rtTA-M2 transgene only, whereas mice of have higher BP than women of the same 33–35 lines 22, 89, and 96 express both the rtTA-M2 and the Cyp4a12 transgenes. Relative age. Androgen has been suggested to copy numbers of the rtTA-M2/Cyp4a12 transgenes for lines 13, 22, 89, and 96 are 0/2, contribute to hypertension in postmeno- 2/1.5, 6/5, and 5/6, respectively. pausal women and in several experimental models.36–40 The postulated mechanisms for androgen modulation of BP are numer- coadministered with DOX or administered to Cyp4a12tg mice ous and include stimulation of renal prohypertensive pro- treated with DOX after 10 days. Cotreatment with 20-HEDE cesses involving the renin-angiotensin-aldosterone system, prevented the BP increase in DOX-treated Cyp4a12tg mice increases in proximal tubular reabsorption, and amplification (93.061.6 mmHg versus 14565.9 mmHg) (Figure 6A). When of vascular tone through an upregulation of vasoconstrictor administered after 10 days of DOX treatment, 20-HEDE signifi- autacoids.31,32,41,42 20-HETE has been recognized as a prohy- cantly attenuated the BP increase (124.464.3 mmHg) (Figure pertensive lipid autacoid43 and several studies,23,27,29,30 in- 6B). On the other hand, 20-HEDE had no effect on the BP of cluding this study, identified 20-HETE as a potential mediator Cyp4a12tg mice without DOX treatment (Figure 6A). Moreover, of androgen-induced hypertension. Given the multiple ac- cessation of DOX administration resulted in a rapid decrease in tions of androgen on several prohypertensive systems, it is BP,which returned to baseline within 5 days. BP began to rise once difficult to separate 20-HETE’s contribution to the BP increase thesemicewereagainplacedonDOXtreatment(Figure6C). from that of androgen. The Cyp4a12tg mice, which overexpress the Cyp4a12 gene Cyp4a12tg Mice Display 20-HETE–Dependent under the control of the nonmammalian, endogenous factor– Increased Vascular Reactivity and Myogenic Response independent tetracycline-sensitive promoter, provided a Renal interlobar arteries from mice treated with DOX for 14 days model to assess the contribution of 20-HETE to hypertension displayed a significant increase in sensitivity to phenylephrine- in the absence of androgen. In these mice, liver and kidney induced vasoconstriction as evidenced by a reduction in EC50 to CYP4A12 expression is rapidly induced upon the addition of phenylephrine (from 1.3060.10 to 0.3760.02 mM). This in- DOX to the drinking water and immunohistochemistry anal- crease in sensitivity was attenuated in arteries from mice cotrea- ysis of kidney sections indicated that CYP4A12 expression is ted with DOX and 20-HEDE (EC50=1.6060.10 mM) (Figure localized not only to tubular structures but also to the vascu- 7A). In addition, endothelial dysfunction measured as impaired lature. Moreover, renal microsomes and PGMVs harvested relaxing responses to acetylcholine was observed in DOX-treated from DOX-treated Cyp4a12tg mice displayed higher levels mice. The relaxation response to 10 mM acetylcholine in arteries of CYP4A12 protein and produced more 20-HETE. More im- from DOX-treated Cyp4a12tg mice was 31.7%63.2% (com- portantly, the DOX-treated Cyp4a12tg mice were hyperten- pared with 68.7%610.9% in arteries from vehicle-treated Cy- sive and showed 20-HETE–dependent increases in vascular p4a12tg mice); this impaired relaxing response was attenuated in reactivity and endothelial dysfunction, along with increased arteries from DOX-treated Cyp4a12tg mice that were coadmi- myogenic responses. nistered 20-HEDE (60.4%65.9% relaxation) (Figure 7B). The increase in BP in DOX-treated Cyp4a12tg mice, not- A similar contribution of 20-HETE to vascular tone was also withstanding the drawbacks of the tail-cuff plethysmography seen in the mesenteric microcirculation of Cyp4a12tg mice. As method, was comparable regarding the time and magnitude of

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mesenteric arteries that was abrogated by 20-HETE antagonist suggests that hypertension in this model is a consequence of increased vascular tone brought about by increased 20-HETE production in renal and extrarenal microvasculature. However, the contribution of tubular CYP4A12 expression to setting the level of BP in this model cannot be excluded. Indeed, immunofluorescence showed a substantial CYP4A12 expression in tubular structures and kidney microsomes from DOX-treated Cyp4a12tg mice displayed high capacity to produce 20-HETE, suggesting the possibility that tubular actions of 20-HETE also contributed to the hypertension in this model. In this regard, Quigley and co-workers,32,42 demonstrated that hypertension in androgen- treated rats and in Cyp4a14(2/2)miceis associated with androgen-driven increases in sodium reabsorption at the level of the proximal tubule, a major site of 20-HETE synthesis along the nephron.44,45 It is possi- ble that chronic upregulation of 20-HETE leads to enhanced tubular reabsorption even though 20-HETE acutely inhibits sodium transport in both the proximal tubule46 and the medullary thick ascending loop of Henle.47,48 Taken together, the mechanism underlying 20-HETE contribution to hypertension in these models may be a combination of elevated peripheral vascular resistance coupled with 20-HETE–dependent increase in tubular sodium reabsorption. This notion is supported by findings by Fi- delis et al.,49 who showed that increased renal vasoconstriction and reduced vasodila- Figure 4. DOX-increased expression of CYP4A12 in Cyp4a12tg mice. Relative ex- tion along with diminished capacity to pression of Cyp4a12 in the liver (A) and kidney (B) of line 13 and line 89 (Cyp4a12tg) excrete sodium contributed to the hyperten- DOX-treated mice. (C) Western blot of CYP4A12 in kidney of line 13 and line 89 sion in the Cyp4a14(2/2) mice. The reports (Cyp4a12tg) DOX-treated mice. (D) 20-HETE synthase activity of microsomal fractions of Quigley et al.42 that deletion of Cyp4a14 isolated from the kidneys of control and DOX-treated mice expressing only the rTA-M2 had no effect on vascular reactivity of the gene (line 13) or the combination of the Cyp4a12 and rTA-Ms transgenes (line 89). afferent arterioles and Holla et al.23 of loss Activity is determined by measuring the conversion of 14C-labeled AA (100 mM) to 20- of myogenic tone of the afferent arteriole HETE. (E) CYP4A12 (red) and CD31 (green) immunofluorescence in kidney sections also suggest resetting of the pressure natri- – from DOX-treated and untreated Cyp4a12tg mice. Panels a and d f are from DOX- uresis independent of elevated renal vascular treated Cyp4a12tg mice; panels b and c are from untreated Cyp4a12tg mice. Original tone similar to what was reported in the magnification, 3100 in E, panels a and b; 3200inE,panelc;3400 in E, panels d–f. SHR.50,51 Taken together, data presented here indicate that these models have a 20- the response to that observed in DHT-treated mice; in both, BP HETE–dependent hypertension driven by increased renal and increased significantly at day 5–6, reaching 140–150 mmHg. In peripheral vascular reactivity. The exact mechanism of the role addition, in both models, the BP increases were 20-HETE de- of the kidney in this model remains to be determined. pendent because a 20-HETE antagonist normalized BP. The The finding that high BP is driven by increasing the demonstration of increased vascular reactivity of the renal inter- expression and activity of the CYP4A12–20-HETE pathway lobar arteries and myogenic tone of the third branch of the has significant implications to human hypertension. In

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Figure 5. DOX-increased expression of CYP4A12 and 20-HETE production in PGMV from Cyp4a12tg mice. (A) A representative Western blot and densitometry analysis of CYP4A12 and (B) 20- HETE levels in PGMV from Cyp4a12tg mice treated with and without DOX and 20-HEDE for 14 days (n=4). *P,0.05 versus water. humans, two CYP isoforms have been identified to be the major 20-HETE synthases, CYP4A11 and CYP4F2.52,53 The T8590C CYP4A11 polymorphism has been shown to be associated with hypertension.52,54 AstudybyWardet al.21 Figure 6. 20-HETE antagonist attenuates DOX-induced increase showed a positive association between the V433M CYP4F2 in BP in Cyp4a12tg mice. (A) Systolic BP in Cyp4a12tg treated with polymorphism and both BP and urinary 20-HETE, and a re- and without DOX in the presence and absence of 20-HEDE. (B) cent study by Hu et al.55 showed increased central and periph- Systolic BP in Cyp4a12tg mice treated with DOX for 10 days eral arterial stiffness that was correlated with increased urinary followed by cotreatment with 20-HEDE or vehicle for 5 additional 20-HETE in patients with the V433M CYP4F2 polymorphism. days. (C) Systolic BP in Cyp4a12tg mice treated with DOX for 9 Thus, the CYP4F2 polymorphism, which results in a mutation days and switched to water; at day 18 DOX is added back to the 6 – , that renders the CYP4F2 enzyme inactive,56 is associated with drinking water. Results are mean SE (n=4 6). *P 0.05 versus vehicle; #P,0.05 versus DOX. increased levels of 20-HETE in humans. This observation suggests that the V433M CYP4F2 polymorphism is somewhat analogous to deletion of the Cyp4a14 in mice; in both, there sex-dependent differences in BP and the hypertension fre- is an increase in 20-HETE levels. Interestingly, a recent study quently seen in menopausal women. These studies suggested by Fava et al.57 demonstrated that diastolic BP is higher only in that targeting androgen synthesis or action may have thera- male carriers of the CYP4F2 V433M allele. In addition, trans- peutic values.34 However, androgen actions are diverse and genic mice expressing the human CYP4F2 in the kidney have recent studies suggest that testosterone has beneficial effects elevated systolic BP associated with increased levels of 20- on several cardiovascular risk factors, including suppression HETE.58 of the inflammatory response and improving patient symp- Although the relationship between androgen, 20-HETE, toms and well-being. Low testosterone in men is a risk factor and hypertension in animal models is well studied, this for cardiovascular/metabolic disease and is associated inde- relationship and its association with either CYP4A11 or pendently with visceral obesity, insulin resistance, hyperglyce- CYP4F2 expression in humans are yet to be established. mia, hypertension, and dyslipidemia.41,59 These opposing Androgen has been implicated as the driving force of effects of androgen call for a better understanding of its

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(1 mg/ml) in their drinking water for 15–60 days. In some experiments, mice were administered the 20-HETE antagonists, 20-HEDE or 20- HEDGE (10 mg/kg of body weight per day in 5% ethanol in saline, intraperitoneally).18,30 C57BL/ 6 mice were implanted with pellets containing placebo or DHT (5 mg/kg per day for 14 days; Innovative Research of America, Sarasota, FL). BP was measured by the tail-cuff method. At the end of the experiments, mice were anesthetized, kidneys were removed, and renal PGMVs or mesenteric arteries were microdissected for bio- chemical and functional analyses.

Measurements of 20-HETE Renal microsomal 20-HETE synthesis from AA was measured as previously described.29 PGMVs were isolated from mice and incubated in oxy- genated Krebs bicarbonate buffer, pH 7.4, with 1 mM nicotinamide adenine dinucleotide phos- phate for 1 hour at 37°C with gentle shaking. Deuterated 20-HETE was added as an internal standard and 20-HETE was extracted and quan- tiﬁed by liquid chromatography with tandem mass spectrometry (Applied Biosystems, Foster City, CA) as previously described.17

Vascular Function Figure 7. Vascular function in microvessels from Cyp4a12tg mice. Cumulative con- Wire myograph was used to measure constrictor 29 25 centration-response curve to phenylephrine (A) and acetylcholine (B) in renal interlobar responses to phenylephrine (10 to 5310 M) 2 2 arteries. Effect of stepwise increments in intraluminal pressure on the absolute (C) and and relaxation to acetylcholine (10 9 to 5310 5 the normalized (D) internal diameter (ID) in third branch of mesenteric arteries (n=4–6). M) of renal interlobar arteries (approximately # *P,0.05 versus water; P,0.05 versus DOX. 100 mm diameter) as previously described.30 Myo- genic responses were measured in freshly isolated segments from the third branch of mesenteric arteries (1–2 mm length) mounted between two mi- mechanism of action in distinct pathophysiologic conditions. cropipettes in the chamber (1 ml) of a pressure myograph as previously Our studies identifying 20-HETE as the mediator of andro- described.60 gen-induced hypertension suggest that targeting 20-HETE may spare the beneficial effects of androgen while abrogating Statistical Analyses androgen-dependent vascular dysfunction and hypertension. The data are presented as mean 6 SE. Statistical significance (P,0.05) between the experimental groups was determined by the Fisher method of analysis for multiple comparisons. For comparison be- CONCISE METHODS tween treatment groups, the null hypothesis was tested by a single- factor ANOVA (Dunnett’s multiple comparison test) for multiple Animal Experimentation groups or unpaired t test for two groups.We thank Dr. Alberto Nasjletti Afulldescriptionofvectorconstructions, verification,andproduction and Dr. Tanush Gupta for their insightful comments in preparing this of transgenic mice is provided in the Supplemental Material. Cyp4a12 manuscript. transgenic mice were produced on a B6D2 background (a cross between female C57BL/6J and male DBA/2J). All experiments were performed following an Institutional Animal Care and Use Commit- tee–approved protocol in accordance with the National Institutes of ACKNOWLEDGMENTS Health Guidelines for the Care and Use of Laboratory Animals. Male and female Cyp4a14(2/2)23 (129/Sv background) were used between 8 This study was supported by National Institutes of Health grants and 14 weeks of age. Cyp4a12tg mice were administered DOX ( HL097402 to C.-C.W., GM31278 to J.R.F., DK038226 to J.H.C.,

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HL034300 to M.L.S.), a Diversity Supplement Award (HL34300- vascular 20-hydroxyeicosatetraenoic acid synthesis in the rat kidney. 26A1S1 to V.G.), an American Heart Association predoctoral fel- Am J Physiol 276: F246–F253, 1998 lowship (0715781T to J.C.), and a Robert A. Welch Foundation grant 16. Wang MH, Zhang F, Marji J, Zand BA, Nasjletti A, Laniado- Schwartzman M: CYP4A1 antisense oligonucleotide reduces mesen- (GL625910 to J.R.F.). teric vascular reactivity and blood pressure in SHR. Am J Physiol Regul Integr Comp Physiol 280: R255–R261, 2001 17. Inoue K, Sodhi K, Puri N, Gotlinger KH, Cao J, Rezzani R, Falck JR, fi DISCLOSURES Abraham NG, Laniado-Schwartzman M: Endothelial-speci cCYP4A2 overexpression leads to renal injury and hypertension via increased pro- None. duction of 20-HETE. Am J Physiol Renal Physiol 297: F875–F884, 2009 18. 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