Androgen-sensitive hypertension associates with upregulated vascular CYP4A12-20-HETE synthase

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 5α-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(-/-) mice, which exhibit androgen-sensitive hypertension in the male mice, produced increased levels of vascular 20-HETE; furthermore, 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.

Figure 1.

Androgen-driven 20-HETE–dependent hypertension in mice. Mice are treated with either vehicle (placebo pellet) or DHT for 18 days. A subgroup of DHT-treated mice is administered 20-HEDE (10 mg/kg per day) starting at day 12 of treatment. (A) CYP4A12 protein levels in renal PGMVs. (B) 20-HETE levels in renal PGMV. (C) Systolic BP (n=4–6). *P<0.05 versus vehicle.

Figure 2.

BP elevation in Cyp4a14(−/−) male mice is 20-HETE dependent. (A) Systolic BP in male and female Cyp4a14(−/−) mice (n=4–6). *P<0.05 versus female mice. (B) 20-HETE levels in renal PGMV from male and female Cyp4a14(−/−) mice (n=4). *P<0.05 versus female mice. (C) Systolic BP in wild-type (WT) and Cyp4a14(−/−) mice treated with and without 20-HEDGE (10 mg/kg per day) for 12 days (n=4–6). (D) Cumulative concentration-response curve to phenylephrine (10⁻⁹ to 10⁻⁴ M) in PGMVs from WT and Cyp4a14(−/−) mice treated with and without 20-HEDGE for 12 days. *P<0.05 versus WT; ^#P<0.05 versus Cyp4a14(−/−).

Figure 3.

BP measurements in four different lines of Cyp4a12tg mice after administration 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 lines 22, 89, and 96 express both the rtTA-M2 and the Cyp4a12 transgenes. Relative copy numbers of the rtTA-M2/Cyp4a12 transgenes for lines 13, 22, 89, and 96 are 0/2, 2/1.5, 6/5, and 5/6, respectively.

Figure 4.

DOX-increased expression of CYP4A12 in Cyp4a12tg mice. Relative expression of Cyp4a12 in the liver (A) and kidney (B) of line 13 and line 89 (Cyp4a12tg) DOX-treated mice. (C) Western blot of CYP4A12 in kidney of line 13 and line 89 (Cyp4a12tg) DOX-treated mice. (D) 20-HETE synthase activity of microsomal fractions isolated from the kidneys of control and DOX-treated mice expressing only the rTA-M2 gene (line 13) or the combination of the Cyp4a12 and rTA-Ms transgenes (line 89). Activity is determined by measuring the conversion of ¹⁴C-labeled AA (100 µM) to 20-HETE. (E) CYP4A12 (red) and CD31 (green) immunofluorescence in kidney sections from DOX-treated and untreated Cyp4a12tg mice. Panels a and d–f are from DOX-treated Cyp4a12tg mice; panels b and c are from untreated Cyp4a12tg mice. Original magnification, ×100 in E, panels a and b; ×200 in E, panel c; ×400 in E, panels d–f.

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.

Figure 6.

20-HETE antagonist attenuates DOX-induced increase in BP in Cyp4a12tg mice. (A) Systolic BP in Cyp4a12tg treated with and without DOX in the presence and absence of 20-HEDE. (B) Systolic BP in Cyp4a12tg mice treated with DOX for 10 days followed by cotreatment with 20-HEDE or vehicle for 5 additional days. (C) Systolic BP in Cyp4a12tg mice treated with DOX for 9 days and switched to water; at day 18 DOX is added back to the drinking water. Results are mean ± SE (n=4–6). *P<0.05 versus vehicle; ^#P<0.05 versus DOX.

Figure 7.

Vascular function in microvessels from Cyp4a12tg mice. Cumulative concentration-response curve to phenylephrine (A) and acetylcholine (B) in renal interlobar arteries. Effect of stepwise increments in intraluminal pressure on the absolute (C) and the normalized (D) internal diameter (ID) in third branch of mesenteric arteries (n=4–6). *P<0.05 versus water; ^#P<0.05 versus DOX.