Androgen-induced Hypertension Research Articles

Androgen-induced hypertension in angiotensinogen deficient mice: Role of 20-HETE and EETS

20-HETE is a potent inducer of endothelial ACE in vitro and administration of lisinopril or losartan attenuates blood pressure in models of 20-HETE-dependent hypertension. The present study was undertaken to further define the relationship between 20-HETE and the renin–angiotensin system in hypertension using an angiotensinogen-deficient mouse (Agt+/−). Treatment of male AGT+/− with 5α-dihydrotestosterone (DHT) increased systolic BP from 102±2 to 125±3mmHg; in comparison, the same treatment raised BP in wild type (WT) from 110±2 to 138±2mmHg. DHT increased vascular 20-HETE levels in AGT+/− and WT from 1.5±0.7 and 2.1±0.6 to 13.0±2.0 and 15.8±4.0ng/mg, respectively. Concurrent treatment with the 20-HETE antagonist, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE) prevented the increases in BP in both AGT+/− and WT mice. Administration of 20-HEDE at the peak of the DHT-induced BP increase (12 days) reduced BP to basal levels after 48h. Interestingly, basal levels of renal microvascular EETs were higher in AGT+/− compared to WT (55.2±9.7 vs 20.0±4.1ng/mg) and treatment of AGT+/− with DHT decreased the levels of EETs (28.4±5.1ng/mg). DHT-mediated changes in vascular EET level were not observed in WT mice. Vascular Cyp4a12 and ACE protein levels were increased in both AGT+/− and WT by 30–40% and decreased with concomitant administration of 20-HEDE. Lisinopril was as effective as 20-HEDE in preventing DHT-mediated increases in BP in both AGT+/− and WT mice. This study substantiates our previous findings that the RAS plays an important role in 20-HETE-mediated hypertension. It also proposes a novel interaction between 20-HETE and EETs.

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

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.

20‐HETE INDUCES REMODELING OF RENAL RESISTANCE ARTERIES INDEPENDENT OF BLOOD PRESSURE ELEVATION IN ANDROGEN‐INDUCED HYPERTENSION

20‐Hydroxyeicosatetraenoic acid (20‐HETE) is a cytochrome P450‐derived arachidonate metabolite that has been shown to increase smooth muscle contractions and proliferation, stimulate endothelial dysfunction and promote hypertension. We examined if 20‐HETE contributes to vascular remodeling in androgen‐induced hypertension. In Sprague‐Dawley (SD) rats treated with 5α‐dihydrotestosterone (DHT), the increase in blood pressure (BP) is prevented by 20‐HETE inhibitor, HET0016, or 20‐HETE antagonist, 20–6,15‐HEDE. Likewise, inhibition of 20‐HETE's biosynthesis or activity abrogated DHT‐induced remodeling in renal interlobar arteries. In cyp4a14−/− mice, which display androgen‐driven and 20‐HETE‐dependent hypertension, treatment with 20–6,15‐HEDE abolished remodeling of renal interlobar arteries decreasing media thickness from 23.67±0.88 to 14.56±0.59μm and M/L from 0.29±0.03 to 0.17±0.01. To assess whether 20‐HETE induces vascular remodeling independent of BP increase, SD rats were administered reserpine (100μg/kg/day) and DHT for 21 days. Reserpine prevented BP elevation but did not affect vascular remodeling; under these conditions, treatment with 20–6,15‐HEDE attenuated vascular remodeling. This study demonstrates that 20‐HETE is a key regulator of vascular remodeling in androgen‐induced hypertension; its effect is independent of BP elevation.

Abstract 263: 20-hete-induced Vascular Remodeling in Models Of Androgen-dependent and Independent Hypertension

20-Hydroxyeicosatetraenoic acid (20-HETE) promotes smooth muscle contraction and proliferation, endothelial dysfunction and activation and hypertension. We have previously demonstrated that 20-HETE mediates androgen-induced hypertension. This study examines if 20-HETE contributes to vascular remodeling in androgen-induced hypertension. Sprague-Dawley rats were treated with vehicle or 5α-dihydrotestosterone (DHT) with and without the 20-HETE synthesis inhibitor (HET0016, 10mg/kg/day) for 14 days. Media-to-lumen ratio (M:L) in renal interlobar arteries from DHT-treated rats was higher (P<0.05) than vehicle-treated rats (0.21±0.01 vs. 0.15±0.02). This was prevented by HET0016 (0.14±0.01). Arteries from DHT-treated rats had increased (p<0.05) collagen type IV that was attenuated by HET0016. To assess whether 20-HETE induces vascular remodeling independent of blood pressure (BP) increase, reserpine (100 μg/kg/day) was administered to DHT- and vehicle-treated rats for 21 days. DHT increased BP (133.7±2.4 vs. 101.40±0.40 mmHg). Reserpine prevented this BP increase (104.20±1.30 mmHg) but had no effect on DHT-induced increase in M:L ratio (0.19±0.02) or in collagen I/IV deposition. These results suggest that DHT-mediated vascular remodeling is 20-HETE-dependent and that 20-HETE may contribute to DHT-induced vascular remodeling independent of BP elevation. Whether 20-HETE induces vascular remodeling in a DHT-independent manner was also examined. Cyp4a12 transgenic mice were treated with doxycycline (DOX) for 42 days to upregulate the cyp4a12 expression. BP of DOX-treated cyp4a12tg mice significantly increased compared to vehicle (139.59±4.06 vs. 92.25±5.25 mmHg) and 20-6,15-HEDE abolished BP increase (88.63±1.13 mmHg). Media thickness and M:L ratio significantly increased in DOX-treated group compared to vehicle (23.40±1.15 vs. 16.25±0.83μm; 0.39±0.04 vs. 0.23±0.02) and decreased by co-treatment with 20-6,15-HEDE (18.42±1.53μm; 0.28±0.03). These results indicate that 20-HETE can induce vascular remodeling in the absence of androgen.

Abstract LB-160: CYP4F isoform expression and 20-HETE synthesis in prostate cancer cells are regulated by androgen and contribute to growth

Abstract 20-hydroxyeicosatetraenoic acid (20-HETE), a CYP450-derived arachidonic acid metabolite in the microcirculation, contributes to vascular tone and stimulates vascular and epithelial cell proliferation, angiogenesis and inflammation, all of which play a role in cancer progression. Our laboratory identified 20-HETE as the mediator of androgen-induced hypertension. Given that androgen contributes to the pathogenesis of prostate cancer, we examined the presence and function of 20-HETE in human prostate cancer cells. Androgen-responsive prostate cancer cells, LNCaP, produced 2-fold more 20-HETE compared to androgen-nonresponsive C4-2 cells (565±139 pg/mg protein vs 223±50 pg/mg protein). Both C4-2 and LNCaP produced greater amount of 20-HETE compared with U87 glioma, MCF-7 breast cancer and ML-1 thyroid cancer cells. 20-HETE levels in prostate primary stromal cells and epithelial RWPE-1 cells were barely detected (16±26 and 23±40 pg/mg protein respectively). Treatment of LNCaP with 5 alpha-dihydrotestosterone (DHT) stimulated 20-HETE production by 65% when compared to vehicle control. DHT treatment did not alter 20-HETE levels in C4-2. Treatment of C4-2 and LNCaP with HET0016, an inhibitor of 20-HETE synthesis, selectively decreased endogenous 20-HETE production while inhibiting cell growth in a dose-dependent manner. Further addition of exogenous 20-HETE stimulated growth. CYP4F2, CYP4F3 and CYP4F8 mRNA levels were several fold higher in LNCaP compared with C4-2, while levels of CYP4A11 and CYP4A22 mRNA were unchanged. In LNCaP, DHT increased CYP4F isoform expression without altering the CYP4A expression; flutamide abrogated DHT-induced expression of CYP4F2 and CYP4F3 but did not affect expression of CYP4F8. Importantly, flutamide inhibited DHT-stimulated 20-HETE production. These results suggest that 20-HETE has an important role in cancer cell proliferation and CYP4F2/3 might be the source of androgen-driven 20-HETE synthesis via androgen receptor dependent pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-160. doi:1538-7445.AM2012-LB-160

Disturbed ratio of renal 20-HETE/EETs is involved in androgen-induced hypertension in cytochrome P450 4F2 transgenic mice

We have previously established a cytochrome P450 4F2 (CYP4F2) transgenic mouse model. The present study elucidated the molecular foundation of hypertension by androgen-induction in this model. The renal expression of CYP4F2 in transgenic mice was highly expressed and strongly induced with 5α-dihydrotestosterone (DHT) treatment determined by Western blot. DHT also increased the renal arachidonic acid ω-hydroxylation and urinary 20-hydroxyeicosatetraenoic acid (20-HETE) excretion (P<0.01), and furthermore elevated the systolic blood pressure by 10 and 22mm Hg (P<0.05) in female and castrated male transgenic mice, respectively. HET0016 completely eliminated the androgen-induced effects (P<0.01). Endogenous Cyp4a ω-hydroxylases, evaluated by real-time quantitative PCR, were significantly suppressed in transgenic mice (P<0.05). Importantly, transgenic mice with increased 20-HETE showed decreased epoxyeicosatrienoic acids (EETs) and increased dihydroxyeicosatetraenoic acids determined by liquid chromatography-tandem mass spectrometry, contributing to significantly raised ratio of 20-HETE/EETs in the urine and kidney homogenate (P<0.01). These data demonstrate that the androgen aggravated hypertension possibly through an altered ratio of 20-HETE/EETs in CYP4F2 transgenic mice.

Vascular Cytochrome P450 4A Expression and 20-Hydroxyeicosatetraenoic Acid Synthesis Contribute to Endothelial Dysfunction in Androgen-Induced Hypertension

Epidemiological evidence suggests a role for sex-dependent mechanisms in the pathophysiology of hypertension. It has been shown that 5alpha-dihydrotestosterone (DHT) administration (56 mg/kg of body weight per day IP for 14 days) increases blood pressure, cytochrome P450 4A expression, and 20-hydroxyeicosatetraenoic acid synthesis in rats. We examined whether increased vascular 20-hydroxyeicosatetraenoic acid synthesis underlies endothelial dysfunction and hypertension in DHT-treated male Sprague-Dawley rats by using HET0016, a selective cytochrome P450 4A inhibitor. Coadministration of HET0016 (10 mg/kg per day IP for 14 days) to DHT-treated rats markedly reduced DHT-induced interlobar arterial production of 20-hydroxyeicosatetraenoic acid (14.3+/-1.5 versus 1.5+/-0.5 ng/mg of protein per hour; P<0.05), superoxide anion (246+/-47 versus 31+/-8 cpm/microg of protein), and the levels of gp91-phox, p47-phox, and 3-nitrosylated proteins. Moreover, the maximal relaxing response to acetylcholine in phenylephrine-preconstricted renal interlobar arteries from DHT-treated rats (42.8+/-4.8%) significantly (P<0.05) increased in the presence of HET0016 (81.5+/-10.8%). Importantly, the administration of HET0016 negated DHT-induced hypertension; systolic blood pressure was reduced from 146+/-2 mm Hg in DHT-treated rats to 130+/-1 mm Hg (P<0.05). The results strongly implicate vascular cytochrome P450 4A-derived 20-hydroxyeicosatetraenoic acid in the development of androgen-induced endothelial dysfunction and hypertension.

Does 20-Hydroxyeicosatetraenoic Acid Contribute to Sex Differences in Cardiovascular Risk by Increasing Oxidative Stress?

Previous studies have indicated that there are large sex differences in the incidence of hypertension, and the role of androgens in enhancing the risk of cardiovascular disease is well recognized.1,2 However, the mechanisms by which androgens increase blood pressure are poorly understood. Recent studies have demonstrated that androgen-sensitive forms of hypertension are associated with increased production of 20-hydroxyeicosatetranoic acid (20-HETE) in the kidney and the vasculature.3 However, the role of 20-HETE in this form of hypertension has not been established, because this pathway has both prohypertensive and antihypertensive actions.4 At the level of the renal tubule, upregulation of the formation of 20-HETE inhibits sodium transport and opposes the development of hypertension, whereas in the vasculature, 20-HETE is a potent vasoconstrictor that promotes hypertension. The study of Singh et al5 in the current issue explores the role of 20-HETE in the development of endothelial dysfunction and hypertension in rats chronically treated with dihydrotestosterone. The results indicate that that the expression cytochrome P450 4A protein and the production of 20 HETE increase in renal arteries of rats treated with dihydrotestosterone. The increase in vascular 20-HETE production is because of upregulation of the expression of the cytochrome P450 4A8 isoform. The development of hypertension in these studies was associated with the development of endothelial dysfunction, as reflected by an impaired vasodilator response to acetylcholine, increased expression of the gp-91 …

Levels of adrenodoxin, NADPH-cytochrome P-450 reductase and cytochromes P-450 11β, P-450 21, P-450 scc, in adrenal zona fasciculata-reticularis tissue from androgen-treated rats

Treatment of rats with methylandrostenediol (MAD), an anabolic androgen, caused a profound reduction (65%) in the level of cytochrome P-450 11 β in rat adrenocortical mitochondria as measured by immunoblots using a specific antibody. The decreases in mitochondrial cytochrome P-450 scc (15%) and adrenodoxin (20%) were much less than that observed for cytochrome P-450 11 β . A 35% decrease in adrenal microsomal cytochrome P-450 21 and NADPH-cytochrome P-450 reductase levels was brought about by the treatment with MAD. The data establish that the preferential decrease in adrenal steroid 11β-hydroxylase activity associated with androgen treatment results from a decrease in cytochrome P-450 11 β . This is consistent with the role of 11-deoxycorticosterone in the pathogenesis of androgen-induced hypertension in rats.

Peripheral serum corticosteroid concentrations in relation to the rat adrenal cortical circadian rhythm in androgen-induced hypertension.

Adrenal function was assessed in control rats and in rats treated for 2 and 4 weeks with 17 alpha-methylandrostenediol (MAD; 17 alpha-methyl-5-androstene-3 beta-diol), a synthetic androgen known to produce hypertensive cardiovascular disease. In both groups and at both time periods, a circadian rhythm of blood corticosteroid concentrations was observed. The high point for serum corticosterone (B), 18-hydroxy-11-deoxycorticosterone (18-hydroxy-DOC), and 11-deoxycorticosterone (DOC) concentrations occurred at the beginning of the dark period (1800 hours), and the low point occurred at the onset of the light period (0600 hours). Serum concentrations of DOC were always found to be higher in MAD-treated rats as compared with controls. The serum concentrations of B and 18-hydroxy-DOC were lower than control values at 1800 hours but higher than control concentrations at 0600 hours. The in vitro 11 beta- and 18-hydroxylation of DOC was markedly reduced with MAD treatment. In contrast, cholesterol side-chain cleavage activity was higher in animals treated with MAD. These in vitro findings correlated with spectral studies that showed a decreased binding of DOC to cytochrome P450(11) beta and increased binding of cholesterol to cytochrome P450scc. These studies suggest that MAD treatment selectively decreases 11 beta- and 18-hydroxylation in adrenal mitochondria, and this results in an increased serum concentration of DOC, a hypertensinogenic steroid. This effect of MAD on peripheral serum DOC concentration is most readily observed in quiescent animals at the high point of the circadian rhythm.