Estrogen receptor-alpha mediates estrogen protection from angiotensin II-induced hypertension in conscious female mice

It has been shown that the female sex hormones have a protective role in the development of angiotensin II (ANG II)-induced hypertension. The present study tested the hypotheses that 1) the estrogen receptor-alpha (ERalpha) is involved in the protective effects of estrogen against ANG II-induced hypertension and 2) central ERs are involved. Blood pressure (BP) was measured in female mice with the use of telemetry implants. ANG II (800 ng.kg(-1).min(-1)) was administered subcutaneously via an osmotic pump. Baseline BP in the intact, ovariectomized (OVX) wild-type (WT) and ERalpha knockout (ERalphaKO) mice was similar; however, the increase in BP induced by ANG II was greater in OVX WT (23.0 +/- 1.0 mmHg) and ERalphaKO mice (23.8 +/- 2.5 mmHg) than in intact WT mice (10.1 +/- 4.5 mmHg). In OVX WT mice, central infusion of 17beta-estradiol (E(2); 30 microg.kg(-1).day(-1)) attenuated the pressor effect of ANG II (7.0 +/- 0.4 mmHg), and this protective effect of E(2) was prevented by coadministration of ICI-182,780 (ICI; 1.5 microg.kg(-1).day(-1), 18.8 +/- 1.5 mmHg), a nonselective ER antagonist. Furthermore, central, but not peripheral, infusions of ICI augmented the pressor effects of ANG II in intact WT mice (17.8 +/- 4.2 mmHg). In contrast, the pressor effect of ANG II was unchanged in either central E(2)-treated OVX ERalphaKO mice (19.0 +/- 1.1 mmHg) or central ICI-treated intact ERalphaKO mice (19.6 +/- 1.6 mmHg). Lastly, ganglionic blockade on day 7 after ANG II infusions resulted in a greater reduction in BP in OVX WT, central ER antagonist-treated intact WT, central E(2) + ICI-treated OVX WT, ERalphaKO, and central E(2)- or ICI-treated ERalphaKO mice compared with that in intact WT mice given just ANG II. Together, these data indicate that ERalpha, especially central expression of the ER, mediates the protective effects of estrogen against ANG II-induced hypertension.     

Contribution of the subfornical organ to angiotensin II-induced hypertension

Previous studies clearly demonstrated acute actions of angiotensin II (ANG II) at one of the central circumventricular organs, the subfornical organ (SFO), but studies demonstrating a role for the SFO in the chronic actions of ANG II remain uncertain. The purpose of this study was to examine the role of the SFO in the chronic hypertensive phase of ANG II-induced hypertension. We hypothesized that the SFO is necessary for the full hypertensive response observed during the chronic phase of ANG II-induced hypertension. To test this hypothesis, male Sprague-Dawley rats were subjected to sham operation (sham rats) or electrolytic lesion of the SFO (SFOx rats). After 1 wk, the rats were instrumented with venous catheters and radiotelemetric transducers for intravenous administration of ANG II and measurement of blood pressure and heart rate, respectively. Rats were then allowed 1 wk for recovery. After 3 days of saline control infusion (7 ml of 0.9% NaCl/day), sham and SFOx rats were infused with ANG II at 10 ng.kg(-1).min(-1) i.v. for 10 consecutive days and then allowed to recover for 3 days. A 0.4% NaCl diet and distilled water were provided ad libitum. At day 5 of ANG II infusion, mean arterial pressure increased 11.7 +/- 3.0 mmHg in sham rats (n = 9) but increased only 3.7 +/- 1.4 mmHg in SFOx rats (n = 9). This trend continued through day 10 of ANG II treatment. These results support the hypothesis that the SFO is necessary for the full hypertensive response to chronic ANG II administration.     

Central estrogen inhibition of angiotensin II-induced hypertension in male mice and the role of reactive oxygen species

It has been shown that reactive oxygen species (ROS) contribute to the central effect of ANG II on blood pressure (BP). Recent studies have implicated an antihypertensive action of estrogen in ANG II-infused female mice. The present study used in vivo telemetry recording and in vitro living mouse brain slices to test the hypothesis that the central activation of estrogen receptors in male mice inhibits ANG II-induced hypertension via the modulation of the central ROS production. In male wild-type mice, the systemic infusion of ANG II induced a significant increase in BP (Delta30.1 +/- 2.5 mmHg). Either central infusion of Tempol or 17beta-estradiol (E2) attenuated the pressor effect of ANG II (Delta10.9 +/- 2.3 and Delta4.5 +/- 1.4 mmHg), and the protective effect of E2 was prevented by the coadministration of an estrogen receptor, antagonist ICI-182780 (Delta23.6 +/- 3.1 mmHg). Moreover, the ganglionic blockade on day 7 after the start of ANG II infusions resulted in a smaller reduction of BP in central Tempol- and in central E2-treated males, suggesting that estrogen inhibits the central ANG II-induced increases in sympathetic outflow. In subfornical organ slices, the application of ANG II resulted in a 21.5 +/- 2.5% increase in ROS production. The coadministration of irbesartan, an ANG II type 1 receptor antagonist, or the preincubation of brain slices with Tempol blocked ANG II-induced increases in ROS production (-1.8 +/- 1.6% and -1.0 +/- 1.8%). The ROS response to ANG II was also blocked by E2 (-3.2 +/- 2.4%). The results suggest that the central actions of E2 are involved in the protection from ANG II-induced hypertension and that estrogen modulation of the ANG II-induced effects may involve interactions with ROS production.     

Sexual dimorphism in angiotensin II-induced hypertension and vascular alterations

Sex differences in the degree of high blood pressure have been described in several forms of experimental animal models of hypertension. However, the influence of sex on angiotensin II-induced hypertension has not been studied. In the present study, we investigated and compared the effects of chronic angiotensin II treatment on blood pressure and vascular function in male and female rats. Chronic treatment with angiotensin II (0.7 mg/kg daily for 10 d) significantly raised arterial blood pressure in male but not female Sprague-Dawley rats; it upregulated the NAD(P)H oxidase gp67 phox subunit in the aorta of male but not female rats; and it exaggerated the vasoconstrictor responses to norepinephrine and serotonin in the mesenteric vascular bed (MVB) of male but not female rats. Vasodilator responses to acetylcholine (ACh) but not papaverine (PPV) or isoprenaline (ISO) were reduced in the MVB of angiotensin II-treated male but not female rats. ACh, but not PPV or ISO dilatory responses were potentiated in the MVB of angiotensin II-treated female rats. The present findings demonstrate that exogenous angiotensin II upregulates aortic NAD(P)H oxidase gp67 phox subunit, and induces hypertension and mesenteric vascular dysfunction only in male rats.     

Prevention of aortic fibrosis by N-acetyl-seryl-aspartyl-lysyl-proline in angiotensin II-induced hypertension

Fibrosis is an important component of large conduit artery disease in hypertension. The endogenous tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) has anti-inflammatory and antifibrotic effects in the heart and kidney. However, it is not known whether Ac-SDKP has an anti-inflammatory and antifibrotic effect on conduit arteries such as the aorta. We hypothesize that in ANG II-induced hypertension Ac-SDKP prevents aortic fibrosis and that this effect is associated with decreased protein kinase C (PKC) activation, leading to reduced oxidative stress and inflammation and a decrease in the profibrotic cytokine transforming growth factor-beta1 (TGF-beta1) and phosphorylation of its second messenger Smad2. To test this hypothesis we used rats with ANG II-induced hypertension and treated them with either vehicle or Ac-SDKP. In this hypertensive model we found an increased collagen deposition and collagen type I and III mRNA expression in the aorta. These changes were associated with increased PKC activation, oxidative stress, intercellular adhesion molecule (ICAM)-1 mRNA expression, and macrophage infiltration. TGF-beta1 expression and Smad2 phosphorylation also increased. Ac-SDKP prevented these effects without decreasing blood pressure or aortic hypertrophy. Ac-SDKP also enhanced expression of inhibitory Smad7. These data indicate that in ANG II-induced hypertension Ac-SDKP has an aortic antifibrotic effect. This effect may be due in part to inhibition of PKC activation, which in turn could reduce oxidative stress, ICAM-1 expression, and macrophage infiltration. Part of the effect of Ac-SDKP could also be due to reduced expression of the profibrotic cytokine TGF-beta1 and inhibition of Smad2 phosphorylation.     

Sex differences in primary hypertension

Men have higher blood pressure than women through much of life regardless of race and ethnicity. This is a robust and highly conserved sex difference that it is also observed across species including dogs, rats, mice and chickens and it is found in induced, genetic and transgenic animal models of hypertension. Not only do the differences between the ovarian and testicular hormonal milieu contribute to this sexual dimorphism in blood pressure, the sex chromosomes also play a role in and of themselves. This review primarily focuses on epidemiological studies of blood pressure in men and women and experimental models of hypertension in both sexes. Gaps in current knowledge regarding what underlie male-female differences in blood pressure control are discussed. Elucidating the mechanisms underlying sex differences in hypertension may lead to the development of anti-hypertensives tailored to one's sex and ultimately to improved therapeutic strategies for treating this disease and preventing its devastating consequences.     

Endothelin mediates superoxide production in angiotensin II-induced hypertension in rats

Angiotensin II and endothelin-1 (ET) are two hormones involved in cardiovascular diseases and well known for their capacity to induce free radical generation in vascular and cardiac tissues. In addition to its prooxidative effect, angiotensin II can increase the synthesis of ET-1 in vascular smooth muscle cells (VSMC). Our objective was to determine whether the ET-1 synthesis in VSMC is involved in angiotensin II-induced superoxide anion production in rats. Our results show that treatments of isolated VSMC with angiotensin II and ET increased superoxide. However, this increase occurred in a bimodal fashion for angiotensin II with a fast transient production (10 min) and a late sustained production (6 h), while ET-1 induced superoxide formation after a delay of 6 h. LU302872 and BQ-123, a nonselective and a selective ETA receptor antagonists, respectively, prevented angiotensin II-induced superoxide anion production only during the late phase. In contrast, BQ-3020, a selective ETB receptor antagonist, had no effect. In vivo, LU302872 reduced the aortic superoxide production induced by angiotensin II administered for 12 days. In conclusion, our results suggest that the superoxide generation induced by chronic angiotensin II infusion may be mediated by ET-1 acting on ETA receptors in VSMC in vitro. Furthermore, this effect appears to contribute to the excess superoxide production during the chronic activation of the renin-angiotensin system in vivo.     

Sex differences in the fetal programming of hypertension

Background: Numerous clinical and experimental studies support the hypothesis that the intrauterine environment is an important determinant of cardiovascular disease and hypertension.Objective: This review examined the mechanisms linking an adverse fetal environment and increased risk for chronic disease in adulthood with an emphasis on gender differences and the role of sex hormones in mediating sexual dimorphism in response to a suboptimal fetal environment.Methods: This review focuses on current findings from the PubMed database regarding animal models of fetal programming of hypertension, sex differences in phenotypic outcomes, and potential mechanisms in offspring of mothers exposed to an adverse insult during gestation. For the years 1988 to 2007, the database was searched using the following terms: fetal programming, intrauterine growth restriction, low birth weight, sex differences, estradiol, testosterone, high blood pressure, and hypertension.Results: The mechanisms involved in the fetal programming of adult disease are multifactorial and include alterations in the regulatory systems affecting the long-tterm control of arterial pressure. Sex differences have been observed in animal models of fetal programming, and recent studies suggest that sex hormones may modulate activity of regulatory systems, leading to a lower incidence of hypertension and vascular dysfunction in females compared with males.Conclusions: Animal models of fetal programming provide critical support for the inverse relationship between birth weight and blood pressure. Experimental models demonstrate that sex differences are observed in the pathophysiologic response to an adverse fetal environment. A role for sex hormone involvement is strongly suggested,with modulation of the renin-angiotensin system as a possible mechanism.     

Resolvin-D1 attenuation of angiotensin II-induced cardiac inflammation in mice is associated with prevention of cardiac remodeling and hypertension

AimsDespite the broad pharmacological arsenal to treat hypertension, chronic patients may develop irreversible cardiac remodeling and fibrosis. Angiotensin II, the main peptide responsible for the Renin-Angiotensin-Aldosterone-System, has been closely linked to cardiac remodeling, hypertrophy, fibrosis, and hypertension, and some of these effects are induced by inflammatory mediators. Resolvin-D1 (RvD1) elicits potent anti-inflammatory and pro-resolving effects in various pathological models. In this study, we aimed to examine whether RvD1 ameliorates cardiac remodeling and hypertension triggered by angiotensin II.Methods and resultsAlzet® osmotic mini-pumps filled with angiotensin II (1.5 mg/kg/day) were implanted in male C57BL/6 J mice for 7 or 14 days. RvD1 (3 μg/kg/day, i.p) was administered one day after the surgery and during the complete infusion period. Blood pressure and myocardial functional parameters were assessed by echocardiography. At the end of the experimental procedure, blood and heart tissue were harvested, and plasma and histological parameters were studied. After 7 and 14 days, RvD1 reduced the increase of neutrophil and macrophage infiltration triggered by angiotensin II, and also reduced ICAM-1 and VCAM-1 expression levels. RvD1 also reduced cytokine plasma levels (IL-1β, TNF-α, IL-6, KC, MCP-1), cardiac hypertrophy, interstitial and perivascular fibrosis, and hypertension.ConclusionsThis study unveils novel cardioprotective effects of RvD1 in angiotensin II-induced hypertension and cardiac remodeling by attenuating inflammation and provides insights into a potential clinical application.     

Role of asymmetric dimethylarginine for angiotensin II-induced target organ damage in mice

The aim of the present study was to investigate the role of the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) and its degrading enzyme dimethylarginine dimethylaminohydrolase (DDAH) in angiotensin II (ANG II)-induced hypertension and target organ damage in mice. Mice transgenic for the human DDAH1 gene (TG) and wild-type (WT) mice (each, n = 28) were treated with 1.0 microg kg(-1) min(-1) ANG II, 3.0 microg kg(-1) min(-1) ANG II, or phosphate-buffered saline over 4 wk via osmotic minipumps. Blood pressure, as measured by tail cuff, was elevated to the same degree in TG and WT mice. Plasma levels of ADMA were lower in TG than WT mice and were not affected after 4 wk by either dose of ANG II in both TG and WT animals. Oxidative stress within the wall of the aorta, measured by fluorescence microscopy using the dye dihydroethidium, was significantly reduced in TG mice. ANG II-induced glomerulosclerosis was similar between WT and TG mice, whereas renal interstitial fibrosis was significantly reduced in TG compared with WT animals. Renal mRNA expression of protein arginine methyltransferase (PRMT)1 and DDAH2 increased during the infusion of ANG II, whereas PRMT3 and endogenous mouse DDAH1 expression remained unaltered. Chronic infusion of ANG II in mice has no effect on the plasma levels of ADMA after 4 wk. However, an overexpression of DDAH1 alleviates ANG II-induced renal interstitial fibrosis and vascular oxidative stress, suggesting a blood pressure-independent effect of ADMA on ANG II-induced target organ damage.     

Inhibitory effects of benzyl benzoate and its derivatives on angiotensin II-induced hypertension

Hypertension is a lifestyle-related disease which often leads to serious conditions such as heart disease and cerebral hemorrhage. Angiotensin II (Ang II) plays an important role in regulating cardiovascular homeostasis. Consequently, antagonists that block the interaction of Ang II with its receptors are thought to be effective in the suppression of hypertension. In this study, we searched for plant compounds that had antagonist-like activity toward Ang II receptors. From among 435 plant samples, we found that EtOH extract from the resin of sweet gum Liquidambar styraciflua strongly inhibited Ang II signaling. We isolated benzyl benzoate and benzyl cinnamate from this extract and found that those compounds inhibited the function of Ang II in a dose-dependent manner without cytotoxicity. An in vivo study showed that benzyl benzoate significantly suppressed Ang II-induced hypertension in mice. In addition, we synthesized more than 40 derivatives of benzyl benzoate and found that the meta-methyl and 3-methylbenzyl 2'-nitrobenzoate derivatives showed about 10-fold higher activity than benzyl benzoate itself. Thus, benzyl benzoate, its derivatives, and benzyl cinnamate may be useful for reducing hypertension.     

Human paraoxonase gene cluster overexpression alleviates angiotensin II-induced cardiac hypertrophy in mice

Cardiac hypertrophy is the strongest predictor of the development of heart failure, and anti-hypertrophic treatment holds the key to improving the clinical syndrome and increasing the survival rates for heart failure. The paraoxonase (PON) gene cluster (PC) protects against atherosclerosis and coronary artery diseases. However, the role of PC in the heart is largely unknown. To evaluate the roles of PC in cardiac hypertrophy, transgenic mice carrying the intact human PON1, PON2, and PON3 genes and their flanking sequences were studied. We demonstrated that the PC transgene (PC-Tg) protected mice from cardiac hypertrophy induced by Ang II; these mice had reduced heart weight/body weight ratios, decreased left ventricular wall thicknesses and increased fractional shortening compared with wild-type (WT) control. The same protective tendency was also observed with an Apoe^-/- background. Mechanically, PC-Tg normalized the disequilibrium of matrix metalloproteinases (MMPs)/tissue inhibitors of MMPs (TIMPs) in hypertrophic hearts, which might contribute to the protective role of PC-Tg in cardiac fibrosis and, thus, protect against cardiac remodeling. Taken together, our results identify a novel anti-hypertrophic role for the PON gene cluster, suggesting a possible strategy for the treatment of cardiac hypertrophy through elevating the levels of the PON gene family.     

Sex differences in prenatal programming of hypertension by dexamethasone

Prenatal dexamethasone has been shown to increase blood pressure in male offspring but the mechanism for the increase in blood pressure is unclear. The present study examined if prenatal programming by maternal injection of dexamethasone on days 15 and 16 of gestation affected the blood pressure comparably in female and male offspring. Our hypothesis was that males would be affected by prenatal dexamethasone to a greater extent than females and that either an increase in renal tubular transporter abundance or an increase in renin or aldosterone system would be associated with hypertension with prenatal programming. Prenatal dexamethasone increased blood pressure at two months and six months of age and resulted in proteinuria and albuminuria at six months in male but not female rat offspring. There was no effect of prenatal dexamethasone on blood pressure and proteinuria at one month in male and in female offspring. While prenatal dexamethasone increased male renal thick ascending limb sodium potassium two chloride cotransporter protein abundance at two months, prenatal dexamethasone on days 15 and 16 of gestation did not affect transporter abundance in males at other ages, nor did it affect proximal tubule sodium/hydrogen exchanger or distal convoluted tubule sodium chloride cotransporter protein abundance at any age. There was no difference in systemic renin or aldosterone in the prenatal dexamethasone group compared to same sex controls. In conclusion, male but not female offspring have an increase in blood pressure and urinary protein excretion with prenatal dexamethasone. The increase in blood pressure with prenatal programming was not associated with a consistent increase in renal tubular transporter protein abundance, nor plasma renin activity and serum aldosterone.     

Role of endothelin ETB receptor activation in angiotensin II-induced hypertension: effects of salt intake

We showed recently that endothelin (ET)A receptors are involved in the salt sensitivity of ANG II-induced hypertension. The objective of this current study was to characterize the role of endothelin ETB receptor activation in the same model. Male rats on fixed normal (2 meq/day) or high (6 meq/day) salt intake received a continuous intravenous infusion of ANG II or salt only for 15 days. During the middle 5 days of the infusion period, rats were given either the selective ETB receptor antagonist A-192621 or the nonselective endothelin receptor antagonist A-182086 (both at 24 mg x kg(-1) x day(-1) intra-arterially). Infusion of ANG II caused a greater rise in arterial pressure in rats on high-salt intake. The administration of A-192621 increased arterial pressure further in all rats. The chronic hypertensive effect of A-192621 was not significantly affected by salt intake or ANG II. The administration of A-182086 lowered arterial pressure chronically only in rats on normal salt intake receiving ANG II. Thus the salt sensitivity of ANG II-induced hypertension is not caused by changes in ETB receptor function.     

Plasminogen activator inhibitor-1 deficient mice are protected from angiotensin II-induced fibrosis

PAI-1 has been shown to be both profibrotic and antifibrotic in animal models of hepatic fibrosis. Although these models have similarities to human fibrotic liver disease, no rodent model completely recapitulates the clinical situation; indeed, transaminase values in most models of hepatic fibrosis are much higher than in chronic liver diseases in humans. Here, wild-type and PAI-1^−/− mice were administered AngII (500 ng/kg/min) for 4 weeks. ECM accumulation was evaluated by Sirius red staining, hydroxyproline content, and fibrin and collagen immunostaining. Induction of pro-fibrotic genes was assessed by real-time RT-PCR. Despite the absence of any significant liver damage, AngII infusion increased the deposition of hepatic collagen and fibrin ECM, with a perisinusoidal pattern. PAI-1^−/− mice were protected from these ECM changes, indicating a causal role of PAI-1 in this fibrosis model. Protection in the knockout strain correlated with a blunted increase in αSMA, and elevated activities of matrix metalloproteinases (MMP2, MMP9). These data suggest that PAI-1 plays a critical role in mediating fibrosis caused by AngII and lends weight-of-evidence to a pro-fibrotic role of this protein in liver. Furthermore, the current study proposes a new model of ‘pure’ hepatic fibrosis in mice with little inflammation or hepatocyte death.     

Sex differences in choline-deficient diet-induced steatohepatitis in mice

Alcoholic steatohepatitis (ASH) and nonalcoholic steatohepatitis (NASH) are common liver diseases in the United States. ASH and NASH occur more frequently in women than in men, and liver injury is also more severe in women. The role of estrogens in ASH has been well established, but their role in NASH has received relatively little study. The purpose of this study was to evaluate the effect of estrogens in methionine-choline deficient diet (MCDD)-induced steatohepatitis in mice. The degree of steatohepatitis was evaluated in males and in intact and ovariectomized females that were fed MCDD for 4 weeks, and in females that were fed MCDD containing tamoxifen. Hematoxylin and eosin-stained sections of livers showed marked steatohepatitis in all experimental groups. Compared to the control group, markers of hepatocyte injury such as aspartate transaminase (AST), alanine transaminase (ALT), and liver triglyceride levels increased significantly in males and in intact and ovariectomized female mice that were fed MCDD. Also, it was interesting that levels of AST and ALT increased much more in the MCDD + tamoxifen group than in the MCDD group. In female mice fed MCDD, hepatocyte proliferative and apoptotic indices increased slightly compared to mice that were fed a normal diet. Based on these results, it can be concluded that MCDD-induced steatohepatitis is comparable in male and female mice, and that ovariectomy or antiestrogen treatment had no protective effect in MCDD-induced steatohepatitis.