AT1, AT2, and AT(1-7) receptor expression in the uteroplacental unit of normotensive and hypertensive rats during early and late pregnancy.

INTRODUCTION: We investigated the expression of angiotensin receptors in early pregnancy and established whether normal pregnancy or preeclampsia alters the expression and distribution of the uteroplacental AT1R, AT2R and mas/AT1-7R at late gestation. METHODS: The percentage of each receptor subtype present in tissues from virgin rats and from normotensive and RUPP hypertensive pregnant rats was established by in vitro receptor autoradiography. Receptor mRNA levels were determined by quantitative PCR at early and late pregnancy. RESULTS: AT1R mRNA levels were up-regulated in the interimplantation (IIS) site at day 7 of gestation. AT2R mRNA levels were decreased at day 5 and 7 in the IIS but increased in the implantation site (IS) at day 5 and 7 as compared to the IIS at day 5. Mas/AT1-7R mRNA was increased in early pregnancy. In normal pregnancy and RUPP the mRNA for all angiotensin receptors was reduced in the uterus at late gestation. The AT1R accounted for the majority of binding in the uterus of virgin and the placenta of pregnant and RUPP. In RUPP pregnancy there was a significant competition with d-Ala in the placenta labyrinth. DISCUSSION AND CONCLUSION: The expression of angiotensin receptors suggests their involvement in the maintenance of early stages of pregnancy. During late gestation down-regulation of Ang receptors in the uterus may arise from feedback down-regulation by Ang II. In the placenta the levels of AT1Rs are equivalent in the RUPP model. The increased binding of mas/AT1-7R at late gestation in RUPP may represent a compensatory mechanism to reduce uteroplacental vascular resistance.

Decidualized pseudopregnant rat uterus shows marked reduction in Ang II and Ang-(1-7) levels.

UNLABELLED: Previous studies showed that angiotensin (Ang) II and Ang-(1-7) concentrations were reduced in the implantation site at day 7 of pregnancy in Sprague-Dawley rats as compared to the site immediately adjacent to it, which does not have the embryo attached, clearly showing the importance of the blastocyst in the regulation of renin-angiotensin system (RAS). OBJECTIVE: The objective of this study was to evaluate the regulation of the RAS in the decidualized uterus in the pseudopregnant rat, a model without the presence of a conceptus. METHODS: Ovariectomized, adult female rats were sensitized for the decidual cell reaction with steroid treatments; decidualization was induced by oil-injection of the right horn; the left horn served as a control. The uterine content of Ang I, Ang II, and Ang-(1-7) was examined in the decidualized and non-decidualized uteri. RESULTS: Both Ang-(1-7) and Ang II and ACE and ACE2 mRNA were significantly reduced in the decidualized horn as compared to the non-decidualized horn. Immunocytochemical characterization of Ang II, Ang-(1-7), ACE and ACE2 demonstrated that Ang-(1-7), Ang II, and ACE2 polarize to the anti-mesometrial pole with decidualization. CONCLUSION: The decidualization process elicits marked reduction in uterine Ang II and Ang-(1-7) content as compared to the non-decidualized horn. The differential immunocytochemical expression of Ang II and Ang-(1-7) with ACE2, but not ACE in the anti-mesometrial pole of the decidualized horn may favor the formation and action of Ang-(1-7) in the anti-mesometrial pole, an area which plays a role in triggering the decidualization process.

Therapeutic targets in liver transplantation: angiotensin II in nonsteatotic grafts and angiotensin-(1-7) in steatotic grafts.

Numerous steatotic livers are discarded as unsuitable for transplantation because of their poor tolerance of ischemia-reperfusion(I/R). The injurious effects of angiotensin (Ang)-II and the benefits of Ang-(1-7) in various pathologies are well documented. We examined the generation of Ang II and Ang-(1-7) in steatotic and nonsteatotic liver grafts from Zucker rats following transplantation. We also studied in both liver grafts the effects of Ang-II receptors antagonists and Ang-(1-7) receptor antagonists on hepatic I/R damage associated with transplantation. Nonsteatotic grafts showed higher Ang II levels than steatotic grafts, whereas steatotic grafts showed higher Ang-(1-7) levels than nonsteatotic grafts. Ang II receptor antagonists protected only nonsteatotic grafts against damage, whereas Ang-(1-7) receptor antagonists were effective only in steatotic grafts. The protection conferred by Ang II receptor antagonists in nonsteatotic grafts was associated with ERK 1/2 overexpression, whereas the beneficial effects of Ang-(1-7) receptor antagonists in steatotic grafts may be mediated by NO inhibition. Our results show that Ang II receptor antagonists are effective only in nonsteatotic liver transplantation and point to a novel therapeutic target in liver transplantation based on Ang-(1-7), which is specific for steatotic liver grafts.

Characterization of vascular reactivity in dorsal hand veins after oral rosiglitazone treatment in healthy subjects.

OBJECTIVE: In clinical studies with diabetic patients thiazolidinediones have been shown to restore abnormal vascular function which might be attributed to improved blood sugar control or to restoration of vascular endothelium and smooth muscle responsiveness. The present study was undertaken to investigate whether rosiglitazone modulates vascular responsiveness to different vasoactive agents and exerts renin-angiotensin-system (RAS)-inhibiting properties in healthy subjects in vivo. METHODS: 24 healthy male subjects were randomized to receive either rosiglitazone or placebo. Venoconstrictor responses to angiotensin II (Ang II) and phenylephrine, and endothelium-dependent response to histamine and insulin, and endothelium-independent response to glyceroltrinitrate were compared using the dorsal hand vein compliance method. Effects on the RAS were investigated by plasma level determinations of Ang II and angiotensin-(1-7). Treatment effects on the systemic arterial system were investigated by standardized pulse-wave-analysis. RESULTS: Rosiglitazone significantly inhibited venoconstrictor responses to Ang II by 19% (-70% vs. -51% constriction, p = 0.034) and in the presence of rosiglitazone the ED80 for phenylephrine was increased (ED80: 317 A+/- 86 ng vs. 531 A+/- 102 ng; p = 0.010). Rosiglitazone treatment was without effect on endothelium-dependent dilation, blood pressure, pulse-wave-velocity and plasma angiotensin peptide levels. CONCLUSIONS: The data of the present study in veins of healthy subjects are consistent with data from in vitro and animal studies supporting a direct effect of rosiglitazone on venous tone by modulation of the vascular smooth muscle response via AT1-receptor-downregulation.

Angiotensin-(1-7) serves as an aquaretic by increasing water intake and diuresis in association with downregulation of aquaporin-1 during pregnancy in rats.

We previously demonstrated that kidney and urine levels of angiotensin-(1-7) [ANG-(1-7)] were increased in pregnancy. To explore the role of ANG-(1-7) on fluid and electrolyte homeostasis during pregnancy, we evaluated the effect of the ANG-(1-7) antagonist D-alanine-[ANG-(1-7)] (A-779) on kidney function. Virgin and pregnant rats received infusion of vehicle or A-779 (48 microg.kg(-1).h(-1)) for 8 days by osmotic minipumps. Metabolic studies were done on treatment day 7-8. Virgin and pregnant rats at day 15 and 19 were killed, and blood and kidneys were collected. Kidneys were prepared for Western blot analysis for aquaporin-1 (AQP1) and aquaporin-2. In virgin female rats, A-779 increased urine volume and decreased urinary osmolality and AQP1 with no change in water intake. In 19-day pregnant rats, A-779 significantly decreased water intake and urine volume and increased urinary osmolality and kidney AQP1 expression. Only in late gestation did A-779 treatment decrease the difference between intake and output (balance). A-779 treatment increased plasma vasopressin in late gestation but did not change vasopressin in virgins. In virgin and pregnant animals, A-779 administration had no effect on blood pressure, plasma volume, blood volume, or urinary electrolytes. These results suggest that ANG-(1-7) produces antidiuresis associated with upregulation of AQP1 in virgin rats, whereas ANG-(1-7) produces diuresis in late gestation with downregulation of AQP1. ANG-(1-7) contributes to the enhanced water intake during pregnancy, allowing maintenance of the normal volume-expanded state despite diuresis produced in part by decreased AVP and AQP1.

Effects of hormone therapy on blood pressure and the renin-angiotensin system in postmenopausal women.

Observational studies have documented an association between lower rates of cardiovascular disease and hormone therapy (HT). Meanwhile, randomized clinical trials have documented increased rates of myocardial infarction and stroke in women receiving hormone therapy. These seemingly discordant findings have stimulated new research to examine estrogen's effects on the cardiovascular system, including its effects on blood pressure, regulation of the renin-angiotensin system (RAS), and the clinical consequences of hypertension. In the last 6 years several studies have better defined the mechanisms by which HT affect the RAS, blood pressure, and the clinical effects of hypertension. Recent studies documented increases in angiotensinogen synthesis and the suppression of active renin with estrogen replacement. Genotype may be a factor in determining the degree of suppression of angiotensin converting enzyme levels that occurs with estrogen therapy. Estrogen supplementation in postmenopausal women increases systemic angiotensin II, a potent vasoconstrictor. This vasopressor effect is attenuated by an estrogen-induced reduction of angiotensin II type 1 receptor expression. Renal blood flow reduction, in the absence of blood pressure changes, have been reported after estrogen replacement, and an increased risk of total stroke has been demonstrated in hypertensive women on HT compared to normotensive women on this therapy. Estrogen replacement affects many components of the RAS, but its effect on this system has little effect on blood pressure. Further studies are needed to describe the effects of estrogen replacement on abnormal vasculature and how these effects relate to myocardial infarction and stroke.

Temporal-spatial expression of ANG-(1-7) and angiotensin-converting enzyme 2 in the kidney of normal and hypertensive pregnant rats.

We recently demonstrated that renin-angiotensin system (RAS) overactivity during late gestation in rats is associated with increased kidney and urine levels of ANG-(1-7) and enhanced kidney immunostaining of ANG-(1-7) and angiotensin-converting enzyme 2 (ACE2). To understand the temporal-spatial changes in normal and hypertensive pregnancies, the renal distribution of ANG-(1-7) and ACE2 in association with kidney angiotensin peptides and ACE2 activity was examined in virgin, normal pregnant (NP; gestational days 5, 15, and 19) and reduced uterine perfusion pressure (RUPP at day 19) pregnant Sprague-Dawley rats. ANG-(1-7) and ACE2 immunocytochemical staining increased 1.8- and 1.9-fold and 1.7- and 1.8-fold, respectively, at days 15 and 19 of NP, compared with virgin rats. ANG-(1-7) and ANG II concentrations were increased in the kidney at 19 days of gestation. ACE2 activity measured using a fluorescent substrate was increased 1.9- and 1.9-fold in the cortex and 1.9- and 1.8-fold in the medulla at days 15 and 19 of NP. In the RUPP animals, ANG-(1-7) immunostaining and concentration were significantly decreased compared with 19-day NP rats. ACE2 activity was unchanged in the cortex and medulla of RUPP rats. In conclusion, during NP, the concurrent changes of ACE2 and ANG-(1-7) suggest that ACE2 plays an important role in regulating the renal levels of ANG-(1-7) at mid to late gestation. However, the decrease in renal ANG-(1-7) content in the absence of a concomitant decrease in ACE2 implicates the participation of other ANG-(1-7) forming or degrading enzymes during hypertensive pregnancy.

Effect of estrogen on neprilysin expression in uterus and kidney of Sprague-Dawley normotensive and heterozygous (mRen2)27-transgenic hypertensive rats.

The present study was designed to determine whether estrogen modulates the angiotensin processing enzymes in membrane homogenates obtained from uterus and kidney cortex and medulla of Sprague-Dawley (SD) and heterozygous (mRen2)27-transgenic hypertensive (Tg(+)) female rats treated with or without 17beta-estradiol (E2). We evaluated estrogen's influence on neprilysin (NEP), an endopeptidase that forms angiotensin-(1-7) [Ang-(1-7)] and on aminopeptidase (AMP), which degrades Ang-(1-7). Renal tissue from normotensive and hypertensive male rats was also evaluated. E2 up-regulated NEP mRNA in the uterus of both SD and Tg(+) and this was associated with increased NEP activity in the uterus of SD (0.31+/-0.03 nmol/min/mg versus 0.18+/-0.04 nmol/min/mg of protein, p<0.05) and Tg(+) (0.26+/-0.04 nmol/min/mg versus 0.13+/-0.02 nmol/min/mg of protein, p<0.05) female). E2 had no significant effect on NEP activity in cortex and medulla of hypertensive and normotensive female. In female animals, cortical NEP activity is two-fold higher than medullary; in males there is a four-fold higher cortical NEP activity as compared to medulla. In male animals, medullary NEP was significantly lower than females with or without E2 treatment; no gender specific effect was found in cortex. E2 treatment also caused a two-fold increase in AMP activity in the uterus and 1.6-fold decrease in kidney cortex of SD and Tg(+) female (p<0.05). Our studies indicate that NEP may be a primary candidate for increased Ang-(1-7) processing in the uterus with estrogen treatment; kidney NEP, on the other hand, showed no modulation by estrogen, suggesting that down regulation of other processing enzymes, like AMP and ACE, may come into play in the kidney with estrogen replacement. In addition, these studies showed that there is tissue-specific regulation of NEP with estrogen treatment that is strain independent.

Distribution of angiotensin-(1-7) and ACE2 in human placentas of normal and pathological pregnancies.

This work was designed to study the expression of the vasodilator peptide angiotensin-(1-7) [Ang-(1-7)] and its generating enzyme (ACE2) in the uteroplacental interface. Placentas were obtained from 11 early pregnancy failures (5 miscarriages and 6 ectopic pregnancies), 15 normotensive, and 10 preeclamptic gestations. In placental villi, the main sites of immunocytochemical expression of Ang-(1-7) and ACE2 were the syncytiotrophoblast, cytotrophoblast, endothelium and vascular smooth muscle of primary and secondary villi. Syncitial Ang-(1-7) expression in samples obtained from miscarriages and ectopic pregnancies was increased compared to normal term pregnancy [2.0 (2.0-2.25 for the 25 and 75% interquartile range) vs 1.3 (1.0-1.9), p<0.01]. In the maternal stroma, Ang-(1-7) and ACE2 were expressed in the invading and intravascular trophoblast and in decidual cells in all 3 groups. Ang-(1-7) and ACE2 staining was also found in arterial and venous endothelium and smooth muscle of the umbilical cord. The expression of Ang-(1-7) and ACE2 was similar in samples obtained from normal term or preeclamptic pregnancies, except for increased expression of ACE2 in umbilical arterial endothelium in preeclampsia [0.5 (0.5-0.8) vs 0.0 (0.0-0.0), p<0.01]. The uteroplacental location of Ang-(1-7) and ACE2 in pregnancy suggests an autocrine function of Ang-(1-7) in the vasoactive regulation that characterizes placentation and established pregnancy.

Enhanced expression of Ang-(1-7) during pregnancy

Pregnancy is a physiological condition characterized by a progressive increase of the different components of the renin-angiotensin system (RAS). The physiological consequences of the stimulated RAS in normal pregnancy are incompletely understood, and even less understood is the question of how this system may be altered and contribute to the hypertensive disorders of pregnancy. Findings from our group have provided novel insights into how the RAS may contribute to the physiological condition of pregnancy by showing that pregnancy increases the expression of both the vasodilator heptapeptide of the RAS, angiotensin-(1-7) [Ang-(1-7)], and of a newly cloned angiotensin converting enzyme (ACE) homolog, ACE2, that shows high catalytic efficiency for Ang II metabolism to Ang-(1-7). The discovery of ACE2 adds a new dimension to the complexity of the RAS by providing a new arm that may counter-regulate the activity of the vasoconstrictor component, while amplifying the vasodilator component. The studies reviewed in this article demonstrate that Ang-(1-7) increases in plasma and urine of normal pregnant women. In preeclamptic subjects we showed that plasma Ang-(1-7) was suppressed as compared to the levels found in normal pregnancy. In addition, kidney and urinary levels of Ang-(1-7) were increased in pregnant rats coinciding with the enhanced detection and expression of ACE2. These findings support the concept that in normal pregnancy enhanced ACE2 may counteract the elevation in tissue and circulating Ang II by increasing the rate of conversion to Ang-(1-7). These findings provide a basis for the physiological role of Ang-(1-7) and ACE2 during pregnancy.

Enhanced expression of Ang-(1-7) during pregnancy.

Pregnancy is a physiological condition characterized by a progressive increase of the different components of the renin-angiotensin system (RAS). The physiological consequences of the stimulated RAS in normal pregnancy are incompletely understood, and even less understood is the question of how this system may be altered and contribute to the hypertensive disorders of pregnancy. Findings from our group have provided novel insights into how the RAS may contribute to the physiological condition of pregnancy by showing that pregnancy increases the expression of both the vasodilator heptapeptide of the RAS, angiotensin-(1-7) [Ang-(1-7)], and of a newly cloned angiotensin converting enzyme (ACE) homolog, ACE2, that shows high catalytic efficiency for Ang II metabolism to Ang-(1-7). The discovery of ACE2 adds a new dimension to the complexity of the RAS by providing a new arm that may counter-regulate the activity of the vasoconstrictor component, while amplifying the vasodilator component. The studies reviewed in this article demonstrate that Ang-(1-7) increases in plasma and urine of normal pregnant women. In preeclamptic subjects we showed that plasma Ang-(1-7) was suppressed as compared to the levels found in normal pregnancy. In addition, kidney and urinary levels of Ang-(1-7) were increased in pregnant rats coinciding with the enhanced detection and expression of ACE2. These findings support the concept that in normal pregnancy enhanced ACE2 may counteract the elevation in tissue and circulating Ang II by increasing the rate of conversion to Ang-(1-7). These findings provide a basis for the physiological role of Ang-(1-7) and ACE2 during pregnancy.

Angiotensin(1-7) potentiates bradykinin-induced vasodilatation in man.

BACKGROUND: It has been clearly demonstrated that angiotensin(1-7) potentiates the vasodilating effect of bradykinin in isolated vessels of animals. OBJECTIVE: To investigate the interaction between angiotensin(1-7) Ang(1-7) and bradykinin in human forearm resistant vessels of normotensive healthy men in vivo, by the measurement of forearm blood flow using venous occlusion, strain-gauge plethysmography with intra-arterial infusions of peptides in a placebo-controlled, double-blind, cross-over design. METHODS: In eight men, bradykinin was infused intra-arterially twice; placebo, Ang(1-7), or angiotensin II was co-infused with the second infusion. The effect of inhibition of nitric oxide synthase on the interaction between Ang(1-7) and bradykinin was also tested in eight other individuals. The effects of Ang(1-7) were analyzed by analysis of variance (ANOVA) and by the ratios of individually derived areas under the dose-response curves (AUC) of bradykinin, adjusted for changes in the AUCs by repeated infusions of bradykinin with placebo. RESULTS: Ang(1-7) (1000 pmol/min) significantly potentiated the vasodilating effect of bradykinin compared with the effect of saline (P = 0.0471, ANOVA) and in a dose-dependent manner (adjusted AUC ratio [95% confidence interval (CI)] 2.75 (1.72 to 3.78) with 1000 pmol/min, 1.62 (1.31 to 1.93) with 100 pmol/min, and 0.98 (0.80, to 1.09) with 10 pmol/min). This effect was completely abolished by co-infusion of NG-monomethyl-l-arginine [AUC ratio 0.98 (0.90 to 1.04)]. Ang(1-7) did not affect the vasodilating effects of either acetylcholine or sodium nitroprusside. CONCLUSIONS: Ang(1-7) potentiates the vasodilating effect of bradykinin, possibly through a mechanism(s) involving nitric oxide release, in human forearm resistance vessels.

Differential effects of E and droloxifene on C-reactive protein and other markers of inflammation in healthy postmenopausal women.

Although increased levels of C-reactive protein have been linked to E therapy, the significance of this finding and whether it occurs with the selective ER modulators are unknown. Thirty-five healthy postmenopausal women were enrolled in a placebo-controlled, two-period cross-over design trial to evaluate the effects of 0.625 mg oral conjugated E and 60 mg droloxifene, a structural analog of tamoxifen, on serum levels of C-reactive protein, IL-6, and endothelial cell adhesion molecules. E treatment resulted in 65.8% higher levels of C-reactive protein (P = 0.0002) and 48.1% higher levels of IL-6 (P < 0.001), but also resulted in a 10.9% reduction in soluble E-selectin (P = 0.002) and borderline reductions in vascular cell adhesion molecule-1. In contrast, droloxifene had no effect on C-reactive protein and IL-6, but did produce a significant 11% reduction in E-selectin (P < 0.00001). However, droloxifene also resulted in an 11.6% increase in vascular cell adhesion molecule-1 (P < 0.007). These data provide additional evidence of a proinflammatory effect of E that may have adverse cardiovascular consequences. However, these changes were also accompanied by a reduction in E-selectin, suggesting an antiinflammatory effect at the level of the endothelium. The net clinical impact of these changes is not yet well established. In contrast, droloxifene had little or no proinflammatory effects on C-reactive protein and IL-6 and had mixed effects on endothelial adhesion molecules. This observation provides additional rationale for continuing to evaluate the potential cardiovascular benefits of selective ER modulators.

The angiotensin II AT1 receptor antagonist irbesartan prevents thromboxane A2-induced vasoconstriction in the rat hind-limb vascular bed in vivo.

OBJECTIVE: We studied the vasoconstrictor effects of the thromboxane A2 (TxA2) analogue U46619 in the perfused hind limb of rats under constant flow before and after intravenous injection of irbesartan, an angiotensin II AT1 receptor antagonist, to test whether irbesartan interacts in vivo with the thromboxane A2/prostaglandin endoperoxidase H2 (TxA2/PGH2) receptor. DESIGN: Male Sprague-Dawley rats (n = 15, body weight 350-420 g) were anesthetized with thiobutabarbital sodium (Inactin, 100 mg/kg intraperitoneally). Regional vascular responses to U46619 (0.5 and 1.0 microg) were investigated in the rat hind quarter under conditions of controlled flow before and after administration of irbesartan (10 mg/kg, intravenously). In addition, to test the specificity of the effect of irbesartan on U46619, phenylephrine (0.5, 1.0 microg) and another AT1 receptor antagonist, candesartan CV11974 (0.3 mg/kg, intravenously) were used. RESULTS: The dose-dependent increases in hind-limb perfusion pressure produced by U46619 were significantly attenuated by prior injection of irbesartan, at a dose that blocked the angiotensin II (Ang II) pressor responses. The specificity for the response was shown with the demonstrations that the increase in vascular resistance produced by phenylephrine was unchanged by irbesartan and, furthermore, that the increase in vascular resistance produced by U46619 was unchanged by another AT1 receptor antagonist, candesartan. CONCLUSION: This study demonstrates that irbesartan interacts at the TxA2/PGH2 receptor in the rat's hind limb in vivo, to modify changes in local regional vascular resistance. The dual antagonistic actions of irbesartan, acting at both AT1 and TxA2 receptors in blood vessels, may overall enhance its therapeutic profile in the treatment of hypertension.

Urinary vasodilator and vasoconstrictor angiotensins during menstrual cycle, pregnancy, and lactation.

Since normal human pregnancy is characterized by normotension in the face of an increased renin-angiotensin-aldosterone system (RAAS), we evaluated the temporal pattern of urinary excretion of a novel vasodilator within this system, angiotensin-(1-7) (Ang-[1-7]), during the menstrual cycle, pregnancy, and lactation. The urinary profiles of Ang I, Ang II, human chorionic gonadotropin, 17beta-estradiol, and progesterone were also determined. During the menstrual cycle, urinary Ang-(1-7) and Ang II remained stable (mean cycle value: 94.6 +/- 11.3 and 11.4 +/- 1.1 pmol/g of creatinine, respectively) in nine females. In 10 normal pregnant women, urinary Ang-(1-7) and Ang II increased throughout gestation, averaging 1499.8 +/- 310 and 224.4 +/- 58 pmol/g of creatinine, respectively (p < 0.05) at wk 35 and falling during lactation to 394.0 +/- 95 and 65.7 +/- 20 pmol/ g of creatinine (p < 0.05), respectively. The Ang-(1-7)/Ang II ratio was unchanged in the different reproductive periods. During the menstrual cycle, Ang II and Ang-(1-7) correlated with 17beta-estradiol and progesterone using multivariate analysis (r = 0.31, p < 0.001) and r = 0.28, p < 0.02, respectively). During gestation, 17beta-estradiol and progesterone correlated with urinary Ang-(1-7) (r = 0.48, p < 0.001 and r = 0.47, p < 0.001, respectively) and Ang II (r = 0.24, p < 0.03 and r = 0.25, p < 0.03, respectively); by multiple regression, only Ang-(1-7) correlated with both steroids (r = 0.49,p < 0.001). The progressive rise of Ang-(1-7) throughout gestation, probably modulated by estrogen and progesterone, suggests a physiologic counterregulation within the RAAS.

Inhibition of platelet aggregability by losartan in essential hypertension.

Most clinical events associated with hypertension have a thrombotic component. Losartan is a selective, competitive antagonist of the thromboxane A2 receptor in experiments performed in isolated vascular strips and in human and rat platelet-enriched plasma. In this study, we investigated for the first time whether losartan at therapeutic doses has an effect on platelet aggregability and indexes of fibrinolysis in essential hypertensive subjects. Changes in the dose-response curve to platelet aggregation induced by the thrombin receptor-activating peptide SFLRRN-NH2 were determined in 9 patients (56% men, 72% white; mean age 52.8 years) with stage I or II essential hypertension and in 9 untreated healthy volunteers. After a 4-week washout period, hypertensive subjects received 2 weeks of placebo followed by 4 weeks of losartan 50 mg/day. Both subjects and end points were blinded for treatment assignment. In addition, plasminogen activator inhibitor type 1 antigen and von Willebrand antigen were studied in all patients and controls. Four weeks of losartan produced a statistically significant (p <0.05) increase in the concentration of SFLRRN-NH2 required to induce a half-maximal response in platelet aggregation extent and rate 4 weeks after initiation of treatment. The decrease in platelet aggregability was independent of blood pressure control and the effects of gender and age. Losartan had no effect on plasma concentrations of plasminogen activator inhibitor-1 and von Willebrand factor in hypertensive subjects. These data demonstrate for the first time a novel antiplatelet effect of losartan at therapeutic doses, which was independent of changes in blood pressure, plasma markers of fibrinolytic activity, and endothelial perturbation.

Effects of estrogen replacement on the progression of coronary-artery atherosclerosis.

BACKGROUND: Heart disease is a major cause of illness and death in women. To understand better the role of estrogen in the treatment and prevention of heart disease, more information is needed about its effects on coronary atherosclerosis and the extent to which concomitant progestin therapy may modify these effects. METHODS: We randomly assigned a total of 309 women with angiographically verified coronary disease to receive 0.625 mg of conjugated estrogen per day, 0.625 mg of conjugated estrogen plus 2.5 mg of medroxyprogesterone acetate per day, or placebo. The women were followed for a mean (+/-SD) of 3.2+/-0.6 years. Base-line and follow-up coronary angiograms were analyzed by quantitative coronary angiography. RESULTS: Estrogen and estrogen plus medroxyprogesterone acetate produced significant reductions in low-density lipoprotein cholesterol levels (9.4 percent and 16.5 percent, respectively) and significant increases in high-density lipoprotein cholesterol levels (18.8 percent and 14.2 percent, respectively); however, neither treatment altered the progression of coronary atherosclerosis. After adjustment for measurements at base line, the mean (+/-SE) minimal coronary-artery diameters at follow-up were 1.87+/-0.02 mm, 1.84+/-0.02 mm, and 1.87+/-0.02 mm in women assigned to estrogen, estrogen plus medroxyprogesterone acetate, and placebo, respectively. The differences between the values for the two active-treatment groups and the value for the placebo group were not significant. Analyses of several secondary angiographic outcomes and subgroups of women produced similar results. The rates of clinical cardiovascular events were also similar among the treatment groups. CONCLUSIONS: Neither estrogen alone nor estrogen plus medroxyprogesterone acetate affected the progression of coronary atherosclerosis in women with established disease. These results suggest that such women should not use estrogen replacement with an expectation of cardiovascular benefit.

Cardiovascular effects of droloxifene, a new selective estrogen receptor modulator, in healthy postmenopausal women.

Selective estrogen receptor modulators, like tamoxifen and related compounds, have mixed estrogen agonistic/antagonistic effects. Tamoxifen may confer significant cardiovascular benefits without the estrogen-associated risks of endometrial and breast cancer. Droloxifene, a structural analogue of tamoxifen, has estrogen agonistic effects on bone and antagonistic effects on endometrial and breast tissue. Its cardiovascular effects in women are unknown. We enrolled 24 healthy postmenopausal women in a randomized, double-blind, 2-period crossover trial comparing the effects of droloxifene (60 mg/d) with conjugated estrogen (0.625 mg/d). Plasma lipids, coagulation and fibrinolytic factors, and brachial flow-mediated vasodilator responses were measured at the beginning and end of each treatment period. Droloxifene and estrogen resulted in 16.6% and 12.0% reductions, respectively, in low density lipoprotein cholesterol (P<0.001) and 13.2% and 9.5% reductions, respectively, in lipoprotein(a) (P<0.05). In contrast, estrogen, but not droloxifene, increased high density lipoprotein (18.5%, P<0.001). Droloxifene also reduced fibrinogen by 17.8% versus a 7.3% reduction with estrogen (P=0.004) but produced no estrogen-like changes in plasminogen, plasminogen activator inhibitor-1, or tissue plasminogen activator. Droloxifene and estrogen produced 36.4% and 27.3% increases, respectively, in flow-mediated vasodilation (percent change from baseline, P<0.05 for both). Droloxifene has estrogen agonistic properties regarding low density lipoprotein and lipoprotein(a) metabolism, certain coagulation factors, and endothelium-dependent vasodilation but, unlike estrogen, has no effect on high density lipoprotein/triglyceride metabolism and the fibrinolytic cascade. It remains unknown whether droloxifene can confer a true cardiovascular benefit.

Novel angiotensin II AT(1) receptor antagonist irbesartan prevents thromboxane A(2)-induced vasoconstriction in canine coronary arteries and human platelet aggregation.

This study was conducted to investigate whether the novel orally active nonpeptide angiotensin II (Ang II) AT(1) receptor antagonist irbesartan interacts with the thromboxane A(2)/prostaglandin endoperoxide H(2) (TxA(2)/PGH(2)) receptor in canine coronary arteries and human platelets. Coronary artery rings were isolated from male dog hearts (n = 18) and isometric tension of vascular rings was measured continuously at optimal basal tension in organ chambers. Autoradiographic binding of [(3)H]SQ29,548, a TxA(2) receptor antagonist, in canine coronary sections was determined. Blood for platelet aggregation studies was collected by venous puncture from healthy human volunteers (n = 6) who were free of aspirin-like agents for at least 2 weeks. Vascular reactivity and platelet aggregation in response to the TxA(2) analogs U46619 and autoradioagraphic receptor binding to the TxA(2) receptor antagonist [(3)H]SQ29,548 were studied with and without irbesartan. The TxA(2) analog U46619 produced dose-dependent vasoconstriction in coronary rings (EC(50) = 11.6 +/- 1.5 nM). Pretreatment with irbesartan inhibited U46619-induced vasoconstriction, and the dose-response curve was shifted to the right in a dose-dependent manner. The EC(50) of U46619 was increased 6- and 35-fold in the presence of 1 and 10 microM of irbesartan without a change of maximal contraction. At 1 microM, irbesartan is 2-fold more potent than the AT(1) receptor antagonist losartan in the inhibition of U46619-induced vasoconstriction in canine coronary arteries. In contrast, neither AT(1) receptor antagonists (CV11974 and valsartan), the AT(2) receptor antagonist PD123319, nor the angiotensin converting enzyme inhibitor lisinopril had any effect on U46619-induced coronary vasoconstriction. Irbesartan did not change potassium chloride-induced vasoconstriction; however, irbesartan did inhibit the vasoconstriction mediated by another TxA(2)/PGH(2) receptor agonist prostaglandin F(2alpha) (PGF(2alpha)). Neither the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester nor the cyclooxygenase inhibitor indomethacin had any effect on irbesartan's attenuation of U46619-induced vasoconstriction. Irbesartan specifically reversed U46619-preconstricted coronary artery rings with and without endothelium in a dose-dependent manner. Irbesartan at high concentrations significantly competed for [(3)H]SQ29,548 binding in canine coronary sections. U46619 stimulated dose-dependent human platelet aggregation of platelet-rich plasma. Preincubation with irbesartan significantly inhibited platelet aggregation in a concentration-dependent manner. In conclusion, the dual antagonistic actions of irbesartan by acting at both the AT(1) and TxA(2) receptors in blood vessels and platelets may overall enhance its therapeutic profile in the treatment of hypertension, atherosclerosis, and arterial thrombosis.

Differential response of angiotensin peptides in the urine of hypertensive animals.

Urinary excretion rates of angiotensin I (Ang I), angiotensin II (Ang II), and angiotensin-(1-7) [Ang-(1-7)] were determined in normotensive Sprague Dawley (SD), spontaneously hypertensive (SHR), and mRen-2 transgenic hypertensive animals before and following blockade of Ang II synthesis or activity for two weeks. This study was performed to determine for the first time whether inhibition of Ang II alters the excretion of angiotensin peptides in the urine. Rats were given either tap water or water medicated with lisinopril, losartan or both agents in combination. Blood pressure was monitored at regular intervals during the experiment by the tail-cuff method, and once again at the end of the study with a catheter implant into a carotid artery. Metabolic studies and 24 h urinary excretion variables and angiotensin peptides were determined before and during the procedures. While all three treatments normalized the blood pressure of hypertensive animals, therapy with either lisinopril or the combination of lisinopril and losartan had a greater antihypertensive effect in both SHR and [mRen-2]27 transgenic hypertensive rats. In the urine, the concentration of the angiotensins (normalized by 24-h creatinine excretion) was several-fold higher in the untreated hypertensive animals than in normotensive SD rats. In SD rats, lisinopril or lisinopril and losartan produced a sustained rise in urinary levels of Ang-(1-7) without changes in the excretion of Ang I and Ang II. In contrast, Ang I and Ang-(1-7) were significantly elevated in SHR medicated with lisinopril alone or in combination with losartan. Only losartan, however, augmented urinary levels of Ang II in the SHR. The antihypertensive effects of the three separate regimens had no effect on the urinary excretion of angiotensin peptides in [mRen-2]27 transgenic hypertensive rats. These data show that Ang I and Ang-(1-7) are excreted in large amounts in the urine of SD, SHR and [mRen-2]27 hypertensive rats. The unchanged Ang-(1-7) excretion in transgenic hypertensive (Tg+) rats after inhibition of the renin-angiotensin system agrees with the previous finding of a reduced plasma clearance of the peptide in this model of hypertension. The data suggest that this form of hypertension may be associated with increased activity of an endogenous converting enzyme inhibitor.