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To address whether differential regulation of the renin-angiotensin-aldosterone system occurs in pre-eclampsia, we performed an analysis of the time course of circulating and urinary profiles of the vasoconstrictor (Ang II) and the vasodilator [Ang-(1–7)] peptides in normal pregnant (NP) and pre-eclamptic (PE) women. Urine and plasma samples from 86 nulliparous women were collected prospectively; 67 subjects continued as NP and 19 developed PE. Subjects were enrolled prior to 12 weeks of gestation and plasma and spot urine samples were obtained throughout gestation. Control samples were obtained at 6 weeks postpartum (PP). Mean blood pressure (p

Preeclampsia is a syndrome with diverse clinical presentation that currently has no cure. The apelin receptor system is a pleiotropic pathway with a potential for therapeutic targeting in preeclampsia. We established the systemic outcomes of (Pyr1)-apelin-13 administration in rats with preeclamptic features (TGA-PE, female transgenic for human angiotensinogen mated to male transgenic for human renin). (Pyr1)-apelin-13 (2 mg/kg/day) or saline was infused in TGA-PE rats via osmotic minipumps starting at day 13 of gestation (GD). At GD20, TGA-PE rats had higher blood pressure, proteinuria, lower maternal and pup weights, lower pup number, renal injury, and a larger heart compared to a control group (pregnant Sprague-Dawley rats administered vehicle). (Pyr1)-apelin-13 did not affect maternal or fetal weights in TGA-PE. The administration of (Pyr1)-apelin-13 reduced blood pressure, and normalized heart rate variability and baroreflex sensitivity in TGA-PE rats compared to controls. (Pyr1)-apelin-13 increased ejection fraction in TGA-PE rats. (Pyr1)-apelin-13 normalized proteinuria in association with lower renal cortical collagen deposition, improved renal pathology and lower immunostaining of oxidative stress markers (4-HNE and NOX-4) in TGA-PE. This study demonstrates improved hemodynamic responses and renal injury without fetal toxicity following apelin administration suggesting a role for apelin in the regulation of maternal outcomes in preeclampsia.

Renal tubular dysgenesis (RTD) is a developmental abnormality of the nephron characterized by fetal anuria, oligohydramnios, and severe postnatal hypotension. Genetic forms have an autosomal recessive inheritance and are caused by mutations in genes encoding key components of the renin-angiotensin pathway. We report three patients from two unrelated families with RTD due to pathogenic variants of the angiotensin-converting enzyme (ACE) gene, in whom RTD was associated with microcolon. We also detail key variations of the renin-angiotensin system in one of these infants. The severe intestinal developmental abnormality culminating in microcolon and early terminal ileum perforation/necrotizing enterocolitis is a novel finding not previously associated with RTD, which points to a role of the renin-angiotensin system in gut development.

To address whether differential regulation of the renin-angiotensin-aldosterone system occurs in pre-eclampsia, we performed an analysis of the time course of circulating and urinary profiles of the vasoconstrictor (Ang II) and the vasodilator [Ang-(1-7)] peptides in normal pregnant (NP) and pre-eclamptic (PE) women.Urine and plasma samples from 86 nulliparous women were collected prospectively; 67 subjects continued as NP and 19 developed PE. Subjects were enrolled prior to 12 weeks of gestation and plasma and spot urine samples were obtained throughout gestation. Control samples were obtained at 6 weeks postpartum (PP).Mean blood pressure (p 0.001) was elevated at 31-37 weeks of gestation in PE subjects as compared with NP subjects. Plasma Ang I and Ang II levels were elevated in NP subjects as early as 16 weeks of gestation and maintained throughout gestation. In PE subjects both plasma Ang I and Ang II were elevated at 16-33 weeks as compared with PP levels. PE subjects showed reduced plasma Ang I and Ang II (at 35-37 weeks of gestation) compared with NP subjects. Plasma Ang-(1-7) was unchanged in both groups. All three urinary peptides increased throughout gestation in NP subjects. In PE subjects urinary Ang I was increased at 23-26 weeks and was maintained throughout gestation. Urinary Ang II was increased at 27-29 and 31-33 weeks of gestation. PE subjects had no change in urinary Ang-(1-7).The activation of the RAS, particularly Ang II throughout normal gestation may contribute to the maintenance of vascular tone during normal pregnancy. However higher sensitivity to Ang II in pre-eclampsia may be potentiated by the higher circulating and urinary levels of Ang II, unopposed by local renal Ang-(1-7), and thus may contribute to the development of pre-eclampsia.

Diets rich in grains and meat and low in fruits and vegetables (acid-producing diets) associate with incident hypertension, whereas vegetarian diets associate with lower blood pressure (BP). However, the pathways that sense and mediate the effects of acid-producing diets on BP are unknown. Here, we examined the impact of the deletion of an acid sensor GPR4 on BP. GPR4 is a proton-sensing G protein-coupled receptor and an acid sensor in brain, kidney, and blood vessels. We found that GPR4 mRNA was higher in subfornical organ (SFO) than other brain regions. GPR4 protein was abundant in SFO and present in capillaries throughout the brain. Since SFO partakes in BP regulation through the renin-angiotensin system (RAS), we measured BP in GPR4−/− and GPR4+/+ mice and found that GPR4 deletion associated with lower systolic BP: 87 ± 1 mmHg in GPR4−/− ( n = 35) vs. 99 ± 2 mmHg ( n = 29) in GPR4+/+; P < 0.0001, irrespective of age and sex. Angiotensin II receptors detected by 125I-Sarthran binding were lower in GPR4−/− than GPR4+/+ mice in SFO and in paraventricular nucleus of hypothalamus. Circulating angiotensin peptides were comparable in GPR4−/− and GPR4+/+ mice, as were water intake and excretion, serum and urine osmolality, and fractional excretion of sodium, potassium, or chloride. A mild metabolic acidosis present in GPR4−/− mice did not associate with elevated BP, implying that deficiency of GPR4 may preclude the effect of chronic acidosis on BP. Collectively, these results posit the acid sensor GPR4 as a novel component of central BP control through interactions with the RAS.

Background. Angiotensin-(1-7) [Ang-(1-7)] is an endogenous antiangiogenic hormone with anticancer activity. In a phase I study of Ang-(1-7), two of three patients with metastatic sarcoma experienced disease stabilization. This phase II study examined clinical and biomarker outcomes for patients with metastatic sarcoma. Methods. Ang-(1-7) was administered by subcutaneous injection at a dose of 20 mg daily. If excessive toxicities occurred in the first cohort, a dose deescalation cohort was allowed. Blood samples were obtained to measure changes in biomarkers. Results. Treatment was well-tolerated and the dose deescalation cohort was not required. Plasma PlGF concentrations following treatment were not statistically significantly changed. A significant increase in plasma Ang-(1-7) was observed at 4 hours after injection. The median progression-free survival was 2.7 months (95% CI; 1.4 to 4.1 months), and the median overall survival was 10.2 months (95% CI; 5.3 to 18.3 months). Two patients with vascular sarcomas demonstrated prolonged disease stabilization of 10 months (hemangiopericytoma) and 19 months (epithelioid hemangioendothelioma). Conclusions. Ang-(1-7) at a dose of 20 mg daily was well-tolerated. This prospective phase II study failed to confirm the PlGF biomarker effect identified in the prior phase I study. Prolonged disease stabilization in hemangiopericytoma and epithelioid hemangioendothelioma may warrant further investigation.

Placental oxygenation varies throughout pregnancy. The detection of early changes in placental oxygenation as pregnancy progresses is important for early identification of preeclampsia or other complications. This invited commentary discusses a recent preclinical study on the application of 3-dimensional photoacoustic imaging (PAI) for assessment of regional variations in placental oxygenation and longitudinal analysis of differences in placental oxygenation throughout normal pregnancy and pregnancy associated with hypertension or placental insufficiency in mice. Three-dimensional PAI more accurately reflects oxygen saturation, hemoglobin concentrations, and changes in oxygen saturation in whole placenta compared to 2-dimensional imaging. These studies suggest that PAI is a sensitive tool to detect different levels of oxygen saturation in the placental and fetal vasculature in pathologic and normal pregnancy in mice.

Hypothesis:Hyperglycemia decreases angiotensin-(1-7), the endogenous counter-regulator of angiotensin II in the retina.Materials and methods:The distribution and levels of retinal angiotensin II (Ang II) and angiotensin-(1-7) (Ang-(1-7)) were evaluated by confocal imaging and quantitative immunohistochemistry during the development of streptozotocin-induced diabetes in rats.Results:In the nondiabetic eye, Ang II was localized to the endfeet of Müller cells, extending into the cellular processes of the inner plexiform layer and inner nuclear layer; Ang-(1-7) showed a wider distribution, extending from the foot plates of the Müller cells to the photoreceptor layer. Eyes from diabetic animals showed a higher intensity and extent of Ang II staining compared with nondiabetic eyes, but lower intensity with a reduced distribution of Ang-(1-7) immunoreactivity. Treatment of the diabetic animals with the angiotensin-converting enzyme inhibitor (ACEI) captopril showed a reduced intensity of Ang II staining, whereas increased intensity and distribution were evident with Ang-(1-7) staining.Conclusions:These studies reveal that pharmacological inhibition with ACEIs may provide a specific intervention for the management of the diabetes-induced decline in retinal function, reversing the profile of the endogenous angiotensin peptides closer to the normal condition.

Angiotensin-converting enzyme 2 (ACE2) knockout is associated with reduced fetal weight at late gestation; however, whether uteroplacental vascular and/or hemodynamic disturbances underlie this growth-restricted phenotype is unknown. Uterine artery reactivity and flow velocities, umbilical flow velocities, trophoblast invasion, and placental hypoxia were determined in ACE2 knockout (KO) and C57Bl/6 wild-type (WT) mice at day 14 of gestation. Although systolic blood pressure was higher in pregnant ACE2 KO vs. WT mice (102.3 ± 5.1 vs. 85.1 ± 1.9 mmHg, n = 5–6), the magnitude of difference was similar to that observed in nonpregnant ACE2 KO vs. WT mice. Maternal urinary protein excretion, serum creatinine, and kidney or heart weights were not different in ACE2 KO vs. WT. Fetal weight and pup-to-placental weight ratio were lower in ACE2 KO vs. WT mice. A higher sensitivity to Ang II [pD2 8.64 ± 0.04 vs. 8.5 ± 0.03 (−log EC50)] and greater maximal contraction to phenylephrine (169.0 ± 9.0 vs. 139.0 ± 7.0% KMAX), were associated with lower immunostaining for Ang II receptor 2 and fibrinoid content of the uterine artery in ACE2 KO mice. Uterine artery flow velocities and trophoblast invasion were similar between study groups. In contrast, umbilical artery peak systolic velocities (60.2 ± 4.5 vs. 75.1 ± 4.5 mm/s) and the resistance index measured using VEVO 2100 ultrasound were lower in the ACE2 KO vs. WT mice. Immunostaining for pimonidazole, a marker of hypoxia, and hypoxia-inducible factor-2α were higher in the trophospongium and placental labyrinth of the ACE2 KO vs. WT. In summary, placental hypoxia and uterine artery dysfunction develop before major growth of the fetus occurs and may explain the fetal growth restricted phenotype.

To address whether differential regulation of the renin-angiotensin system (RAS) occurs in preelampsia, we performed an extensive analysis of the time course of circulating and urinary profiles of the vasoconstrictor (Ang II) and the vasodilator [Ang-(1-7)] components in pregnant and preeclamptic women. Plasma and spot urine samples were collected prospectively from nulliparous normal pregnant (n=67) and preeclamptic (n=19) women between the ages of 18-40, at

Accurate analysis of placental and fetal oxygenation is critical during pregnancy. Photoacoustic imaging (PAI) combines laser technology with ultrasound in real time. We tested the sensitivity and accuracy of PAI for analysis of placental and fetal oxygen saturation (sO2) in mice. The placental labyrinth (L) had a higher sO2 than the junctional zone plus decidua region (JZ+D) in C57Bl/6 mice. Changing maternal O2 from 100 to 20% in C57Bl/6 mice lowered sO2 in these regions. C57Bl/6 mice were treated with the NO synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME) from gestational day (GD) 11 to GD18 to induce hypertension. L-NAME decreased sO2 in L and JZ+D at GD14 and GD18 in association with fetal growth restriction and higher blood pressure. Hypoxia-inducible factor 1α immunostaining was higher in L-NAME vs. control mice at GD14. Fetal sO2 levels were similar between l-NAME and control mice at GD14 and GD18. In contrast to untreated C57Bl/6, L-NAME decreased placental sO2 at GD14 and GD18 vs. GD10 or GD12. Placental sO2 was lower in fetal growth restriction in an angiotensin-converting enzyme 2 knockout mouse model characterized by placental hypoxia. On phantom studies, patterns of sO2 measured directly correlated with those measured by PAI. In summary, PAI enables the detection of placental and fetal oxygenation during normal and pathologic pregnancies in mice.—Yamaleyeva, L. M., Sun, Y., Bledsoe, T., Hoke, A., Gurley, S. B., Brosnihan, K. B. Photoacoustic imaging for in vivo quantification of placental oxygenation in mice.

The renin-angiotensin system (RAS) is a complex circulating and tissue-based system. There are multiple pathways for the formation and degradation of peptides. In order to understand the functions of the system, characterization of angiotensin peptides (products and substrates) is important. Radioimmunoassays with the requisite specificity and sensitivity have been developed to allow for the characterization and quantification of circulating and tissue angiotensins. Here, we describe the appropriate methods for collecting the tissue and blood, the extractions steps required to partially purify and remove larger molecular weight-interfering proteins from tissue and plasma, and the radioimmunoassay of three of the peptides of this system (Ang I, Ang II, and Ang-(1-7)), as well as the verification of immunoreactive identity for Ang II and Ang-(1-7) by combined high-performance liquid chromatography-RIA analysis.

Summary Aims Obesity is associated with metabolic dysfunctions, which may be mediated by changes in adipose tissue signaling factors. These molecules are denoted as A dipose T issue G enerated M ediators of C ardio V ascular R isk (ATGMCVR) here, and include leptin, adiponectin, C-reactive protein (CRP), interleukin 6 (IL-6), tumor necrosis factor alpha (TNFα), and plasminogen activator inhibitor 1 (PAI-1). This study examined the effect of a weight loss program on ATGMCVR in obese adults with prediabetes. Materials and methods Subjects were randomized to usual care (UC; n = 15) or lifestyle weight loss groups (LWL; n = 15). LWL was a community-based weight loss intervention to promote physical activity and healthy eating. ATGMCVR at 1-year were compared between groups by analysis of covariance; baseline value of the mediator was the covariate. Baseline means for ATGMCVR were compared between those with (n = 21) and without (n = 9) metabolic syndrome (MetS). Results At baseline, subjects were 58 ± 9 (SD) years, 70% female, with a BMI of 34 ± 4 kg/m2. One-year weight loss (%) was 7.8 ± 6.0% for LWL and 1.7 ± 4.5% for UC. Group differences at 1-year were noted (adjusted means [95%CI] for UC and LWL, respectively) for adiponectin (8526.3 [7397.7, 9827]; 10,870.9 [9432.0, 12,529.3] ng/ml; p = 0.02), leptin (30.4 [26.1, 35.4]; 23.7 [20.3, 27.5] ng/ml; p = 0.02), IL-6 (0.4 [0.3, 0.5]; 0.2 [0.1, 0.2] pg/ml; p = 0.001), and PAI-1 (50 [42.7, 58.7]; 36.2 [30.8, 42.4] pg/ml; p = 0.01). No differences in baseline ATGMCVR were seen between subjects with and without MetS. Conclusion These findings suggest ATGMCVR can be improved with weight loss; larger studies are needed to determine if improvements in metabolic dysfunction are related to changes in ATGMCVR.

Increased vascular sensitivity to angiotensin II (Ang II) is a marker of a hypertensive human pregnancy. Recent evidence of interactions between the renin–angiotensin system and the endocannabinoid system suggests that anandamide and 2-arachidonoylglycerol may modulate Ang II contraction. We hypothesized that these interactions may contribute to the enhanced vascular responses in hypertensive pregnancy. We studied Ang II contraction in isolated uterine artery (UA) at early gestation in a rat model that mimics many features of preeclampsia, the transgenic human angiotensinogen×human renin (TgA), and control Sprague–Dawley rats. We determined the role of the cannabinoid receptor 1 by blockade with SR171416A, and the contribution of anandamide and 2-arachidonoylglycerol degradation to Ang II contraction by inhibiting their hydrolyzing enzyme fatty acid amide hydrolase (with URB597) or monoacylglycerol lipase (with JZL184), respectively. TgA UA showed increased maximal contraction and sensitivity to Ang II that was inhibited by indomethacin. Fatty acid amide hydrolase blockade decreased Ang II MAX in Sprague–Dawley UA, and decreased both Ang II MAX and sensitivity in TgA UA. Monoacylglycerol lipase blockade had no effect on Sprague–Dawley UA and decreased Ang II MAX and sensitivity in TgA UA. Blockade of the cannabinoid receptor 1 in TgA UA had no effect. Immunolocalization of fatty acid amide hydrolase and monoacylglycerol lipase showed a similar pattern between groups; fatty acid amide hydrolase predominantly localized in endothelium and monoacylglycerol lipase in smooth muscle cells. We demonstrated an increased Ang II contraction in TgA UA before initiation of the hypertensive phenotype. Anandamide and 2-arachidonoylglycerol reduced Ang II contraction in a cannabinoid receptor 1–independent manner. These renin–angiotensin system-endocannabinoid system interactions may contribute to the enhanced vascular reactivity in early stages of hypertensive pregnancy.

To understand the role of Angiotensin-(1-7) (Ang-(1-7)) in vasculature of pregnant women, we compared cardiac output (CO), total peripheral resistance (TPR) and forearm blood flow (FBF) responses to Ang-(1-7) infusion between normotensive pregnant women in their third trimester and healthy age matched non-pregnant women. The responses of skin microcirculation to Ang-(1-7) were tested in preeclamptic, normotensive pregnant and non-pregnant women. Responses to Angiotensin II (Ang II) were also determined.Non-invasive methods for systemic (bioimpedance and VascuMAP), FBF (venous occlusion strain gauge plethysmography), and skin (laser Doppler) hemodynamics assessments were used.Compared to non-pregnant women, systemic infusion of Ang-(1-7) (2000 pmol/min) resulted in a greater increase in CO (9.4 ± 6.4 versus -3.3 ± 2.1%, n = 9-10) in normotensive pregnant women. Brachial local infusion of Ang-(1-7) had no effect on FBF in either group. In non-pregnant and normotensive pregnant women, local Ang II induced a dose-dependent decrease in FBF and increase in forearm resistance at 50 and 100 pmol/min (p 0.05 versus corresponding baseline, n = 7-10). Following iontophoretic application of 5 mmol/l dose of Ang-(1-7), the change in skin flow was higher in normotensive pregnant versus preeclamptic women (182.5 ± 93 versus 15.76 ± 19.46%, n = 14-15). Skin flow was lower in normotensive pregnant versus preeclamptic women (-46.5 ± 48.7 versus 108.7 ± 49.1%, n = 14-15) following Ang II infusion at 1.0 pmol/min.In the third trimester of pregnancy, Ang-(1-7) induces alterations in CO and differentially regulates micro- and macro-circulations, depending on the dose. Dysregulation in skin vasculature may contribute to the development of vascular dysfunction and hypertension in preeclampsia.

Although the mechanism of action of angiotensin II (Ang II) receptor (AT1-R) blockers (ARBs) is well established, data suggest that their antihypertensive effects may be limited in part by the overriding effect of compensatory increases in plasma renin activity (PRA) and Ang II.1 Contrasting with the mechanism of action of other ARBs, Agata et al. 2 reported that the antihypertensive and cardiac antihypertrophic actions of 4-week olmesartan medoxomil administration were not associated with the expected increases in circulating Ang II in adult stroke-prone spontaneously hypertensive rats (SHR). Agata et al. 2 speculated that the failure of plasma Ang II to rise in response to blockade of AT1-R could be explained by angiotensin-(1–7) [Ang-(1–7)]–induced inhibition of angiotensin converting enzyme (ACE) activity, given previous observations of increased Ang-(1–7) after treatment with ARBs,3,4 as well as an inhibitory role of the heptapeptide at the C- and N-terminus catalytic activity of the enzyme.5,6 The intriguing possibility of a differential effect of olmesartan on plasma Ang II levels compared with other ARBs was also consistent with 2 other studies in human essential hypertensive subjects in whom prolonged therapy with olmesartan was accompanied by either decreases or no changes in plasma Ang II levels.7,8 The effects of olmesartan on Ang II levels may also result from increased conversion of the octapeptide to Ang-(1–7) because ACE2 has been shown to be upregulated by AT1-R blockade.4,9,10 This has been demonstrated in vivo by recent work showing that the infusion of soluble human recombinant ACE2 efficiently lowered plasma Ang II while increasing Ang-(1–7).10 Furthermore, in isolated cardiac myocytes, ACE2 messenger RNA expression and activity were not affected by Ang-(1–7); however, the inhibitory effects of Ang II on ACE2 were blocked by Ang-(1–7).11 The heptapeptide modulatory effect was prevented by the Ang-(1–7) mas receptor antagonist [D-ALA7]-Ang-(1–7) (A-779), indicating that the Ang-(1–7) response was mediated by a specific Ang-(1–7) receptor. A-779 is a selective blocker of the mas receptor that has been identified to mediate vasodilatory, antitrophic, and antiproliferative effects of Ang-(1–7).12–14 The long-term effects of Ang-(1–7) antagonism in the presence of concomitant Ang II receptor blockade have not been determined. With this in mind, we investigated the Ang-(1–7)–mediated effects of olmesartan on blood pressure, plasma, renal, and cardiac Ang II as well as ACE2 in mRen2.Lewis congenic hypertensive rats. This monogenetic hypertensive rat strain was developed in our laboratory through a backcross of the hypertensive (mRen2)27 transgenic rats with normotensive Lewis rats. The aim of this backcross was to offset the heterogeneity of the parent strain that contributed to the genetic variability found within the original transgenic strain.15,16 Because the malignant phase of hypertension is not observed in mRen2.Lewis rats, the longer life span of this experimental model provides a better opportunity to investigate the function and regulation of tissue renin–angiotensin system (RAS) and its contribution to the etiology of hypertension and target organ damage.

We examined the antihypertensive effects of valsartan, aliskiren, or both drugs combined on circulating, cardiac, and renal components of the renin–angiotensin system in congenic mRen2.Lewis hypertensive rats assigned to: vehicle (n=9), valsartan (via drinking water, 30 mg/kg per day; n=10), aliskiren (SC by osmotic mini-pumps, 50 mg/kg per day; n=10), or valsartan (30 mg/kg per day) combined with aliskiren (50 mg/kg per day; n=10). Arterial pressure and heart rate were measured by telemetry before and during 2 weeks of treatment; trunk blood, heart, urine, and kidneys were collected for measures of renin–angiotensin system components. Arterial pressure and left-ventricular weight/tibia length ratio were reduced by monotherapy of valsartan, aliskiren, and further reduced by the combination therapy. Urinary protein excretion was reduced by valsartan and further reduced by the combination. The increases in plasma angiotensin (Ang) II induced by valsartan were reversed by the treatment of aliskiren and partially suppressed by the combination. The decreases in plasma Ang-(1–7) induced by aliskiren recovered in the combination group. Kidney Ang-(1–12) was increased by the combination therapy whereas the increases in urinary creatinine mediated by valsartan were reversed by addition of aliskiren. The antihypertensive and antiproteinuric actions of the combined therapy were associated with marked worsening of renal parenchymal disease and increased peritubular fibrosis. The data show that despite improvements in the surrogate end points of blood pressure, ventricular mass, and proteinuria, dual blockade of Ang II receptors and renin activity is accompanied by worsening of renal parenchymal disease reflecting a renal homeostatic stress response attributable to loss of tubuloglomerular feedback by Ang II.

Background Cyclooxygenase (COX)-derived prostanoids (PGE2, PGI2) are important contributors to the process of decidualization. Previous studies showed the presence of Ang-(1-7) in the primary and secondary decidualized zones of the implantation site at early pregnancy. Decreased concentrations of Ang-(1-7) were found in the decidualized uterus compared to the non-decidualized uterus of pseudopregnant rats, suggesting that low levels of Ang-(1-7) are required for successful decidualization at early pregnancy. Methods To understand the role of Ang-(1-7) in prostaglandin production in a decidualized uterus, induced by a bolus injection of sesame oil, Ang-(1-7) (24 μg/kg/h) or vehicle was then infused directly into the decidualized uterine horn using an osmotic minipump. The right horns were not injected or infused and served as non-decidualized uterine horns in both groups of animals. Results Decidualization increased PGE2 concentration in the uterus (0.53 ± 0.05 vs. 12.0 ± 3.2 pmol/mg protein, p

Placental hypoxia/ischemia induces abnormal maternal and neonatal outcomes including preeclampsia and intrauterine growth restriction (IUGR). The ability to accurately determine placental oxygenation in a non-invasive way and in real-time is highly important during pregnancy as it may allow for the early diagnosis of IUGR and preeclampsia. Photoacoustic imaging (PA) is a novel preclinical and emerging clinical tool that combines optical contrast of photoacoustic laser technology with high spatial resolution of ultrasound. PA measures tissue oxygen saturation (sO2) that reflects differences in absorption spectra for oxygenated and deoxygenated hemoglobin. By using photoacoustic features of VEVO LAZR high resolution ultrasound system (VisualSonics) in a three-dimensional mode we investigated the sensitivity and accuracy of PA for placental oxygenation in C57Bl/6 mice at day 14 of gestation. Furthermore, since nitric oxide deficiency is associated with upregulation of circulatory hypoxia markers, C57Bl/6 mice were chronically treated with the nitric oxide inhibitor, L-NAME via osmotic minipumps (50 mg/kg/day; days 13 to 18 of gestation). The comparisons between scanned vs. not scanned uteroplacental units showed that PA had no effect on fetal (scanned: 0.028±0.001 vs. not scanned: 0.03±0.001 g/maternal body weight; p>0.05) or placental (scanned: 0.002±0.003 vs. not scanned: 0.002±0.002 g/maternal body weight; p>0.05) weights in C57Bl/6 mice. Changing inhaled O2 from 100- to 20% resulted on average in 12.5% reduction in total placental sO2. Systolic blood pressures were higher in L-NAME-treated vs. C57Bl/6 mice (215.8±0.8 vs. sham 99.3±4.4 mmHg; p

We examined the impact of early diabetes on the circulating and kidney renin-angiotensin system (RAS) in male and female mRen2.Lewis (mRen2) hypertensive rats. Diabetes (DB) was induced by streptozotocin (STZ; 65 mg/kg) at 11 wk of age for 4 wk without insulin replacement. Systolic blood pressures were not increased in DB males or females compared with controls (CON). Circulating angiotensin-converting enzyme 2 (ACE2) increased ninefold ( P < 0.05) in DB females and threefold ( P < 0.05) in DB males, but circulating ACE and ANG II were higher in the DB groups. Serum C-reactive protein was elevated in DB females but not DB males, and the vascular responses to acetylcholine and estradiol were attenuated in the DB females. Proteinuria, albuminuria, and angiotensinogen excretion increased to a similar extent in both DB females and males. Glomerular VEGF expression also increased to a similar extent in both DB groups. Renal inflammation (CD68+cells) increased only in DB females although males exhibited greater inflammation that was not different with DB. Cortical ACE2 did not change in DB females but was reduced (30%) in DB males. Renal neprilysin activity (>75%, P < 0.05) was markedly reduced in the DB females to that in the DB and CON males. ACE activity was significantly lower in both female (75%, P < 0.05) and male (50%; P < 0.05) DB groups, while cortical ANG II and Ang-(1-7) levels were unchanged. In conclusion, female mRen2 rats are not protected from vascular damage, renal inflammation, and kidney injury in early STZ-induced diabetes despite a marked increase in circulating ACE2 and significantly reduced ACE within the kidney.

The current study assessed whether activation of the novel estrogen receptor GPR30 ameliorates salt-dependent renal damage in intact mRen2.Lewis (mRen2) females. Hemizygous mRen2 rats were maintained on either a normal salt (0.5% Na) or high-salt (HS; 4.0% Na) diet for 10 weeks (5 to 15 weeks of age), and HS animals were treated with the GPR30 agonist G-1 or vehicle for 2 weeks. Systolic blood pressure markedly increased with HS diet (149±3 to 219±5 mm Hg; P P =0.42). G-1 and estradiol induced relaxation of preconstricted mesenteric vessels from normal salt mRen2 rats, but both responses were attenuated in the HS group. Despite the lack of an effect on blood pressure, G-1 decreased renal hypertrophy, proteinuria, urinary 8-isoprostane excretion, and tubular 4-hydroxynonenal staining. HS diet significantly increased GPR30 mRNA (1.01±0.04 versus 1.59±0.13; P P

The renin-angiotensin (Ang) system is important during placental development. Dysregulation of the renin-Ang system is important in preeclampsia (PE). Female rats transgenic for the human angiotensinogen gene crossed with males transgenic for the human renin gene develop the PE syndrome, whereas those of the opposite cross do not. We used this model to study the role of Ang II in trophoblast invasion, which is shallow in human PE but deeper in this model. We investigated the following groups: PE rats, opposite-cross rats, Ang II–infused rats (1000 ng/kg per day), and control rats. Ang II infusion increased only circulating Ang II levels (267.82 pg/mL), opposite cross influenced only uteroplacental Ang II (13.52 fmol/mg of protein), and PE increased both circulating (251.09 pg/mL) and uteroplacental (19.24 fmol/mg of protein) Ang II. Blood pressure and albuminuria occurred in the models with high circulating Ang II but not in the other models. Trophoblast invasion increased in PE and opposite-cross rats but not in Ang II–infused rats. Correspondingly, uterine artery resistance index increased in Ang II–infused rats but decreased in PE rats. We then studied human trophoblasts and villous explants from first-trimester pregnancies with time-lapse microscopy. Local Ang II dose-dependently increased migration by 75%, invasion by 58%, and motility by 282%. The data suggest that local tissue Ang II stimulates trophoblast invasion in vivo in the rat and in vitro in human cells, a hitherto fore unrecognized function. Conceivably, upregulation of tissue Ang II in the maternal part of the placenta represents an important growth factor for trophoblast invasion and migration.

Angiotensin (Ang)-converting enzyme 2 (ACE2) cleaves Ang II to form Ang-(1-7). Here we examined whether soluble human recombinant ACE2 (rACE2) can efficiently lower Ang II and increase Ang-(1-7) and whether rACE2 can prevent hypertension caused by Ang II infusion as a result of systemic versus local mechanisms of ACE2 activity amplification. rACE2 was infused via osmotic minipumps for 3 days in conscious mice or acutely in anesthetized mice. rACE2 caused a dose-dependent increase in serum ACE2 activity but had no effect on kidney or cardiac ACE2 activity. After Ang II infusion (40 pmol/min), rACE2 (1 mg/kg per day) resulted in normalization of systolic blood pressure and plasma Ang II. In acute studies, rACE2 (1 mg/kg) prevented the rapid hypertensive effect of Ang II (0.2 mg/kg), and this was associated with both a decrease in Ang II and an increase in Ang-(1-7) in plasma. Moreover, during infusion of Ang II, the effect of rACE2 on blood pressure was unaffected by a specific Ang-(1-7) receptor blocker, A779 (0.2 mg/kg), and infusing supraphysiologic levels of Ang-(1-7) (0.2 mg/kg) had no effect on blood pressure. We conclude that, during Ang II infusion, rACE2 effectively degrades Ang II and, in the process, normalizes blood pressure. The mechanism of rACE2 action results from an increase in systemic, not tissue, ACE2 activity and the lowering of plasma Ang II rather than the attendant increase in Ang-(1-7). Increasing ACE2 activity may provide a new therapeutic target in states of Ang II overactivity by enhancing its degradation, an approach that differs from the current focus on blocking Ang II formation and action.

Background: We investigated renal effects of nebivolol, a selective β1-receptor blocker with additional antioxidative ability, in spontaneously hypertensive rats (SHR) where increased salt intake induces oxidative stress and worsens renal function as a result of further activation of the renin-angiotensin and sympathetic nervous systems. Methods: Male SHR were given an 8% salt diet (HS; n = 22) for 5 weeks; their age-matched controls (n = 9) received standard chow. Nebivolol was given at a dose of 10 mg/kg/day for 5 weeks in 11 HS rats. Results: HS increased blood pressure, plasma renin concentration, urinary protein excretion, and renal nitroxidative stress while decreasing renal blood flow and angiotensin 1–7 receptor (mas) protein expression. There was no change in angiotensin II type 1 receptor expression among the experimental groups. Nebivolol did not alter the salt-induced increase in blood pressure but reduced urinary protein excretion, plasma renin concentration, and nitroxidative stress. Nebivolol also increased neuronal NOS expression while preventing the salt-induced decrease in renal blood flow and mas protein expression. Conclusion: Nebivolol prevented salt-induced kidney injury and associated proteinuria in SHR through a blood pressure-independent mechanism. Its protective effects may be related to reduction in oxidative stress, increases in neuronal NOS and restoration of angiotensin II type 1/mas receptor balance.

Objective: Angiotensin II has a critical role in the regulation of blood pressure and cell growth and excess activity of the peptide is implicated in the pathogenesis of salt-induced cardiovascular injury. On the other hand, the role of counteracting angiotensin-(1-7) in cardiac structural and functional responses to high salt diet has not been elucidated. Therefore, the present study examined the changes in cardiac angiotensin-(1-7), its forming enzyme angiotensin converting enzyme 2 (ACE2) and receptor mas in response to a high salt diet in spontaneously hypertensive rats (SHR). Methods: Eight-week-old male spontaneously hypertensive rats (SHR) were given an 8% salt diet for 5 weeks ( n = 8). Age- and gender-matched controls received standard chow ( n = 6). Results: Salt excess increased arterial pressure ( p < 0.05) and plasma renin and angiotensin II concentrations ( p < 0.05). Salt-induced left ventricular remodeling and diastolic dysfunction were associated with diminished levels of angiotensin-(1-7) in the heart ( p < 0.05) and no changes in cardiac angiotensin II levels. Exposure to high salt intake decreased cardiac ACE2 mRNA and protein level ( p < 0.05). There was no difference in the protein levels of angiotensin II type 1 and mas receptors between the two experimental groups. Conclusion: The adverse cardiac effects of excessive salt intake may result not only from the undesirable action of angiotensin II but may also be a consequence of diminished protective effects of the angiotensin-(1-7).

Growth-differentiation factor 15 (GDF-15), a stress-responsive transforming growth factor-β–related cytokine, is emerging as a new risk marker in patients with cardiovascular disease. We explored GDF-15 in preeclampsia and in diabetic pregnancies, because these conditions are associated with augmented risk for cardiovascular disease, both in mother and in offspring. Plasma from pregnant women (n=267; controls: n=59, preeclampsia: n=85, diabetes mellitus: n=112, and superimposed preeclampsia in diabetes mellitus: n=11), fetal plasma (n=72), and amniotic fluid (n=99) were analyzed by immunoassay for GDF-15. Placental GDF-15 mRNA and protein expression levels were analyzed by quantitative real-time PCR and immunoblots in 78 and 18 pregnancies, respectively. Conditioned media from preeclamptic (n=6) and control (n=6) villous placenta explants were analyzed by immunoassay for GDF-15. Median maternal GDF-15 concentration was elevated in those with diabetes mellitus, as compared with controls (91 549 versus 79 875 ng/L; P =0.02). Median GDF-15 concentration was higher in patients with preeclampsia than in controls in term maternal blood samples (127 061 versus 80 319 ng/L; P P =0.002). Placenta immunoblots confirmed a single GDF-15 protein band, and a time-dependent increase in GDF-15 protein was detected in the conditioned media. Our study is the first to show that GDF-15 is dysregulated, both in preeclampsia and in diabetic pregnancies. The mechanisms and diagnostic implications of these findings remain to be explored.

We previously demonstrated that angiotensin-(1-7) [Ang-(1-7)], which is increased in the kidney and urine during pregnancy, influences normal fluid expansion of pregnancy. These previous studies were completed by chronic administration of the Ang-(1-7) receptor antagonist D-Alanine-[Ang-(1-7)] (A-779) at a dose of 48 μg/kg/hour after the start of pregnancy (gestational days 11 to 19). To further explore the role of Ang-(1-7) on kidney function during early, middle, and late pregnancy, Sprague Dawley rats were chronically pretreated 8 days prior to pregnancy and throughout pregnancy (gestational days 0 to 19) with vehicle or A-779 at a dose of 24 μg/kg/hour. Metabolic studies were completed in virgin animals and throughout pregnancy (gestational days 4 to 5, 14 to 15, and 18 to 19). Chow consumption and water intake increased throughout pregnancy while the difference between intake and output (balance) was increased only at late (day 19) pregnancy with both vehicle and A-779 administration. Urine volume and urinary osmolality were significantly increased and decreased, respectively, throughout pregnancy in vehicle-treated rats only. In late (19 days) pregnancy, A-779 administration significantly decreased chow consumption and water intake. In virgin animals, A-779 administration significantly increased urine volume, while during late pregnancy (19 days), urine volume was significantly decreased with A-779 administration. These studies using pretreatment with a lower dose of A-779 prior to pregnancy confirm results of higher dose A-779 administration after the start of pregnancy. These studies show that Ang-(1-7) produces antidiuresis in virgin rats and diuresis in late gestation. Ang-(1-7) also contributes to the enhanced water intake during pregnancy allowing maintenance of the normal volume expanded state despite diuresis.

Pregnancy is characterized by an increase in many of the different components of the circulating renin—angiotensin system (RAS). However, the physiological mechanisms of stimulated RAS activity during pregnancy are unknown. Even less understood is how this system may be altered in pre-eclampsia, a hypertensive disorder of pregnancy. Additional studies have shown the presence of a local tissue specific RAS in the uteroplacental unit of normal and pre-eclamptic pregnancies. Differences in normal pregnant and pre-eclamptic RAS component regulation may provide insight into the mechanisms responsible for the clinical pathological features of pre-eclampsia. Specifically, this review summarizes the key findings in the circulating and uteroplacental RAS in normal and pre-eclamptic pregnancies.

Sex differences in blood pressure are evident in experimental models and human subjects, yet the mechanisms underlying this disparity remain equivocal. The current study sought to define the extent of male-female differences in the circulating and tissue renin-angiotensin aldosterone systems (RAASs) of congenic mRen( 2 ).Lewis and control Lewis rats. Male congenics exhibited higher systolic blood pressure than females [200 ± 4 vs. 146 ± 7 mmHg, P < 0.01] or Lewis males and females [113 ± 2 vs. 112 ± 2 mmHg, P > 0.05]. Plasma ANG II levels were twofold higher in male congenics [47 ± 3 vs. 19 ± 3 pM, P < 0.01] and fivefold higher than in male or female Lewis rats [6 ± 1 vs. 6 ± 1 pM]. ANG I levels were also highest in the males; however, plasma ANG-(1-7) was higher in female congenics. Male congenics exhibited greater circulating renin and angiotensin-converting enzyme (ACE) activities, as well as angiotensinogen, than female littermates. Renal cortical and medullary ANG II levels were also higher in the male congenics versus all the other groups; ANG I was lower in the males. Cortical ACE2 activity was higher in male congenics, yet neprilysin activity and protein were greater in the females, which may contribute to reduced renal levels of ANG II. These data reveal that sex differences in both the circulating and renal RAAS are apparent primarily in the hypertensive group. The enhanced activity of the RAAS in male congenics may contribute to the higher pressure and tissue injury evident in the strain.

Clinical trials revealed that estrogen may result in cardiovascular risk in patients with coronary heart disease, despite earlier studies demonstrating that estrogen provided cardiovascular protection. It is possible that the preexisting condition of hypertension and the ability of estrogen to activate the renin-angiotensin system could confound its beneficial effects. Our hypothesis is that the attenuation of estrogen to agonist-induced vasoconstrictor responses through the activation of nitric oxide (NO) synthase (NOS) is impaired by hypertension. We investigated the effects of 17β-estradiol (E2) replacement in normotensive Sprague-Dawley (SD) and (mRen2)27 hypertensive transgenic (TG) rats on contractile responses to three vasoconstrictors, angiotensin II (ANG II), serotonin (5-HT), and phenylephrine (PE), and on the modulatory role of vascular NO to these responses. The aorta was isolated from ovariectomized SD and TG rats treated chronically with 5 mg E2 or placebo (P). The isometric tension of the aortic rings was measured in organ chambers, and endothelial NOS (eNOS) in the rat aorta was detected using Western blot analysis. E2 treatment increased eNOS expression in the SD and TG aorta and reduced ANG II- and 5-HT- but not PE-induced contractions in SD and TG rats. The inhibition of NOS with Nω-nitro-l-arginine methyl ester enhanced ANG II-, 5-HT-, and PE-induced contractions in P-treated and ANG II and PE responses in E2-treated SD and TG rats. Only the responses to 5-HT were augmented in hypertensive rats. In conclusion, this study shows that the preexisting condition of hypertension augmented the vascular responsiveness of 5-HT, whereas the attenuation of estrogen by ANG II and 5-HT vascular responses was not impaired by hypertension. The adrenergic agonist was unresponsive to estrogen treatment. The contribution of NO as a factor contributing to the relative refractoriness of the vascular responses is dependent on the nature of the vasoconstrictor and/or the presence of estrogen.

The chorionic villi in the placenta are responsible for the regulation of fetal oxygen and nutrient transport. Although the peripheral renin-angiotensin system is activated during normal pregnancy, the regulation of the local chorionic villi renin-angiotensin system remains unknown. Therefore, placental chorionic villous tissue was collected from nulliparous third-trimester normotensive or preeclamptic subjects and was analyzed for angiotensin peptide content, angiotensinogen, renin, angiotensin-converting enzyme (ACE), ACE2, neprilysin, angiotensin II type 1 (AT 1 ), angiotensin II type 2, Mas receptor mRNAs, and angiotensin receptor density and subtype. Angiotensin II in chorionic villi was significantly higher in preeclamptic subjects, whereas angiotensin (1-7) was not different. Angiotensinogen and AT 1 receptor gene expression was significantly higher in preeclamptic subjects. No differences were observed in renin, ACE, ACE2, or neprilysin gene expression. Mas receptor mRNA in preeclamptic subjects was decreased. The AT 1 receptor was the predominant receptor subtype in normal and preeclamptic chorionic villi. There was no difference in the density of the AT 1, angiotensin II type 2, and angiotensin (1-7) receptors. These results indicate that enhanced chorionic villous expression of angiotensin II may result from increased angiotensinogen. Elevated angiotensin II, acting through the AT 1 receptor, may favor vasoconstriction in placental chorionic villi and contribute to impaired fetal blood flow and decreased fetal nutrition observed during preeclampsia.

Interleukin-10 (IL-10) is a cytokine with pleiotropic properties. Limited biochemical and clinical evidence suggest a link between IL-10 and coronary heart disease (CHD). However, more data are needed to clarify the relationship between IL-10 and risk for CHD events. Methods The present study was a secondary analysis of the estrogen replacement and atherosclerosis (ERA) trial, a randomized clinical trial that examined the effects of hormone replacement therapy on post-menopausal women with known coronary atherosclerosis. IL-10 concentration, measured at baseline, was treated as both a continuous and categorical variable. Cox proportional hazards models were used to compute hazard ratios as estimates of relative risk for CHD events. Results There were 71 events over an average 3.2 year follow-up. Incident rates were higher for individuals with IL-10 concentrations equal to or greater than the median level (1.04 pg/mL) compared to those individuals below the median level (30% versus 18.5%, p = 0.02). The cumulative incidence of CHD events was significantly greater in individuals with IL-10 concentrations ≥1.04 pg/mL ( p = 0.01). A one standard deviation increase in baseline IL-10 concentration was associated with a 34% greater risk of a CHD event (HR 1.34 [1.06 − 1.68], p = 0.01). This elevated risk was not altered by interleukin-6, C-reactive protein, or additional cardiovascular risk factors. IL-10 concentration and risk for CHD events was most pronounced in diabetics (HR 2.4 [1.46 − 3.83], p = 0.0005). Conclusion In the ERA trial, elevated IL-10 concentration was associated with an increased risk for future cardiovascular events in post-menopausal women with established coronary atherosclerosis. Further study of the relationship between IL-10 and the pathogenesis and progression of atherosclerosis and cardiovascular events is warranted.

The present study was designed to determine ANG peptide content [ANG I, ANG II, ANG-(1-7)], ACE2 mRNA, and the immunocytochemical distribution of ANG-(1-7) and ACE2 in the uteroembryonic unit during early and late gestation in Sprague-Dawley rats and in a rat model of pregnancy-induced hypertension, the reduced uterine perfusion pressure (RUPP) model. At early pregnancy ANG-(1-7) and ACE2 staining were localized in the primary and secondary decidual zone and luminal and glandular epithelial cells. During late gestation, ANG-(1-7) and ACE2 staining was visualized in the labyrinth placenta and amniotic and yolk sac epithelium. Uterine ANG II concentration at early pregnancy was significantly decreased by 21–55% in the implantation and interimplantation sites compared with virgin rats, whereas ANG-(1-7) levels were maintained at prepregnancy levels. At late gestation, uterine concentrations of ANG I and ANG II were significantly increased (30% and 25%, respectively). In RUPP animals, ANG-(1-7) concentration is significantly reduced in the uterus (181 ± 16 vs. 372 ± 74 fmol/g of tissue) and placenta (143 ± 26 vs. 197 ± 20 fmol/g of tissue). ACE2 mRNA increased in the uterus of early pregnant compared with virgin rats, yet within the implantation site it was downregulated. At late pregnancy, ACE2 mRNA is elevated by 58% in the uterus and decreased by 59% in RUPP animals. The regulation of ANG-(1-7) and ACE2 in early and late pregnancy supports the hypothesis that ANG-(1-7) and ACE2 may act as a local autocrine/paracrine regulator throughout pregnancy, participating in the early (angiogenesis, apoptosis, and growth) and late (uteroplacental blood flow) events of pregnancy.

Experimental studies point to an interplay between hypercholesterolemia and hypertension, acting through the renin angiotensin system. In a crossover study design with 8 healthy subjects, the authors tested the hypothesis that statin treatment exerts renin angiotensin system-modulating effects in veins by down-regulation of AT1-receptors, resulting in reduced Angiotensin II (Ang II)-induced venoconstriction and by increasing the pleiotropic Ang II-metabolite Ang-(1-7). Irbesartan was used as positive control. Ang II-induced venoconstriction was 49% +/- 9% before and 64% +/- 10% after 30 days of atorvastatin treatment compared to 50% +/- 8% before and 15% +/- 9% after irbesartan (P = .004). Plasma angiotensin levels significantly increased only after irbesartan treatment (Ang II: 35 +/- 4 vs 329 +/- 101 pg/mL [P = .02]; Ang-(1-7): 10 +/- 3 vs 35 +/- 6 pg/mL [P = .01]) compared to atorvastatin treatment (Ang II: 26 +/- 5 vs 31 +/- 4 pg/mL [P = ns]; Ang-(1-7): 9 +/- 2 vs 11 +/- 3 pg/mL [P = ns]). The data indicate that atorvastatin does not inhibit Ang II-induced venoconstriction in vivo and point toward a supportive role of Ang-(1-7) in contributing to the antihypertensive and beneficial vascular effects of irbesartan.

Preeclampsia is associated with maternal perinatal morbidity and mortality and a high risk of premature birth and intrauterine growth restriction. The apelin system is a novel pleiotropic pathway with a potential for therapeutic targeting in preeclampsia. We have previously reported that total apelin content is lower in human preeclamptic chorionic villi. In this study, we determined whether (Pyr1)-apelin-13 improves hypertension, proteinuria, fetal characteristics, uteroplacental hemodynamics, and the autonomic function in preeclamptic rats (TgA, female transgenic for human angiotensinogen mated to male transgenic for human renin). (Pyr1)-apelin-13 (2 mg/kg/day) (n=7) or saline (n=5) was infused in TgA via osmotic minipumps starting at day 13 of gestation, when blood pressure begins to increase in these animals. Pregnant SD (n=6) rats were used as controls. At the 20th day of pregnancy, TgA rats had higher MAP (138±6 vs. 79±3 mmHg in SD, p

The role of the endogenous apelin system in pregnancy is not well understood. Apelin's actions in pregnancy are further complicated by the expression of multiple forms of the peptide. Using radioimmunoassay (RIA) alone, we established the expression of apelin content in the chorionic villi of preeclamptic (PRE) and normal pregnant women (NORM) at 36–38 wk of gestation. Total apelin content was lower in PRE compared with NORM chorionic villi (49.7 ± 3.4 vs. 72.3 ± 9.8 fmol/mg protein; n = 20–22) and was associated with a trend for lower preproapelin mRNA in the PRE. Further characterization of apelin isoforms by HPLC-RIA was conducted in pooled samples from each group. The expression patterns of apelin peptides in NORM and PRE villi revealed little or no apelin-36 or apelin-17. Pyroglutamate apelin-13 [(Pyr1)-apelin-13] was the predominant form of the peptide in NORM and PRE villi. Angiotensin-converting enzyme 2 (ACE2) activity was higher in PRE villi (572.0 ± 23.0 vs. 485.3 ± 24.8 pmol·mg−1·min−1; n = 18–22). A low dose of ANG II (1 nM; 2 h) decreased apelin release in NORM villous explants that was blocked by the ANG II receptor 1 (AT1) antagonist losartan. Moreover, losartan enhanced apelin release above the 2-h baseline levels in both NORM and PRE villi ( P < 0.05). In summary, these studies are the first to demonstrate the lower apelin content in human placental chorionic villi of PRE subjects using quantitative RIA. (Pyr1)-apelin-13 is the predominant form of endogenous apelin in the chorionic villi of NORM and PRE. The potential mechanism of lower apelin expression in the PRE villi may involve a negative regulation of apelin by ANG II.

Background/Objective: Angiotensinogen is the substrate for renin, which then converts angiotensinogen to angiotensin I. Sex differences in multiple components of the renin angiotensin system (RAS) have been described. We examined sex differences in angiotensinogen (Aogen) and renin activity in middle aged men and women at risk for stroke, accounting for sex hormone binding globulin and use of RAS-influencing drugs. Methods: The Sex Age and Variation in Vascular functionalitY (SAVVY) study measured sex differences in subclinical vascular disease and biomarkers in men and women aged 45 to 64 with at least one stroke risk factor, but free of cardiovascular events using a cross-sectional design. Women were oversampled to assess pre- and post-menopausal time points. Aogen, renin activity, and sex hormone binding globulin (SHBG) were performed using commercial ELISAs/RIAs. We compared biomarker levels by sex using parametric statistical tests, and multiple linear regression models were developed to determine if sex, SHBG, and other RAS-related factors were associated with Aogen level and renin activity. Results: In total, 113 subjects (78 women, 35 men) were included. Men (mean 55 y; std 5.3 y) and women (56.6 y; std 5.8 y) were similar in age; Black race (9% in men and 14% in women) and risk factors: hypertension (men 63% vs. women 58%), diabetes (20% vs 18%), hyperlipidemia (80% vs. 76%), and smoking (6% vs. 10%). Systolic and diastolic blood pressures were similar. Women had significantly higher levels of Aogen (mean 35,123 ng/ml; std 12,557) than men (mean 26,601 ng/ml; std 4578; p Conclusions: Aogen, but not renin, is influenced by female sex after accounting for SHBG, hypertension, and use of RAS-influencing drugs. This suggests that potentially different mechanisms may contribute to the identified risk factors for stroke in men and women.

Summary Elevated C-reactive protein (CRP) levels are associated with both prevalent and incident cardiovascular disease. In this study, familial aggregation was estimated, and we tested for association between serum CRP levels and polymorphisms within the CRP and APOE genes in sib-ships with type 2 diabetes mellitus, a population at increased risk for cardiovascular disease. CRP levels were determined in 461 diabetes-affected subjects from 224 sibships from the Diabetes Heart Study (DHS). Heritability estimates of CRP levels were obtained using variance component models. Genetic influence on serum CRP levels by single nucleotide polymorphisms (SNPs) in the CRP and APOE genes was evaluated by association analysis using mixed models. Subjects were Caucasian American (84%) and African-American (16%), 53% female, and had an average age of 62.2 ± 9.2 years. The median CRP level was 3.3 mg/L (range 0 to 59.3 mg/L), and estimated heritability for CRP was approximately 40%. Estimates of heritability were significantly greater than zero (P < 0.0001) and relatively constant, despite adjustments for important modifiers (age, sex, ethnicity, diabetes duration, statin-use and anti-inflammatory use) of CRP. There was no significant evidence for association of CRP levels with CRP gene SNPs; however, consistent with previous reports, there was significant evidence of association of CRP levels with polymorphisms within the APOE gene. These data indicate CRP levels are significantly influenced by genetic (and/or environmental) factors that are shared within DHS families. While the APOE locus shows evidence of contributing to CRP levels, no evidence of CRP gene polymorphism association with CRP levels was observed.

The VIOS (Vascular Improvement with Olmesartan medoxomil Study) study is a randomized, parallel study to determine the relative effects of suppressing the renin-angiotensin system (RAS) with the angiotensin receptor antagonist olmesartan medoxomil versus suppressing sympathetic drive with the beta-adrenoceptor antagonist atenolol on remodeling of the subcutaneous small resistance vessel. Remodeling of small resistance vessels may be the earliest pathologic finding associated with hypertension. It may predate the onset of clinically apparent hypertension.In this study, 100 patients with stage I hypertension are characterized at baseline before being treated for 1 year to obtain a goal BP of less than 140/90 mm Hg as defined by Joint National Committee (JNC)-7. Resistance vessel remodeling is determined using the gluteal fat biopsy technique in the hypertensive patients and a group of normotensive healthy volunteers. Additionally, efforts will be made to define whether noninvasive hemodynamic parameters, retinal vessel measurement changes, or biologic markers may predict and track the underlying vascular morphologic and physiologic changes induced by either regimen during the 12-month treatment period.The primary endpoint will be the degree of vascular remodeling as obtained from percutaneous biopsy of gluteal subcutaneous resistance vessels in each of two treatment arms compared with the normal volunteers. The design of the study and the pertinent baseline characteristics of these patients with uncomplicated essential hypertension are presented.The suppression of the RAS by the blockade of angiotensin II type 1 (AT(1)) receptors may demonstrate remodeling effects on the ubiquitous small resistance vessels similar to that seen in the myocardium and renal glomeruli, thus affording more complete end-organ protection.

The pregnant female human angiotensinogen (hAGN) transgenic rat mated with the male human renin (hREN) transgenic rat is a model of preeclampsia (TgA) with increased blood pressure, proteinuria, and placenta alterations of edema and necrosis at late gestation. We studied vascular responses and the role of COX-derived prostanoids in the uterine artery (UA) at early gestation in this model. TgA UA showed lower stretch response, similar smooth muscle α-actin content, and lower collagen content compared with Sprague-Dawley (SD) UA. Vasodilation to acetylcholine was similar in SD and TgA UA (64 ± 8 vs. 75 ± 6% of relaxation, P > 0.05), with an acetylcholine-induced contraction in TgA UA that was abolished by preincubation with indomethacin (78 ± 6 vs. 83 ± 11%, P > 0.05). No differences in the contraction to phenylephrine were observed (159 ± 11 vs. 134 ± 12 %KMAX, P > 0.05), although in TgA UA this response was greatly affected by preincubation with indomethacin (179 ± 16 vs. 134 ± 9 %KMAX, P < 0.05, pD25.92 ± 0.08 vs. 5.85 ± 0.03, P < 0.05). Endothelium-independent vasodilation was lower in TgA UA (92 ± 2 vs. 74 ± 5% preconstricted tone, P < 0.05), and preincubation with indomethacin restored the response to normal values (90 ± 3 vs. 84 ± 3%). Immunostaining showed similar signals for α-actin, COX-2, and eNOS between groups ( P > 0.05). Plasma thromboxane levels were similar between groups. In summary, TgA UA displays functional alterations at early gestation before the preeclamptic phenotype is established. Inhibition of COX enzymes normalizes some of the functional defects in the TgA UA. An increased role for COX-derived prostanoids in this model of preeclampsia may contribute to the development of a hypertensive pregnancy.

Effects of renin-angiotensin system blockade on renal angiotensin-(1-7) forming enzymes and receptors. Background Angiotensin-converting enzyme (ACE)2, a homologue of ACE, which is insensitive to ACE inhibitors and forms angiotensin-(1-7) [Ang-(1-7)] from angiotensin II (Ang II) with high efficiency was investigated in response to chronic blockade with lisinopril, losartan, and both drugs combined. Methods Thirty-six adult Lewis rats were assigned to receive these medications in their drinking water for 2 weeks while their arterial pressure, water intake, and urine volume were recorded throughout the study. Measures of renal excretory variables included assessing excretion rates of angiotensin I (Ang I), Ang II and Ang-(1-7) while blood collected at the completion of the study was used for measures of plasma angiotensin concentrations. Samples from renal cortex were assayed for renin, angiotensinogen (Aogen), neprilysin, angiotensin types 1 and 2 (AT 1 and AT 2 ) and mas receptor mRNAs by semiquantitative reverse transcriptase (RT) real-time polymerase chain reaction (PCR). ACE2 activity was determined as the rate of Ang II conversion into Ang-(1-7). Results Comparable blood pressure reductions were obtained in rats medicated with either lisinopril or losartan, whereas both drugs produced a greater decrease in arterial pressure. Polyuria was recorded in all three forms of treatment associated with reduced osmolality but no changes in creatinine excretion. Lisinopril augmented plasma levels and urinary excretion rates of Ang I and Ang-(1-7), while plasma Ang II was reduced with no effect on urinary Ang II. Losartan produced similar changes in plasma and urinary Ang-(1-7) but increased plasma Ang II without changing urinary Ang II excretion. Combination therapy mimicked the effects obtained with lisinopril on plasma and urinary Ang I and Ang-(1-7) levels. Renal cortex Aogen mRNA increased in rats medicated with either lisinopril or the combination, whereas all three treatments produced a robust increase in renal renin mRNA. In contrast, ACE, ACE2, neprilysin, AT 1 , and mas receptor mRNAs remained unchanged with all three treatments. Renal cortex ACE2 activity was significantly augmented in rats medicated with lisinopril or losartan but not changed in those given the combination. Conclusion Our data revealed a role for ACE2 in Ang-(1-7) formation from Ang II in the kidney of normotensive rats as primarily reflected by the increased ACE2 activity measured in renal membranes from the kidney of rats given either lisinopril or losartan. The data further indicate that increased levels of Ang-(1-7) in the urine of animals after ACE inhibition or AT 1 receptor blockade reflect an intrarenal formation of the heptapeptide.

We investigated whether prevention of cardiac and vascular remodeling associated with inhibition of angiotensin II is independent of the blood pressure (BP)-lowering action of angiotensin II type 1 (AT1) receptor blockade. Spontaneously hypertensive rats, 8 weeks old, were treated with olmesartan, atenolol, or vehicle in their drinking water for 56 days. At the end of each treatment, arterial pressure and heart rate were measured, the ratio of heart weight to body weight was calculated, collagen deposition in the heart was determined histochemically using picrosirius red staining, and wall-to-lumen ratio in isolated mesenteric arteries was measured by a videographic approach. At 3 weeks after the initiation of treatment, rats medicated with olmesartan showed lower values of systolic BP compared with rats given atenolol or vehicle, whereas no difference in directly measured BP were observed at the end of study in anesthetized rats given olmesartan or atenolol. Rats given atenolol showed sustained bradycardia, whereas cardiac hypertrophy and collagen deposition was prevented only in spontaneously hypertensive rats given olmesartan. Olmesartan or atenolol reduced arteriolar wall-to-lumen ratio (olmesartan: 11.5+/-0.4%; atenolol: 13.3+/-0.6%; vehicle: 18.4%+/-1.1); however, this effect was greatest in rats medicated with the angiotensin II type 1 antagonist. Although control of BP is a factor in the prevention of cardiac and vascular hypertrophy, our studies suggest that blockade of angiotensin II receptors may attenuate the structural changes in the heart and blood vessels of hypertensive animals independent of a reduction in BP.

OBJECTIVE The aim of this study was to evaluate the effect of conjugated equine oestrogen (CEE), and droloxifene, a selective oestrogen receptor modulator (SERM) on individual components of renin-angiotensin-aldosterone (RAAS) and blood pressure (BP) in postmenopausal women. DESIGN AND PATIENTS Twenty-one normotensive (NT) and 10 hypertensive (HT) postmenopausal women received either CEE (0.625 mg/day) or droloxifene (60 mg/day) for 6 weeks and, after a 4-week washout, were restudied on the alternate medication. MEASUREMENTS Hormones of the RAAS and supine BP were measured prior to and at the end of each drug treatment in all subjects. In a subgroup of the NT (n = 10), 24 h ambulatory BP was performed and renal blood flow was determined by PAH clearance both basally and in response to 45-min angiotensin II (Ang II) infusion (3 ng/kg/min). RESULTS CEE significantly increased angiotensinogen, decreased active renin and angiotensin-converting enzyme (ACE), and maintained plasma immunoreactive (ir) angiotensin I (Ang I) levels compared to baseline. With droloxifene, angiotensinogen, active renin and Ang I remained unchanged. Both CEE and droloxifene significantly increased plasma ir-Ang II levels (pmol/l) in NT (baseline: 25.7 +/- 2.5, CEE: 36.6 +/- 3.4, droloxifene: 33.3 +/- 3.1, P < 0.002) and HT women (baseline: 17.9 +/- 2.3, CEE: 27.9 +/- 3.2, droloxifene: 25.9 +/- 4.9, P < 0.005). Renal blood flow was lower on CEE (P = 0.02) compared with baseline. Systemic BP (supine and 24-h ambulatory) was unchanged during both treatments. CONCLUSION This study demonstrates higher circulating levels of ir-Ang II with CEE and droloxifene.

Mesenteric arteries (230-290 microm) were isolated from virgin female rats at diestrous and proestrous phases of the estrous cycle and from ovariectomized (OVX) rats with or without estrogen (E2) replacement (17beta-estradiol, 7.5 + 5 mg pellets, 21 d release). Arteries were mounted in a pressurized myograph system. Angiotensin-(1-7) [Ang-(1-7)] concentration-dependent responses (10(-10)-10(-5) M) were determined in arteries preconstricted with endothelin-1 (10(-7) M). Mesenteric arteries were pretreated with the specific Ang-(1-7) antagonist, D-[Ala7]-Ang-(1-7) (10(-7) M) to assess the Ang-(1-7) receptor-mediated dilator effect. Ang-(1-7) did not dilate mesenteric arteries from virgin rats at diestrus and placebo-treated OVX female rats as compared to the time control; however, Ang-(1-7) elicited a modest dilation at proestrus as compared to diestrus, which reached statistical significance at 10(-8) M concentrations. Ang-(1-7) caused a concentration-dependent vasodilation in mesenteric arteries of females with E2 replacement, with an EC50 of 21 nM. D-[Ala7]-Ang-(1-7) blocked the vasodilator effect of Ang-(1-7). Our results demonstrate that during proestrus Ang-(1-7) elicits modest vasodilation as compared to diestrus, but lacks vasodilatory properties in vessels from diestrous and ovariectomized rats. Estrogen replacement restores a significant dilator response to Ang-(1-7) in OVX rats that is mediated by a D-[Ala7]-Ang-(1-7) sensitive site.

Angiotensin-(1-7) (Ang-[1-7]) is a bioactive component of the renin-angiotensin system, which has depressor, vasodilatory, and antihypertensive actions. In normal pregnancy, we questioned whether the known rise in plasma angiotensin II (Ang II) is counterbalanced by an increase in plasma Ang-(1-7) and whether Ang-(1-7) levels are decreased in preeclampsia and may thus be a factor involved in the development of hypertension. Nulliparous preeclamptic subjects, third-trimester normotensive pregnant subjects, and a nonpregnant group were enrolled (n = 15/group). Preeclamptic subjects had no previous history of hypertension or renal, connective-tissue, or metabolic disease, but at the time of delivery had significant hypertension (159 +/- 3/98 +/- 3 mmHg) andor = 3+ proteinuria. Plasma Ang-(1-7) was increased by 51% in normal pregnancy (p0.05). Plasma Ang I, Ang II, and renin activity were also significantly elevated in normal pregnancy. In preeclamptic subjects, Ang-(1-7) was significantly decreased (p0.01) compared with normal pregnant subjects. All other components of the renin-angiotensin-aldosterone system, except serum angiotensin-converting enzyme, were reduced in preeclamptic subjects compared with normal pregnant subjects; only plasma Ang II remained elevated in preeclamptic compared with nonpregnant subjects. These studies demonstrate, for the first time, increased plasma Ang-(1-7) in normal pregnant subjects compared with nonpregnant subjects and decreased Ang-(1-7) in preeclamptic subjects compared with normal pregnant subjects. In preeclampsia the decreased plasma Ang-(1-7) in the presence of elevated Ang II is consistent with the development of hypertension.

We previously demonstrated the substantial capacity for angiotensin (ANG) II formation in the interstitium of the dog heart in vivo. The current study tested the hypothesis that interstitial fluid (ISF) bradykinin (BK) is influenced by ANG II formation. Four microdialysis probes were inserted into the left ventricular myocardium of eight open-chest anesthetized dogs. The probe effluent was collected during four stages in each dog. Probes 1 and 3 sequentially delivered: 1) buffer; 2) ANG I (15 microM); 3) ANG II type 1 receptor antagonist (AT(1)-ant; irbesartan, 50 microM) or AT(2)-ant (PD123319, 50 microM); and 4) ANG I + AT(1)-ant or ANG I + AT(2)-ant. Probes 2 and 4 used the same protocol, substituting ANG II for ANG I in a concentration (0.5 microM) equivalent to that achieved during ANG I infusion. ISF BK levels increased 15-fold during ANG I (p < 0.001) but not during ANG II infusion. Co-infusion of selective AT(1)- and AT(2)-ants or nonselective AT-ant did not block the increase in ISF BK. ISF infusions of ANG I also produced a greater than 400-fold rise in ISF ANG-(1-7) over baseline. ISF infusion of ANG-(1-7) (10 microM) produced a 15-fold increase in ISF BK (p < 0.001). The metabolic machinery exists for the formation of BK and ANG-(1-7) in the cardiac ISF space that is not blocked by an AT receptor antagonist. The differential increase in ISF BK during ANG I and ANG-(1-7) but not during ANG II infusions suggests the possibility of decreased catabolism of ISF BK by an angiotensin-converting enzyme due to active site occupation by ANG I and ANG-(1-7).