Comprehensive Echocardiographic Assessment of Right Ventricle Function in a Rat Model of Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease caused by vasoconstriction and remodeling of the small arteries in the lungs. This remodeling leads to increased pulmonary vascular resistance, worsened right ventricular function, and premature death. Currently approved therapies for PAH largely target pulmonary vasodilator pathways; however, recent emerging therapeutic modalities are focused on other novel pathways involved in the pathogenesis of the disease, including right ventricle (RV) remodeling. Imaging techniques that allow longitudinal assessment of novel therapeutics are very useful for determining the efficacy of new drugs in preclinical studies. Noninvasive trans-thoracic echocardiography remains the standard approach to evaluating heart function and is widely used in rodent models. However, echocardiographic evaluation of the RV can be challenging due to its anatomical position and structure. In addition, standardized guidelines are lacking for echocardiography in preclinical rodent models, making it difficult to carry out a uniform assessment of RV function across studies in different laboratories. In preclinical studies, the monocrotaline (MCT) injury model in rats is widely used to evaluate drug efficacy for treating PAH. This protocol describes the echocardiographic evaluation of the RV in naïve and MCT-induced PAH rats.

Local intra-uterine Ang-(1-7) infusion attenuates PGE2 and 6-keto PGF1α in decidualized uterus of pseudopregnant rats.

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 < 0.001, non-decidualized vs. decidualized horns); Ang-(1-7) infusion attenuated the increase of PGE2 (12.0 ± 3.2 vs. 5.1 ± 1.3 pmol/mg protein, p < 0.01 control vs. Ang-(1-7) treated decidualized horns). The stable metabolite of PGI2 (6-keto PGF1α) was increased with decidualization (0.79 ± 0.17 vs. 3.5 ± 0.82 pmol/mg protein, p < 0.001, non-decidualized vs. decidualized horns). Ang-(1-7) infusion attenuated the increase in 6-keto PGF1α in the decidualized horn (3.5 ± 0.82 vs 1.8 ± 0.37 pmol/mg protein, p < 0.05 control vs. Ang-(1-7) treated decidualized horns). The circulating levels of 6-keto-PGF1a and TXB2 were decreased by Ang-(1-7) infusion, while no difference was observed in circulating PGE2. Although the global assessment of cleaved caspase 3 immunostaining, a marker of apoptosis, was unchanged within the Ang-(1-7) decidualized horn, there were localized decreases in cleaved caspase 3 staining in the luminal region in the decidualized uterus of Ang-(1-7)-treated rats. CONCLUSIONS: These studies show that increased local uterine Ang-(1-7) alters the uterine prostaglandin environment, possibly leading to disruptions of early events of decidualization.

Local uterine Ang-(1-7) infusion augments the expression of cannabinoid receptors and differentially alters endocannabinoid metabolizing enzymes in the decidualized uterus of pseudopregnant rats.

BACKGROUND: Endocannabinoids (ECs) are important contributors to implantation and decidualization and are suppressed in early pregnancy. Elevated levels of anandamide (AEA), the endogenous ligand for the CB1 and CB2 receptors (R), interfere with receptivity of the blastocyst. Ang-(1-7) is down-regulated in the implantation site (IS) in normal pregnancy at day 7 of gestation. We determined the effects of intra-uterine angiotensin-(1-7) [Ang-(1-7)] (24 microg/kg/h) or vehicle given into the left uterine horn on the ECs in the decidualized uterus. METHODS: Ovariectomized rats were sensitized for the decidual cell reaction by steroid treatment and decidualization was induced by a bolus of oil injected into the left horn; the right horn served as a control. RESULTS: Decidualization increased endometrial permeability (3.1+/-0.2 vs. 7.1+/-0.5 uterus/muscle of cpm of (125)I-BSA, p < 0.0001). VEGF mRNA was increased by the decidualization (1.4-fold, p < 0.05) and by Ang-(1-7) (2.0-fold, p < 0.001). CB1R mRNA was reduced by decidualization (2.7-fold, p < 0.001), but increased by Ang-(1-7) (1.9-fold, p < 0.05). CB2R mRNA was increased by decidualization (4-fold, p < 0.05) and by Ang-(1-7) (2.4-fold, p < 0.001). The enzyme metabolizing AEA, fatty acid amide hydrolase (FAAH), was reduced by decidualization (7.8 fold, p < 0.001) and unchanged by Ang-(1-7) (p > 0.05), whereas the enzyme metabolizing 2-arachidonoylglycerol, monoacyl glycerol lipase (MAGL), was unchanged by decidualization (p > 0.05) and increased by Ang-(1-7) (1.7 fold, p < 0.001). CONCLUSIONS: These findings report for the first time that Ang-(1-7) augments the expression of CB1R, CB2R and MAGL in the decidualized uterus and thus may interfere with the early events of decidualization.

C-122, a novel antagonist of serotonin receptor 5-HT2B, prevents monocrotaline-induced pulmonary arterial hypertension in rats.

Pulmonary arterial hypertension (PAH) is a chronic disease characterized by sustained elevation of pulmonary arterial pressure that leads to right ventricle failure and death. Pulmonary resistance arterioles in PAH undergo progressive narrowing and/or occlusion. Currently approved therapies for PAH are directed primarily at relief of symptoms by interfering with vasoconstrictive signals, but do not halt the microvascular cytoproliferative process. In this study we show that C-122 (2-amino-N-(2-{4-[3-(2-trifluoromethyl-phenothiazin-10-yl)-propyl]-piperazin-1-yl}-ethyl)-acetamide trihydrochloride, a novel antagonist of serotonin receptor 5-HT(2B) (Ki=5.2 nM, IC(50)=6.9 nM), when administered to rats for three weeks in daily oral 10mg/kg doses, prevents not only monocrotaline (MCT)-induced elevations in pressure in the pulmonary arterial circuit (19 ± 0.9 mmHg vs. 28 ± 2 mmHg in MCT-vehicle group, P<0.05) and hypertrophy of the right ventricle (right ventricular wt./body wt. ratio 0.52 ± 0.02 vs. 0.64 ± 0.04 in MCT-vehicle group, P<0.05), but also muscularization of pulmonary arterioles (23% vs. 56% fully muscularized in MCT-vehicle group, P<0.05), and perivascular fibrosis in the lung. C-122 is orally absorbed in the rat, and partitions strongly into multiple tissues, including heart and lung. C-122 has significant off-target antagonist activity for histamine H-1 and several dopamine receptors, but shows no evidence of crossing the blood-brain barrier after a single 10mg/kg oral dose in rats. We conclude that C-122 can prevent microvascular remodeling and associated elevated pressures in the rat MCT model for PAH, and offers promise as a new therapeutic entity to suppress vascular smooth muscle cell proliferation in PAH patients.

Angiotensin II and angiotensin-(1-7) decrease sFlt1 release in normal but not preeclamptic chorionic villi: an in vitro study.

BACKGROUND: During preeclampsia, placental angiogenesis is impaired. Factors released from the placenta including vascular endothelial growth factor (VEGF), placental growth factor (PLGF), soluble VEGF receptor 1 (sFlt1), and soluble endoglin (sEng) are regulatory molecules of placental development and function. While the renin angiotensin system has been shown to regulate angiogenic factors in other research fields, these mechanisms have not been extensively studied during pregnancy. METHODS: We evaluated the effects of angiotensin II (Ang II) and angiotensin-(1-7) [Ang-(1-7)] on the release of VEGF, PLGF, sFlt1, and sEng from placental chorionic villi (CV). CV were collected from nulliparous third-trimester normotensive and preeclamptic subjects. CV were incubated for 0, 2, 4, and 16 hours with or without Ang II (1 nM and 1 microM) or Ang-(1-7) (1 nM and 1 microM). The release of VEGF, PLGF, sFlt1, sEng, lactate dehydrogenase (LDH), and human placenta lactogen (HPL) was measured by ELISA. RESULTS: The release of sFlt1, PLGF, sEng from normal and preeclamptic CV increased over time. Release of sFlt1 and sEng was significantly higher from preeclamptic CV. VEGF was below the detectable level of the assay in normal and preeclamptic CV. After 2 hours, sFlt1 release from normal CV was significantly inhibited with Ang II (1 nM and 1 microM) and Ang-(1-7) (1 nM and 1 microM). There was a time-dependent increase in HPL indicating that the CV were functioning normally. CONCLUSIONS: Our study demonstrates a critical inhibitory role of angiotensin peptides on sFlt1 in normal pregnancy. Loss of this regulation in preeclampsia may allow sFlt1 to increase resulting in anti-angiogenesis and end organ damage in the mother.

The uterine placental bed Renin-Angiotensin system in normal and preeclamptic pregnancy.

Previously, we demonstrated activation of the renin-angiotensin system in the fetal placental chorionic villi, but it is unknown whether the immediately adjacent area of the maternal uterine placental bed is regulated similarly. This study measured angiotensin peptides, renin-angiotensin system component mRNAs, and receptor binding in the fundus from nonpregnant subjects (n = 19) and in the uterine placental bed from normal (n = 20) and preeclamptic (n = 14) subjects. In the uterine placental bed from normal pregnant women, angiotensin II peptide levels and angiotensinogen, angiotensin-converting enzyme, angiotensin receptor type 1 (AT(1)), AT(2), and Mas mRNA expression were lower as compared with the nonpregnant subjects. In preeclamptic uterine placental bed, angiotensin II peptide levels and renin and angiotensin-converting enzyme mRNA expression were significantly higher than normal pregnant subjects. The AT(2) receptor was the predominant receptor subtype in the nonpregnant fundus, whereas all angiotensin receptor binding was undetectable in normal and preeclamptic pregnant uterine placental bed compared with nonpregnant fundus. These findings suggest that the maternal uterine placental bed may play an endocrine role by producing angiotensin II, which acts in the adjacent placenta to vasoconstrict fetal chorionic villi vessels where we have shown previously that AT(1) receptors predominate. This would lead to decreased maternal-fetal oxygen exchange and fetal nutrition, a known characteristic of preeclampsia.

Vascular perfused segments of human intestine as a tool for drug absorption.

Blood-based vascular perfusion of isolated segments of human jejunum was developed as a tool for drug absorption studies before clinical trials. Acceptance criteria for viable human gut preparations included stable blood flow, arterial pressure, glucose utilization, active peristalsis, oxygen uptake, less than 3% absorption of a 70,000 mol. wt. dextran, and a ratio of first-order absorption rate constants (k(a)) of antipyrine to terbutaline of > or =1.4. Mannitol absorption was less than that of antipyrine but larger than that of terbutaline and could not be used as a negative control in absorption studies with human intestine. In separate perfusions (n = 3) a cassette of nine drugs was administered into the gut lumen, and the net absorption of each drug into the circulation was measured over 75 min. Using the mean values of k(a), the test compounds could be ranked into four groups: group 1: sulfasalazine and furosemide, k(a) = 3.9 to 4.0 x 10(-3) min(-1); group 2: cimetidine, timolol, nadolol, and ranitidine, k(a) = 6.4 to 8.3 x 10(-3) min(-1); group 3: atenolol and metoprolol, k(a) = 9.6 x 10(-3) min(-1); and group 4: theophylline, k(a) = 17.5 x 10(-3) min(-1). The rationale for evaluating yet another oral absorption system was as follows: first, a human gut segment with an intact vascular system is the closest system available to a clinical trial without performing one; and second, the data generated would be a direct measure of net drug transport from the gut lumen into the vascular circulation under near physiological conditions, which is not possible in models lacking a blood supply.

Activation of local chorionic villi angiotensin II levels but not angiotensin (1-7) in preeclampsia.

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.

ACE2 and ANG-(1-7) in the rat uterus during early and late gestation.

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.

Angiotensin-(1-7) inhibits in vitro endothelial cell tube formation in human umbilical vein endothelial cells through the AT(1-7) receptor.

Angiotensin-(1-7) is increased in the circulation during human pregnancy, but its functional role is unknown. Recent studies suggested that it opposes angiotensin II mediated vascular growth. Because angiogenesis is critical to normal embryonic development during human pregnancy, this study assessed the in vitro effects of angiotensin-(1-7) on human umbilical vein endothelial cell tube formation. The blocking effects of the angiotensin-(1-7) receptor antagonist, D-[Alanine7]-Ang-(1-7), and angiotensin II receptor AT1 and AT2 antagonists, losartan and PD123319, on tube formation were measured by counting tube branch points. Human umbilical vein endothelial cells were cultured in EGM-2 medium and treated with angiotensin-(1-7) (0.17 nM-17 microM) for 18 h. Angiotensin-(1-7) inhibited tube formation by 24% (P < 0.01) at all doses tested. Treatment with 1.7 microM angiotensin-(1-7) plus 17 microM D-[Alanine7]-Ang-(1-7) resulted in the reversal of angiotensin-(1-7) mediated inhibition of tube formation (P < 0.05). Losartan (17 microM) also reversed the angiotensin-(1-7) mediated inhibition of tube formation (P < 0.05). Tube formation was unaffected by PD123319. These results suggest that angiotensin-(1-7) has an anti-angiogenic effect on human umbilical vein endothelial cells through a unique AT(1-7) receptor that is sensitive to losartan, indicating that angiotensin-(1-7) may play an important role in the regulation of vascular growth in the placenta during pregnancy.

Vascular responses to Angiotensin-(1-7) during the estrous cycle.

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.

Pregnancy enhances the angiotensin (Ang)-(1-7) vasodilator response in mesenteric arteries and increases the renal concentration and urinary excretion of Ang-(1-7).

The vasoactive effect of angiotensin (Ang)-(1-7) in mesenteric resistance arteries together with its plasma and kidney concentration and urinary excretion was assessed in pregnant and virgin rats. Mesenteric arteries (230-290 microm) were mounted in a pressurized myograph system and Ang-(1-7) concentration-dependent response curves (10(-10)-10(-5) M) were determined in arteries preconstricted with endothelin-1 (10(-7) M). The Ang-(1-7) response was investigated in vessels with and without pretreatment with the Ang-(1-7) antagonist [D-[Ala(7)]-Ang-(1-7)] (10(-7) M). Ang-(1-7) caused a significantly enhanced, concentration-dependent dilation of mesenteric vessels (EC(50) = 2.7 nM) from pregnant compared with virgin female rats. D-[Ala(7)]-Ang-(1-7) eliminated the vasodilator effect of Ang-(1-7). There was no significant change in plasma concentration of Ang-(1-7) in pregnant animals. On the other hand, 24 h urinary excretion and kidney concentration of Ang-(1-7) were significantly higher in pregnant animals. The increased mesenteric dilation to Ang-(1-7) with enhanced kidney concentration and 24 h urinary excretion rate of Ang-(1-7) suggests an important role for this peptide in cardiovascular regulation during pregnancy.

Enhanced renal immunocytochemical expression of ANG-(1-7) and ACE2 during pregnancy.

Previously we demonstrated that kidney concentration and urinary excretion of angiotensin-(1-7) are increased during normal pregnancy in rats. Since this finding may reflect local kidney production of angiotensin-(1-7), we determined the immunocytochemical distribution of angiotensin-(1-7) and its newly described processing enzyme, ACE2, in kidneys of virgin and 19-day-pregnant Sprague-Dawley rats. Sprague-Dawley rats were killed at the 19th day of pregnancy, and tissues were prepared for immunocytochemical by using a polyclonal antibody to angiotensin- (1-7) or a monoclonal antibody to ACE2. Angiotensin-(1-7) immunostaining was predominantly localized to the renal tubules traversing both the inner cortex and outer medulla. ACE2 immunostaining was localized throughout the cortex and outer medulla and was visualized in the renal tubules of both virgin and pregnant rats. The quantification of angiotensin-(1-7) and ACE2 immunocytochemical staining showed that in pregnant animals, the intensity of the staining increased by 56% and 117%, respectively (P<0.05). This first demonstration of the immunocytochemical distribution of angiotensin-(1-7) and ACE2 in kidneys of pregnant rats shows that pregnancy increases angiotensin-(1-7) immunocytochemical expression in association with increased ACE2 intensity of staining. The findings suggest that ACE2 may contribute to the local production and overexpression of angiotensin-(1-7) in the kidney during pregnancy.

Characterization of angiotensin-(1-7) receptor subtype in mesenteric arteries.

Mesenteric arteries from male Sprague-Dawley rats were mounted in a pressurized myograph system. Ang-(1-7) concentration-dependent responses were determined in arteries preconstricted with endothelin-1 (10(-7)M). The receptor(s) mediating the Ang-(1-7) evoked dilation were investigated by pretreating the mesenteric arteries with specific antagonists of Ang-(1-7), AT(1) or AT(2) receptors. The effects of Ang-(3-8) and Ang-(3-7) were also determined. Ang-(1-7) caused a concentration-dependent dilation (EC(50): 0.95 nM) that was blocked by the selective Ang-(1-7) receptor antagonist D-[Ala(7)]-Ang-(1-7). Administration of a specific antagonist to the AT(2) receptor (PD123319) had no effect. On the other hand, losartan and CV-11974 attenuated the Ang-(1-7) effect. These results demonstrate that Ang-(1-7) elicits potent dilation of mesenteric resistance vessels mediated by a D-[Ala(7)]-Ang-(1-7) sensitive site that is also sensitive to losartan and CV-11974.