Rebecca Lee Flower

Enhanced Angiotensin II Type 2 Receptor Mechanisms Mediate Decreases in Arterial Pressure Attributable to Chronic Low-Dose Angiotensin II in Female Rats

The renin-angiotensin system is a far more complex enzymatic cascade than realized previously. Mounting evidence suggests sex-specific differences in the regulation of the renin-angiotensin system and arterial pressure. We examined the hemodynamic responses, angiotensin II receptor subtypes, and angiotensin-converting enzyme 2 gene expression levels after graded doses of angiotensin II in males and females. Mean arterial pressure was measured via telemetry in male and female rats in response to a 2-week infusion of vehicle, low-dose (50 ng/kg per minute SC) or high-dose (400 ng/kg per minute SC) angiotensin II. The effect of concurrent infusion of the angiotensin II type 2 receptor (AT2R) blocker (PD123319) was also examined. The arterial pressure response to high-dose angiotensin II was attenuated in females compared with males (24±8 mm Hg versus 42±5 mm Hg; P for the interaction between sex and treatment <0.002). Remarkably, low-dose angiotensin II decreased arterial pressure (11±4 mm Hg; P for the interaction between sex and treatment <0.02) at a dose that did not have an effect in males. This decrease in arterial pressure in females was abolished by AT2R blockade. Renal AT2R, angiotensin-converting enzyme 2, and left ventricular AT2R mRNA gene expressions were markedly greater in females than in males with a renal angiotensin II type 1a receptor:AT2R ratio of ≈1 in females. Angiotensin II infusion did not affect renal AT2R mRNA expression but resulted in significantly less left ventricular mRNA expression. Renal angiotensin-converting enzyme 2 mRNA expression levels were greater in females than in males treated with high-dose angiotensin II (≈2.5 fold; P for the interaction between sex and treatment <0.05). In females, enhancement of the vasodilatory arm of the renin-angiotensin system, in particular, AT2R and angiotensin-converting enzyme 2 mRNA expression, may contribute to the sex-specific differences in response to renin-angiotensin system activation.

EFFECT OF ENDOTHELIN‐1 ON REGIONAL KIDNEY BLOOD FLOW AND RENAL ARTERIOLE CALIBRE IN RABBITS

1. Medullary blood flow (MBF) is important in the long‐term control of arterial pressure. However, it is unclear which vascular elements regulate MBF.

Adult Rabbit Offspring of Mothers With Secondary Hypertension Have Increased Blood Pressure

Abstract—Preexisting chronic hypertension complicates up to 5% of pregnancies and is associated with an increased risk of low-birth-weight babies. Studies suggest that an adverse intrauterine environment leading to low birth weight is linked to an increased risk of cardiovascular disease, including hypertension, in the adult. In this study, the blood pressure of offspring from mothers with hypertension were followed up into adulthood. Two-kidney, 1-wrapped hypertension was induced in 7 female rabbits; 5 other rabbits underwent sham surgery. Four weeks later, rabbits were mated, at which time mean arterial pressure was 118±3 and 87±5 mm Hg in the hypertensive and sham groups, respectively (P <0.001). The blood pressure of 30-week-old females was 89±2 mm Hg in the offspring of hypertensive (n=14) and 79±1 mm Hg in the offspring of normotensive (n=13) mothers (P <0.005). Also, plasma renin activity was significantly lower in the female offspring of hypertensive mothers at 10 weeks of age (P <0.05), suggesting that development of the renin-angiotensin system was altered. In contrast, male offspring from hypertensive and normotensive mothers had similar mean arterial pressure and plasma renin activity. In conclusion, maternal secondary hypertension can “program” hypertension in female adult offspring. The results also suggest that there are gender-specific differences in sensitivity to altered in utero environmental influences.

Ontogeny of Placental Structural Development and Expression of the Renin–Angiotensin System and 11β-HSD2 Genes in the Rabbit

Common pregnancy complications are associated with impaired placental development. This study aimed to characterise the ontogeny of structural correlates of rabbit placental function, its expression of genes encoding components of the renin-angiotensin system (RAS), as well as 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) mRNA since these are known to be expressed by the placenta and are associated with pregnancy complications, including preeclampsia and intrauterine programming. Placentae were collected at gestational age (GA) 14, 21 and 28 (term=32 days). Gene expression was analysed using real time PCR and placental structures were quantified via image analyses. The volume densities and volumes of trophoblast, fetal capillaries, maternal blood space, surface density and surface area of trophoblast all progressively increased, while the arithmetic mean barrier thickness of trophoblast decreased across gestation. Maternal plasma renin activity (PRA) was positively correlated with volumes of trophoblast and maternal blood space, surface density and surface area of trophoblast. Placental renin mRNA declined ( downward arrow62%; P<0.01) across gestation and was negatively correlated with maternal PRA (GA0), fetal and placental weights, placental angiotensin type 1 and 2 receptors (AT(1)R and AT(2)R) mRNA and volume of trophoblast. AT(1)R mRNA expression was increased by 92% (P<0.001) across gestation. AT2R mRNA expression was approximately 81% (P<0.01) greater at GA14 compared to GA21. Placental 11beta-HSD2 mRNA expression was approximately 74% greater (P<0.01) at GA21 than GA14, but by GA28 was similar to that at GA14. These data show that changes in placental gene expression are associated with key events in placental and fetal development, indicating that the rabbit provides a good model for investigations of pregnancy perturbations that alter the RAS or programme the fetus.

Chronic maternal hypertension characterized by renal dysfunction is associated with reduced placental blood flow during late gestation in rabbits

Maternal hypertension associated with renal disease is a common pregnancy complication. Previously, we have shown in a rabbit model of mild hypertension that offspring from hypertensive mothers have increased blood pressure as adults. In human pregnancy, hypertension has been associated with decreased utero-placental blood flow. The aim of this study was to determine placental blood flow (PBF) in mild (2-kidney-1-wrapped; 2K-1W) and moderate (2-kidney-2-wrapped; 2K-2W) rabbit models of maternal hypertension. We hypothesized that PBF would be inversely related to the severity of the hypertension. PBF and renal blood flow (RBF) were measured using microspheres on day 28 of a 32-day gestation, in normotensive (sham), 2K-1W, and 2K-2W hypertensive groups. Mean arterial pressure (MAP, approximately 7 mmHg, P < 0.05) was increased, and RBF ( approximately 35%, P < 0.05) was reduced in the 2K-1W and 2K-2W (MAP approximately 20 mmHg, P < 0.01; RBF approximately 53%, P < 0.05) groups compared with the sham group. In the 2K-1W group, PBF fell by approximately 12% (P = 0.08) and fetal-to-placental weight ratio increased by approximately 12% (P < 0.01) compared with the sham group, reflecting an increase in the functional capacity of the placenta to deliver nutrients to the fetus. In the 2K-2W group, PBF decreased approximately 51% (P < 0.05) compared with the sham group, without changes in placental efficiency. Thus, in late gestation, placental blood flow was significantly reduced in the moderate hypertension group, without accompanying changes in fetal or placental weight or placental efficiency. In contrast, mild hypertension resulted in an increase in placental efficiency, without significant changes in placental blood flow. These findings suggest that mild and moderate hypertension may alter placental delivery of nutrients via differing mechanisms dependent upon the severity of the hypertension.

Chronic maternal hypertension affects placental gene expression and differentiation in rabbits

Background Previously, we have shown that adult offspring from hypertensive rabbits develop hypertension. Method We aimed to determine the effects of mild (+15 mmHg) and moderate (+25 mmHg) increases in maternal blood pressure and plasma renin activity on placental differentiation and expression of components of the renin–angiotensin system and 11β-hydroxysteroid dehydrogenase type 2 mRNA in rabbits. Placentas were collected from normotensive (sham), mild (2-kidney-1-cellophane wrapped; 2K-1W) and moderate (2-kidney-2-cellophane wrapped; 2K-2W) hypertensive groups at gestational age of 14, 21 and 28 days. Placental gene expression was quantified by reverse transcriptase-PCR, and morphometry was assessed by videoimage analyses of placental sections. Results Fetal weight was similar between groups across gestation. In the 2K-1W group at gestational age day 14, fetal-to-placental weight ratio was increased (∼34%) as were volumes of fetal capillaries (↑56%) and maternal blood space at gestational age day 21 (↑55%) compared with sham (all P < 0.05). In the 2K-2W group, fetal-to-placental weight ratio was increased at gestational age day 21 (∼25%; P < 0.01) with an accompanying reduction in placental weight, and at gestational age day 28, volume density of fetal capillaries was increased (∼22%; P < 0.05). Placental renin mRNA was lower in both the 2K-1W (∼88%) and 2K-2W (∼98%) groups at gestational age day 28 (all P < 0.01). Placental 11β-hydroxysteroid dehydrogenase type 2 mRNA was lower in the 2K-1W (∼36%) and 2K-2W (∼31%) groups at gestational age day 14 and greater (∼36%) in the 2K-2W group at gestational age day 21 (all P < 0.01). Associations between placental AT1R and AT2R mRNA and placental differentiation were disturbed by hypertension. Conclusion Mild and moderate maternal hypertension differentially alters placental structure and gene expression that may affect placental functional capacity and contribute to programming of hypertension in offspring.

Alterations in Renal Function Occur Prior to the Increase in Arterial Pressure in Rat Offspring Following Short-Term Exposure to Maternal Corticosterone

### EXCESS PRENATAL CORTICOSTERONE EXPOSURE PROGRAMS HYPOTENSION, VASCULAR REMODELLING AND ALTERS THE RESPONSE TO RESTRAINT STRESS IN ADULT MALE MICE O’Sullivan L, Cuffe JSM, Koning A, Singh RR, Moritz KM, Paravicini TM School of Biomedical Sciences, The University of Queensland, Brisbane, Queensland, Australia Background: Exposure to excess glucocorticoids in utero is known to program susceptibility to cardiovascular disease in later life. Endogenous levels of glucocorticoids are increased during periods of stressThe long-term cardiovascular implications for the offspring of women who suffer from stress during pregnancy are, however, poorly understood. Aim: To examine the direct effects of prenatal corticosterone (CORT) exposure on cardiovascular and renal outcomes in adult offspring. Methods: Pregnant C57Bl6 mice received CORT (33 g/kg/h) for 60 h from embryonic day 12.5 and allowed to litter naturally. Nephron number was determined in the offspring at postnatal day 30, and radiotelemetry was used to measure blood pressure and heart rate (both basal and during restraint stress) at 12 months. Pressurized myography was used to assess vascular function, structure and mechanics. Results: Excess prenatal CORT reduced nephron endowment by 33% and 20% in male and female offspring (respectively) compared to untreated controls (n=6–7; P <0.05). At 12 months, CORT-exposed male offspring were hypotensive: MAP 104.3 11.0 vs 115.6 5.5 mmHg in untreated controls (n=8–9; P <0.05). These mice also showed a blunted tachycardiac response to restraint stress. Power spectral density analysis of heart rate suggested that this may be due to an altered vagal/sympathetic balance. Prenatal CORT reduced activity levels of female offspring at night, but did not alter any cardiovascular parameters. In male offspring (but not females), prenatal CORT caused inward remodeling of mesenteric arteries (luminal diameter = 202 7 m vs 249 9 m in controls) without affecting vascular reactivity or the elasticity of vascular wall components. Conclusion: Prenatal CORT exposure programs hypotension in male offspring, despite the reduced nephron number and inward vascular remodeling in these animals. In …

1008 ANGIOTENSIN TYPE-2 RECEPTOR CONTRIBUTES TO REDUCED VASCULAR RESPONSE TO ANGIOTENSIN-II IN YOUNG RATS

Background: The angiotensin type-2 receptor (AT2R) is known to oppose the vasoconstrictive actions of angiotensin II (AngII). During fetal development AT2R expression is very high, which upon maturation regresses. The present study investigates the sensitivity to acute AngII in young rats and the contribution of the AT2R in attenuating the vasoconstrictor response to AngII. Methods: Male Sprague-Dawley rats at 3 and 6 weeks of age were anesthetized. Mean arterial pressure (MAP) and renal blood flow (RBF; transonic flowmeter), was measured in response to i.v. administration of AngII at 0 (basal) 10 (10AngII) and 100ng/kg/hr (100AngII). The AT2R inhibitor, PD-123319, was given and responses to 100AngII were repeated. Results: In the 6wk rats, 10 and 100AngII increased MAP (89 ± 4 mmHg) by 10 ± 7 and 18 ± 5 mmHg, respectively. Likewise, RBF (4.0 ± 0.6 ml/min/gkw) was reduced by 8 ± 3% and 34 ± 3%. In the 3wk rats basal MAP and RBF were 72 ± 4 mmHg and 2.9 ± 0.3 ml/min/gkwt, The 10AngII dose had no effect on MAP, while 100AngII increased MAP by 6 ± 2 mmHg. RBF in the 3wk rats was not was not affected by the 10ng AngII dose (-2 ± 1% change), while the higher dose produced a 10 ± 3% decrease. PD-123319 potentiated the change in RBF to 100AngII in the 3wk animals (23 ± 4% reduction), but had no additional effect in the 6wk rats. Conclusions: These data indicate that during development, the presence of the AT2R in young rodents contributes to the attenuated vasoconstrictor response to AngII. These findings may have implication for the use of drugs that inhibit the renin-angiotensin system for the treatment of pediatric hypertension and renal failure.

245 INCREASED AGE-RELATED RISE IN ARTERIAL PRESSURE FROM WEANING IN RATS EXPOSED TO PRENATAL GLUCOCORTICOIDS

Background: Offspring exposed to prenatal glucocorticoids have reduced nephron endowment and develop hypertension in adulthood. We aimed to examine early life changes in arterial pressure. We hypothesised that prenatal glucocorticoid exposure increases the age-related postnatal rise in arterial pressure and that this would be exacerbated by high-salt diet. Methods: In rat offspring of dams given corticosterone (CORT; 0.4 mg/kg i.p.bid) or vehicle (VEH; 0.2 ml 0.9% NaCl) on gestational days 14 and 15, mean arterial pressure (MAP) was recorded from 26 to 42 days of age by telemetry. The pups were fed from 22 days of age either a normal (NS: 0.26% NaCl) or a high-salt (HS; 5% NaCl) diet. Results: Birth weight was not different between groups. At 26 days of age MAP was similar between the VEH-NS (n = 6) and CORT-NS (n = 5) groups but MAP was greater in the VEH-HS (n = 6) and CORT-HS (n = 4) groups after 5 days HS (85±1; 85±2; 91±3, 93±1 mmHg, respectively; PDIET<0.005). The age-related increase in MAP (PAGE<0.001) was greater in the CORT as compared to the VEH offspring (PGROUP = 0.04) and the increase in MAP was greater in the HS groups (VEH-NS 96±1 mmHg; CORT-NS 109±5; VEH-HS 100±3 mmHg; CORT-HS 114±4 mmHg; PGROUPxAGE = 0.04). Conclusion: Arterial pressure was not increased at 26 days of age in offspring exposed to maternal glucocorticoids but the trajectory of the normal age-related rise in arterial pressure was increased, an effect exacerbated by a diet high in salt. Our studies identify the early postnatal period as a critical window when steps may be taken to prevent the programming of hypertension.