Anne Monique Nuyt

Altered Vascular Function in Fetal Programming of Hypertension

Background and Purpose— Reduced endothelium-dependent vasorelaxation partly due to loss of nitric oxide (NO) bioavailability occurs in most cases of chronic hypertension. Intrauterine nutritional deprivation has been associated with increased risk for hypertension and stroke, associated with relaxant dysfunction and decreased vascular compliance, but the underlying mechanisms are not known. The present studies were undertaken to investigate whether endothelial dysfunction associated with altered NO-dependent vasodilatation pathways is also observed in a model of in utero programming of hypertension. Methods— Pregnant Wistar rats were fed a normal (18%), low (9%), or very low (6%) protein isocaloric diet during gestation. Vasomotor response of resistance cerebral microvessels (<50 &mgr;m) was studied in adult offspring of dams fed the 18% and 9% protein diets by a video imaging technique. Endothelial NOS (eNOS), soluble guanylate cyclase (sGC), and KCa channel expression were measured by Western blot. NO synthase (NOS) activity was measured enzymatically as well as in situ by NADPH diaphorase staining. Results— Litter size and survival to adulthood were not affected by the diets. Birth weights of offspring of dams fed the 6% diet were markedly lower than those of dams fed the 9% diet, which were marginally lower than those of controls. Systolic blood pressures of adult offspring of mothers in the 6% and 9% groups were comparably greater (156±2 and 155±1 mm Hg, respectively) than that of control offspring (137±1 mm Hg); we therefore focused on the 9% and 18% groups. Cerebral microvessel constriction to thromboxane A2 mimetic and dilation to carba-prostaglandin I2 did not differ between diet groups. In contrast, vasorelaxation to the NO-dependent agents substance P and acetylcholine was diminished by 50% in low protein-exposed offspring, but eNOS expression and activity were similar between the 2 diet groups. Vasorelaxant response to the NO donor sodium nitroprusside was also decreased and was associated with reduced (by 50% to 65%) cGMP levels and sGC expression. cGMP analogues caused comparable vasorelaxation in the 2 groups. Expression of KCa (another important mediator of NO action) and relaxation to the KCa opener NS1619 were unchanged by antenatal diet. Conclusions— Maternal protein deprivation, which leads to hypertension in the offspring, is associated with diminished NO-dependent relaxation of major organ (cerebral) microvasculature, which seems to be largely attributed to decreased sGC expression and cGMP levels. The study provides an additional explanation for abnormal vasorelaxation in nutrient-deprived subjects in utero.

Role of brain and peripheral angiotensin II in hypertension and altered arterial baroreflex programmed during fetal life in rat.

Intrauterine programming of hypertension is associated with evidence of increased renin-angiotensin system (RAS) activity. The current study was undertaken to investigate whether arterial baroreflex and blood pressure variability are altered in a model of in utero programming of hypertension secondary to isocaloric protein deprivation and whether activation of the RAS plays a role in this alteration. Pregnant Wistar rats were fed a normal-protein (18%) or low-protein (9%) diet during gestation, which had no effect on litter size, birth weight, or pup survival. Mean arterial blood pressure (MABP; 126 ± 3 mm Hg 9%versus 108 ± 4 mm Hg 18%; p < 0.05) and blood pressure variability were significantly greater in the adult offspring of the 9% protein–fed mothers. Arterial baroreflex control of heart rate, generated by graded i.v. infusion of phenylephrine and nitroprusside, was significantly shifted toward higher pressure; i.v. angiotensin-converting enzyme inhibitor normalized MABP and shifted the arterial baroreflex curve of the 9% offspring toward lower pressure without affecting the 18% offspring. For examining whether brain RAS is also involved in programming of hypertension, angiotensin-converting enzyme inhibitor and losartan (specific AT1 receptor antagonist) were administered intracerebroventricularly; both significantly reduced MABP of the 9% but not the 18% offspring. Autoradiographic receptor binding studies demonstrated an increase in brain AT1 expression in the subfornical organ and the vascular organ of the lamina terminalis in the 9% offspring. These data demonstrate a major tonic role of brain and peripheral RAS on hypertension associated with antenatal nutrient deprivation.

Neonatal Oxygen Exposure in Rats Leads to Cardiovascular and Renal Alterations in Adulthood

Long-term vascular and renal consequences of neonatal oxidative injury are unknown. Using a rat model, we sought to investigate whether vascular function and blood pressure are altered in adult rats exposed to hyperoxic conditions as neonates. We also questioned whether neonatal O2 injury causes long-term renal damage, important in the pathogenesis of hypertension. Sprague-Dawley pups were kept with their mother in 80% O2 or room air from days 3 to 10 postnatal, and blood pressure was measured (tail cuff) from weeks 7 to 15. Rats were euthanized, and vascular reactivity (ex vivo carotid rings), oxidative stress (lucigenin chemiluminescence and dihydroethidium fluorescence), microvascular density (tibialis anterior muscle), and nephron count were studied. In male and female rats exposed to O2 as newborns, systolic and diastolic blood pressures were increased (by an average of 15 mm Hg); ex vivo, maximal vasoconstriction (both genders) and sensitivity (males only) specific to angiotensin II were increased; endothelium-dependant vasodilatation to carbachol but not to NO-donor sodium nitroprussiate was impaired; superoxide dismutase analogue prevented vascular dysfunction to angiotensin II and carbachol; vascular superoxide production was higher; and capillary density (by 30%) and number of nephrons per kidney (by 25%) were decreased. These data suggest that neonatal hyperoxia leads in the adult rat to increased blood pressure, vascular dysfunction, microvascular rarefaction, and reduced nephron number in both genders. Our findings support the hypothesis of developmental programming of adult cardiovascular and renal diseases and provide new insights into the potential role of oxidative stress in this process.

Nitric oxide in retinal and choroidal blood flow autoregulation in newborn pigs: interactions with prostaglandins.

The role of nitric oxide (NO) as well as its interaction with prostaglandins (PG) in setting the limits of autoregulation of retinal blood flow (RBF) and choroidal blood flow (ChBF) were studied in newborn pigs (1-5 d old). Blood flows were measured by the microsphere technique. Low and high ocular perfusion pressures (OPP) were induced by inflating balloon-tipped catheters placed at the aortic root and isthmus, respectively. Animals were treated with the NO synthase inhibitors, NG-nitro-L-arginine methyl ester (L-NAME, 1 mg/kg followed by 50 μg/kg/min; n = 12) or NG-monomethyl-L-arginine (L-NMMA, same dose as L-NAME;n = 3), or with saline (n = 12). In separate animals(n = 42), guanosine 3′,5′-cyclic monophosphate (cGMP), the second messenger for NO, and PG were measured at an average OPP of 90 mm Hg and 125 ± 6 mm Hg; cGMP levels served as an index of NO release. The effect of the NO donor sodium nitroprusside on choroidal vessel diameter was determined using video imaging of isolated eyecup preparations. In control animals RBF was constant only within a range of 30 to 80 mm Hg OPP(r = 0.03, p > 0.9). There was no autoregulation of ChBF which increased as a function of OPP (τ = 0.58-0.72, p < 0.01). L-NAME and L-NMMA prevented a change in RBF and ChBF from 30 to 146 mm Hg [the highest OPP studied (r < 0.3, p > 0.15)] and caused an increase in retinal as well as choroidal vascular resistance as OPP was raised; these agents did not affect ocular blood flow at OPP < 30 mm Hg. Elevated OPP caused increases in cGMP, 6-keto-PGF1α, and PGE2 in the choroid (a vascular tissue), which were prevented by L-NAME and L-NMMA. Sodium nitroprusside caused a dilatation of choroidal vessels in isolated eyecup preparations, which was significantly attenuated by indomethacin. Data suggest a role for NO in the autoregulation of RBF and ChBF in the newborn such that a release of NO during a rise in OPP prevents adequate constriction necessary for maintaining RBF and ChBF constant; data also suggest that the vasodilator effect of NO might in part be mediated through a release of PG.

Preterm Birth and Hypertension Risk: The Oxidative Stress Paradigm

The majority of epidemiological studies in developmental programming have explored the influence of low birth weight (irrespective of gestational age) on long-term chronic disease in individuals born during the first half of the 20th century.1,2 Low birth weight neonates may represent infants born at term with intrauterine growth restriction (IUGR) or born preterm with or without IUGR. As such, there is emerging interest in the effects of preterm birth alone (beyond birth weight and IUGR) on specific aspects of human development and long-term health. Approximately 10% of all births worldwide are preterm (before 37 completed weeks of gestation).3 Besides being of low birth weight, preterm neonates are suddenly and prematurely exposed to the extrauterine environment at a time when organogenesis is incomplete. Exposure postnatally to factors such as high oxygen concentrations,4 medications5 (including glucocorticoids),6 and inadequate nutrition7 likely adversely influence postnatal growth and ongoing organ development. In addition to possible genetic and epigenetic factors that may contribute to hypertension risk (including hypertension-related complications of pregnancy), a multitude of aspects related to both intrauterine and extrauterine growth, as well as the postnatal environment, may all play an important role in the programming of hypertension in individuals born preterm. In this review, we will highlight, in particular, the potential effect of oxidative stress associated with preterm birth on neonatal development and future disease risk. The survival of neonates born at low and very low gestational ages is recent in the history of medicine and has increased remarkably over the last few decades. The first generations of survivors of very preterm birth are currently just reaching adulthood and as such are providing emerging evidence of chronic health conditions, such as hypertension. The link between preterm birth and hypertension risk (independent of birth weight) has been …

New Reference Curves for Head Circumference at Birth, by Gestational Age

BACKGROUND: The measurement of head circumference (HC) at birth reflects intrauterine brain development. HC charts currently used in Canada are either dated, mixed-gender, nonrepresentative of lower gestational ages (GAs), or reflective of other populations. METHODS: To create both birth weight and HC curves, we combined weight and HC data from the Canadian Neonatal Network (CNN) database (admissions in NICUs across Canada) with McGill’s Obstetrical Neonatal Database (MOND; all births at a tertiary hospital in Montreal, Canada). We included CNN data for GAs of 23 to 34 weeks (2003–2007) and MOND data for GAs of 35 to 41 weeks (1995–2006). Nonsingletons, congenital anomalies, and measurements greater than ±4 SD from the mean were excluded. Distributions of birth weight and HC at each GA were statistically (penalized spline regression) smoothed. Birth weight curves were compared with recent Canadian reference curves and HC curves with historical and/or frequently used curves. RESULTS: We included 39 896 births (3121 births at <30 weeks’ GA) to generate the curves. Current weight curves were similar to Canadian reference charts for both genders. Weight and HC measurements in boys were higher than in girls. When classified according to recent international references, the proportion of CNN-MOND infants at ≥32 weeks’ GA with HCs <10th percentile was significantly underestimated. When classified according to historical reference curves, a significant number of CNN-MOND infants of all GAs with HCs <10th and >90th percentiles were misclassified. CONCLUSIONS: We developed recent gender-specific reference curves for HC at birth for singletons at 23 to 41 completed weeks’ GA, which included a large number of very premature infants, reflecting the current geotemporal Canadian population.

Pregnancy complications among women born preterm

Background: Adults who were born with low birth weights are at increased risk of cardiovascular and metabolic conditions, including pregnancy complications. Low birth weight can result from intrauterine growth restriction, preterm birth or both. We examined the relation between preterm birth and pregnancy complications later in life. Methods: We conducted a population-based cohort study in the province of Quebec involving 7405 women born preterm (554 < 32 weeks, 6851 at 32–36 weeks) and a matched cohort of 16 714 born at term between 1976 and 1995 who had a live birth or stillbirth between 1987 and 2008. The primary outcome measures were pregnancy complications (gestational diabetes, gestational hypertension, and preeclampsia or eclampsia). Results: Overall, 19.9% of women born at less than 32 weeks, 13.2% born at 32–36 weeks and 11.7% born at term had at least 1 pregnancy complication at least once during the study period (p < 0.001). Women born small for gestational age (both term and preterm) had increased odds of having at least 1 pregnancy complication compared with women born at term and at appropriate weight for gestational age. After adjustment for various factors, including birth weight for gestational age, the odds of pregnancy complications associated with preterm birth was elevated by 1.95-fold (95% confidence interval [CI] 1.54–2.47) among women born before 32 weeks’ gestation and 1.14-fold (95% CI 1.03–1.25) among those born at 32–36 weeks’ gestation relative to women born at term. Interpretation: Being born preterm, in addition to, and independent of, being small for gestational age, was associated with a significantly increased risk of later having pregnancy complications.

Circulating docosahexaenoic acid levels are associated with fetal insulin sensitivity.

Background Arachidonic acid (AA; C20∶4 n-6) and docosahexaenoic acid (DHA; C22∶6 n-3) are important long-chain polyunsaturated fatty acids (LC-PUFA) in maintaining pancreatic beta-cell structure and function. Newborns of gestational diabetic mothers are more susceptible to the development of type 2 diabetes in adulthood. It is not known whether low circulating AA or DHA is involved in perinatally “programming” this susceptibility. This study aimed to assess whether circulating concentrations of AA, DHA and other fatty acids are associated with fetal insulin sensitivity or beta-cell function, and whether low circulating concentrations of AA or DHA are involved in compromised fetal insulin sensitivity in gestational diabetic pregnancies. Methods and Principal Findings In a prospective singleton pregnancy cohort, maternal (32-35 weeks gestation) and cord plasma fatty acids were assessed in relation to surrogate indicators of fetal insulin sensitivity (cord plasma glucose-to-insulin ratio, proinsulin concentration) and beta-cell function (proinsulin-to-insulin ratio) in 108 mother-newborn pairs. Cord plasma DHA levels (in percentage of total fatty acids) were lower comparing newborns of gestational diabetic (n = 24) vs. non-diabetic pregnancies (2.9% vs. 3.5%, P = 0.01). Adjusting for gestational age at blood sampling, lower cord plasma DHA levels were associated with lower fetal insulin sensitivity (lower glucose-to-insulin ratio, r = 0.20, P = 0.036; higher proinsulin concentration, r = −0.37, P <0.0001). The associations remained after adjustment for maternal and newborn characteristics. Cord plasma saturated fatty acids C18∶0 and C20∶0 were negatively correlated with fetal insulin sensitivity, but their levels were not different between gestational diabetic and non-diabetic pregnancies. Cord plasma AA levels were not correlated with fetal insulin sensitivity. Conclusion Low circulating DHA levels are associated with compromised fetal insulin sensitivity, and may be involved in perinatally “programming” the susceptibility to type 2 diabetes in the offspring of gestational diabetic mothers.

Ontogeny of angiotensin II type 1 receptor mRNAs in fetal and neonatal rat brain.

Studies have demonstrated a specific function of the angiotensin II (Ang II) type 1 receptor (AT1) in regulation of adult central cardiovascular, fluid, and pituitary hormone release and a predominant role of the renin‐angiotensin system in fetal and neonatal cardiovascular homeostasis. The pattern of brain AT1 mRNA expression during fetal and neonatal development is currently unknown. We used radiolabeled cRNA probes for in situ hybridization histochemistry to determine the ontogenic development of the two AT1 subtypes (AT1a and AT1b) mRNA in rat brain, from 11 days of gestation (E11) to 28 days after birth (P28). No AT1b mRNA was detected in the developing brain, whereas AT1a mRNA was first detected at E19. The age at which AT1a mRNA is first detected varied among different brain areas and expression predominates in areas involved in fluid homeostasis, pituitary hormone release, and cardiovascular regulation, where it persists until P28. AT1a mRNA expression is present from E19 onward in the median preoptic nucleus, the vascular organ of the lamina terminalis, the paraventricular nucleus, the periaqueductal gray, the nucleus raphe pallidus, the motor facial nucleus, and very weakly in the nucleus of the solitary tract and the ambiguus nucleus, and at E21 in the subfornical organ, the anterior olfactory nucleus and the piriform cortex. AT1a mRNA expression is present after birth in many regions, including the preoptic and lateral hypothalamic areas, the area postrema and medullary reticular nuclei. In conclusion, during brain development, expression of AT1a mRNA, appears in late gestation at E19, predominantly in forebrain areas involved in fluid homeostasis and cardiovascular regulation. In contrast, AT1a mRNA expression is absent or present only in very small amounts until after birth in many medullary nuclei, known to play an important role in cardiovascular modulation. Our results suggest that, in perinatal life, AT1a is involved in fluid and perhaps cardiovascular homeostasis and that the role of Ang II in modulating medullary cardiovascular centers matures later in postnatal life. J. Comp. Neurol. 440:000–000, 2001. J. Comp. Neurol. 440:192–203, 2001.

Transient Neonatal High Oxygen Exposure Leads to Early Adult Cardiac Dysfunction, Remodeling, and Activation of the Renin–Angiotensin System

Perinatal conditions (such as preterm birth) can affect adult health and disease, particularly the cardiovascular system. Transient neonatal high O2 exposure in rat in adulthood (a model of preterm birth–related complications) leads to elevated blood pressure, vascular rigidity, and dysfunction with renin–angiotensin system activation. We postulate that neonatal hyperoxic stress also affects myocardial structure, function, and expression of renin–angiotensin system components. Sprague-Dawley pups were kept with their mother in 80% O2 or in room air (control) from days 3 to 10 of life. Left ventricular function was assessed in 4-, 7-, 12-week-old (echocardiography) and in 16-week-old (intraventricular catheterization) male O2-exposed versus control rats. At 16 weeks, hearts from O2-exposed rats showed cardiomyocyte hypertrophy, enhanced fibrosis, and increased expression of transforming growth factor-&bgr;1, senescence-associated proteins p53 and Rb, upregulation of angiotensin II type 1 (AT1) receptor expression (protein and AT1a/b mRNA), and downregulation of AT2 receptors. At 4 weeks (before blood pressure increase), the expression of cardiomyocyte surface area, fibrosis, p53, and AT1b was significantly increased and AT2 decreased in O2-exposed animals. After 4 weeks of continuous angiotensin II infusion (starting at 12 weeks), O2-exposed rats developed severe heart failure, with impaired myocardial mechanical properties compared with saline-infused rats. Transient neonatal O2 exposure in rats leads to left ventricular hypertrophy, fibrosis and dysfunction, and increased susceptibility to heart failure under pressure overload. These results are relevant to the growing population of individuals born preterm who may be at higher risk of cardiac dysfunction when faced with increased peripheral resistance associated with hypertension, vascular diseases, and aging.