Samantha Louey

Fetal growth restriction has long-term effects on postnatal lung structure in sheep.

We have previously shown that fetal growth restriction (FGR) during late gestation in sheep affects lung development in the near-term fetus and at 8 wk after birth. In the present study, our aim was to determine the effects of FGR on the structure of the lungs at 2 y after birth; our hypothesis was that changes observed at 8 wk after birth would persist until maturity. FGR was induced in sheep by umbilicoplacental embolization, which was maintained from 120 d until delivery at term (approximately 147 d); birth weights of FGR lambs were 41% lower than in controls. At 2 y after birth, body and lung weights were not different, but there were 28% fewer alveoli and alveoli were significantly larger than in controls; hence there was a 10% reduction in the internal surface area relative to lung volume in FGR sheep compared with controls. The lungs of FGR sheep, compared with controls, had thicker interalveolar septa as a result of increased extracellular matrix deposition; the alveolar blood–air barrier was also thicker, largely because of an 82% increase in basement membrane thickness. These changes are qualitatively similar to those observed at 8 wk. Our data show that structural alterations in the lungs induced by FGR that were apparent at 8 wk were still evident at 2 y after birth, indicating that FGR may result in permanent changes in the structure of the lungs of the offspring and may affect respiratory health and lung aging later in life.

Placental insufficiency and fetal growth restriction lead to postnatal hypotension and altered postnatal growth in sheep.

Low birth weight has been associated with elevated arterial pressure in later life but mechanisms are unknown. Our aim was to determine the effects of low birth weight resulting from intrauterine growth restriction (IUGR) on fetal and postnatal arterial pressures and the potential roles of circulating cortisol and renin. We induced IUGR by umbilico-placental embolization (UPE) in fetal sheep from 120 d of gestation until birth (approximately 147 d); postnatal lambs (8 IUGR, 8 controls) were studied for 8 wk. Fetal and postnatal arterial pressures were measured and blood samples taken for measurement of gas tensions, cortisol concentrations and renin activity. In IUGR fetuses, mean arterial pressure (MAP) initially increased with UPE, but near term was not different to values in controls. IUGR lambs weighed 33% less than controls at birth and remained lighter than controls during the 8 postnatal weeks; their growth pattern was different to that of controls. IUGR lambs had lower MAP than controls, and this relative hypotension (−4 mm Hg) persisted throughout the 8 postnatal weeks. Covariate analysis showed that the relative hypotension of IUGR lambs could have resulted from their smaller size. Plasma cortisol concentrations were not different between IUGR and control animals before or after birth. Plasma renin activity was not different in postnatal IUGR lambs compared with controls. Thus, postnatal cortisol and renin levels were not consistent with the development of hypotension or hypertension. We conclude that late gestational IUGR in sheep leads to relative hypotension in the early postnatal period, probably a result of reduced body size.

Placental insufficiency decreases cell cycle activity and terminal maturation in fetal sheep cardiomyocytes.

Umbilicoplacental embolization (UPE) in sheep has been used to investigate the effects of placental insufficiency on fetal development. However, its specific effects on the heart have been little studied. The aim of this study was to determine the effects of placental insufficiency, induced by UPE, on cardiomyocyte size, maturation and proliferation. Instrumented fetal sheep underwent UPE for either 10 or 20 days. Hearts were collected at 125 ± 1 days (10 day group) or 136 ± 1 days (20 day group) of gestation (term ∼145 days). Cell size, maturational state (as measured by the proportion of binucleated myocytes) and cell cycle activity (as measured by positive staining of cells for Ki‐67) were determined in dissociated cardiomyocytes. UPE fetuses were hypoxaemic, but mean arterial pressures were not different from controls. UPE fetuses were lighter than control fetuses (10 days: −21%, P < 0.05; 20 days: −27%, P < 0.01) and had smaller hearts, but heart weight was appropriate for body weight. Neither lengths nor widths were different between control and UPE cardiomyocytes at either age. Ten days of UPE did not significantly alter the proportion of binucleated myocytes or cell cycle activity in either ventricle. However, 20 days of UPE reduced cell cycle activity in both ventricles by ∼70% (P < 0.05); the proportion of binucleated myocytes was also lower in UPE fetuses at this age (left ventricle: 31.1 ± 12.0 versus 46.0 ± 6.6%, P < 0.05; right ventricle: 29.4 ± 12.3 versus 46.3 ± 5.3%, P < 0.05). It is concluded that in the absence of fetal arterial hypertension, placental insufficiency is associated with substantially depressed growth of the heart through suppressed proliferation and maturation of cardiomyocytes.

Compromised Respiratory Function in Postnatal Lambs after Placental Insufficiency and Intrauterine Growth Restriction

Epidemiologic studies have shown persistent effects of low birth weight on respiratory function and lung health, but underlying mechanisms are not understood. Our aim was to determine the effects of intrauterine growth restriction (IUGR), a major cause of low birth weight, on postnatal respiratory function. IUGR was induced by umbilico-placental embolization during late gestation in chronically catheterized sheep. Umbilico-placental embolization was performed between 120 d of gestation and term (∼146 d) during which fetuses were hypoxemic and hypoglycemic relative to controls. Umbilico-placental embolization led to a 48% reduction in birth weight compared with controls, and throughout the postnatal study period IUGR lambs (n = 8) remained lighter than controls (n = 8). Respiratory function was repeatedly studied in lambs for 8 wk after birth; during this period, IUGR lambs were mildly hypoxemic and tended to be hypercapnic compared with controls. In IUGR lambs, relative to controls, O2 consumption (mL/min/kg) and minute ventilation (mL/kg) were increased and pulmonary diffusing capacity (adjusted for functional residual capacity) was decreased. Functional residual capacity, measured by helium dilution, and total lung capacity (measured at 30 cm H2O) were smaller in IUGR lambs than in controls. When adjusted for functional residual capacity, static lung compliance was reduced and chest wall compliance was increased in IUGR lambs. At 8 wk, pulmonary DNA and protein concentrations were decreased in IUGR lambs relative to controls. We conclude that restriction of fetal growth by placental insufficiency induces alterations in the lungs and chest wall that result in persistent impairments in respiratory function during early postnatal life.

Thyroid hormone drives fetal cardiomyocyte maturation

Tri‐iodo‐≤sc≥l≤/sc≥‐thyronine (T3) suppresses the proliferation of near‐term serum‐stimulated fetal ovine cardiomyocytes in vitro. Thus, we hypothesized that T3 is a major stimulant of cardiomyocyte maturation in vivo. We studied 3 groups of sheep fetuses on gestational days 125‐130 (term ~145 d): a T3‐infusion group, to mimic fetal term levels (plasma T3 levels increased from ~0.1 to ~1.0 ng/ml; t½~24 h); a thyroidectomized group, to produce low thyroid hormone levels; and a vehicle‐infusion group, to serve as intact controls. At 130 d of gestation, sections of left ventricular freewall were harvested, and the remaining myocardium was enzymatically dissociated. Proteins involved in cell cycle regulation (p21, cyclin D1), proliferation (ERK), and hypertrophy (mTOR) were measured in left ventricular tissue. Evidence that elevated T3 augmented the maturation rate of cardiomyocytes included 14% increased width, 31% increase in binucleation, 39% reduction in proliferation, 150% reduction in cyclin D1 protein, and 500% increase in p21 protein. Increased expression of phospho‐mTOR, ANP, and SERCA2a also suggests that T3 promotes maturation and hypertrophy of fetal cardiomyocytes. Thyroidectomized fetuses had reduced cell cycle activity and binucleation. These findings support the hypothesis that T3 is a prime driver of prenatal cardiomyocyte maturation.—Chattergoon, N. N., Giraud, G. D., Louey, S., Stork, P., Fowden, A. L., Thornburg, K. L. Thyroid hormone drives fetal cardiomyocyte maturation FASEB J. 26, 397–408 (2012). www.fasebj.org

Regulation of the Cardiomyocyte Population in the Developing Heart

During fetal life the myocardium expands through replication of cardiomyocytes. In sheep, cardiomyocytes begin the process of becoming terminally differentiated at about 100 gestation days out of 145 days term. In this final step of development, cardiomyocytes become binucleated and stop dividing. The number of cells at birth is important in determining the number of cardiomyocytes for life. Therefore, the regulation of cardiomyocyte growth in the womb is critical to long term disease outcome. Growth factors that stimulate proliferation of fetal cardiomyocytes include angiotensin II, cortisol and insulin-like growth factor-1. Increased ventricular wall stress leads to short term increases in proliferation but longer-term loss of cardiomyocyte generative capacity. Two normally circulating hormones have been identified that suppress proliferation: atrial natriuretic peptide (ANP) and tri-iodo-L-thyronine (T₃). Atrial natriuretic peptide signals through the NPRA receptor that serves as a guanylate cyclase and signals through cGMP. ANP powerfully suppresses mitotic activity in cardiomyocytes in the presence of angiotensin II in culture. Addition of a cGMP analog has the same effect as ANP. ANP suppresses both the extracellular receptor kinases and the phosphoinositol-3 kinase pathways. T₃ also suppresses increased mitotic activity of stimulated cardiomyocytes but does so by increasing the cell cycle suppressant, p21, and decreasing the cell cycle activator, cyclin D1.

Nephron Endowment and Filtration Surface Area in the Kidney after Growth Restriction of Fetal Sheep

Low birth weight is associated with adult-onset diseases including hypertension and renal disease; altered renal development after intrauterine growth restriction (IUGR) may underlie such prenatal programming. Our aim was to investigate nephron endowment and renal filtration surface area in fetal sheep in which IUGR resulted from late gestational umbilico-placental embolization (UPE) or natural twinning. UPE was performed between 120 and 140 d of gestation (term ∼147 d). At autopsy (140 d), body weights of UPE and twin fetuses were, respectively, 34% and 28% lower than controls. Kidneys were sampled using a smooth fractionator approach and glomerular number was estimated using a physical disector/fractionator technique. Glomerular capillary length and filtration surface area were estimated using unbiased stereological techniques. Although relative kidney weights (grams per kilogram body weight) were not different between groups, nephron endowment was 40% lower in twin fetuses compared with controls (34.3 ± 10.6 × 104 and 55.9 ± 19.8 × 104, respectively; p < 0.05); UPE did not alter nephron number (50.7 ± 13.2 × 104). There was no difference in the glomerular capillary length or surface area between the UPE and control fetuses. IUGR due to twinning leads to reduced nephron endowment whereas late gestational IUGR does not, suggesting that reduced nephron endowment is dependent on the timing of the growth restriction. Our findings demonstrate that reduced birth weight per se does not necessarily imply reduced nephron endowment.

Atrial natriuretic peptide inhibits angiotensin II-stimulated proliferation in fetal cardiomyocytes

The role of atrial natriuretic peptide (ANP) in regulating fetal cardiac growth is poorly understood. Angiotensin II (Ang II) stimulates proliferation in fetal sheep cardiomyocytes when growth is dependent on the activity of the mitogen‐activated protein kinase (MAPK) and phosphoinositol‐3‐kinase (PI3K) pathways. We hypothesized that ANP would suppress near‐term fetal cardiomyocyte proliferation in vitro and inhibit both the MAPK and PI3K pathways. Forty‐eight hour 5‐bromodeoxyuridine (BrdU) uptake (used as an index of proliferation) was measured in cardiomyocytes isolated from fetal sheep (135 day gestational age) in response to 100 nm Ang II with or without ANP (0.003–100 nm) or 1 μm 8‐bromo‐cGMP. The effects of these compounds on the MAPK and PI3K pathways were assessed by measuring extracellular signal‐regulated kinase (ERK) and AKT phosphorylation following 10 min of treatment with Ang II, ANP or 8‐bromo‐cGMP. In right ventricular myocytes (RV), the lowest dose of ANP (0.003 nm) inhibited Ang II‐stimulated BrdU uptake by 68%. Similarly, 8‐bromo‐cGMP suppressed Ang II‐stimulated proliferation by 62%. The same effects were observed in left ventricular (LV) cardiomyoytes but the RV was more sensitive to the inhibitory effects of ANP than the LV (P < 0.0001). Intracellular cGMP was increased by 4‐fold in the presence of 100 nm ANP. Ang II‐stimulated ERK and Akt phosphorylation was inhibited by 100 nm ANP. The activity of ANP may in part be cGMP dependent, as 8‐bromo‐cGMP had similar effects on the cardiomyocytes.

Restriction of placental vasculature in a non-human primate: A unique model to study placental plasticity

The limits of placental plasticity, i.e., the ability of the placenta to adapt and alter its growth trajectory in response to altered fetal requirements, are not known. We report fetal and placental hemodynamic adaptations in a novel non-human primate model in which the fetal inter-placental bridging vessels were surgically ligated. Doppler ultrasound studies showed that the rhesus placenta compensates for an approximate 40% reduction in functional capacity by increased growth and maintenance of umbilical volume blood flow. This unique experimental animal model has applications for mechanistic studies of placental plasticity and the impact on fetal development.

Cardiomyocyte enlargement, proliferation and maturation during chronic fetal anaemia in sheep

Chronic anaemia increases the workload of the growing fetal heart, leading to cardiac enlargement. To determine which cellular process increases cardiac mass, we measured cardiomyocyte sizes, binucleation as an index of terminal differentiation, and tissue volume fractions in hearts from control and anaemic fetal sheep. Fourteen chronically catheterized fetal sheep at 129 days gestation had blood withdrawn for 9 days to cause severe anaemia; 14 control fetuses were of similar age. At postmortem examination, hearts were either enzymatically dissociated or fixed for morphometric analysis. Daily isovolumetric haemorrhage reduced fetal haematocrit from a baseline value of 35% to 15% on the final day (P < 0.001). At the study conclusion, anaemic fetuses had lower arterial pressures than control fetuses (P < 0.05). Heart weights were increased by 39% in anaemic fetuses compared with control hearts (P < 0.0001), although the groups had similar body weights; the heart weight difference was not due to increased ventricular wall water content or disproportionate non‐myocyte tissue expansion. Cardiomyocytes from anaemic fetuses tended to be larger than those of control fetuses. There were no statistically significant differences between groups in the cardiomyocyte cell cycle activity. The degree of terminal differentiation was greater in the right ventricle of anaemic compared with control fetuses by ∼8% (P < 0.05). Anaemia substantially increased heart weight in fetal sheep. The volume proportions of connective and vascular tissue were unchanged. Cardiomyocyte mass expanded by a balanced combination of cellular enlargement, increased terminal differentiation and accelerated proliferation.