Lowell Davis

Cardiac hypertrophy in chronically anemic fetal sheep: Increased vascularization is associated with increased myocardial expression of vascular endothelial growth factor and hypoxia-inducible factor 1

OBJECTIVE Our purpose was to determine whether the increase in fetal cardiac mass and cardiac output in chronic anemia is accompanied by changes in capillary density or size or changes in levels of vascular endothelial growth factor and hypoxia-inducible factor 1, a basic helix-loop-helix transcription factor that has previously been shown to activate vascular endothelial growth factor gene transcription when cultured cells are subjected to hypoxia. STUDY DESIGN Anemia was induced in near-term ovine fetuses by daily isovolemic hemorrhage. In five fetuses the heart was arrested in diastole, isolated, and fixed at physiologic pressures with adenosine-paraformaldehyde, and morphometric measurements of capillaries were made. In six fetuses cardiac expression of vascular endothelial growth factor and hypoxia-inducible factor 1 protein was detected by Western analysis and vascular endothelial growth factor messenger ribonucleic acid by Northern blot analysis. Eleven age-matched fetuses served as controls. RESULTS The anemic fetuses compared with controls had a lower hematocrit (14.8% +/- 0.7% vs 35.3% +/- 1.5%) and a greater heart-to-body weight ratio (10.5 +/- 1.1 vs 7.7 +/- 0.5 gm/kg). The minimal capillary diameter was increased and the intercapillary distance was decreased in both right and left ventricles of anemic fetuses compared with controls. Vascular endothelial growth factor protein was increased 4.5-fold, vascular endothelial growth factor messenger ribonucleic acid 3.2-fold, and hypoxia-inducible factor 1alpha protein 3.8-fold in ventricular tissue from anemic fetuses. CONCLUSIONS In chronic fetal anemia cardiac hypertrophy is accompanied by anatomic changes in myocardial capillary morphometry along with induction of hypoxia-inducible factor 1 and vascular endothelial growth factor. These results provide evidence for a pathway by which anemia-hypoxia may stimulate myocardial vascularization.

Validation of the Myocardial Performance Index by Echocardiography in Mice: A Noninvasive Measure of Left Ventricular Function

BACKGROUND The myocardial performance index (MPI) is a Doppler-based measure of left ventricular (LV) function. It is noninvasive, independent of LV shape, and does not require dimensional measurements. However, it has never been validated in mice. METHODS A total of 29 anesthetized mice with LV pressure catheters underwent echocardiography (2-dimensional, M-mode, and Doppler) at baseline and during manipulations of beta-adrenergic tone, temperature, preload, and afterload. The maximum derivative of LV pressure with respect to time (dP/dt(max)) was compared with MPI, fractional shortening (FS), mean velocity of circumferential fiber shortening, and the FS/MPI ratio. RESULTS MPI (baseline 0.44 +/- 0.07) correlated strongly with dP/dt(max) (R = -.779, P <.001), as did FS and mean velocity of circumferential fiber shortening. MPI differed significantly with contractility, preload, and afterload manipulation. FS/MPI showed the best correlation with dP/dt(max). CONCLUSIONS MPI strongly correlates with dP/dt(max) over a range of hemodynamic conditions in mice. It can be used as a noninvasive index of LV function in this species.

Renal and placental secretion of erythropoietin during anemia or hypoxia in the ovine fetus

OBJECTIVE The source of the erythropoietin (EPO) that circulates in the fetus is unknown although it is known that EPO does not cross the placenta and that fetal kidneys, liver, and placenta express the EPO gene. This study tested to what extent in vivo EPO secretion by the fetal kidneys and placenta can be demonstrated under normoxic and hypoxic conditions. STUDY DESIGN Renal arterial and venous EPO concentrations were determined in eight late-gestation chronically catheterized fetal sheep made progressively anemic by exchange transfusion with saline solution over 5 to 8 days. In a separate additional series of experiments, umbilical arterial and venous EPO concentrations were determined in nine normoxic fetuses and in nine fetuses subjected to 12 hours of hypoxia induced by lowering maternal-inspired oxygen content. Organ secretion rates were calculated as the product of plasma flow rate and the arteriovenous concentration differences. RESULTS Renal vein plasma EPO concentration was higher than the arterial concentration in 36 of 40 paired samples (P<.0001) by 16.3%+/-2.7% (mean+/-SE). This difference was concentration independent over a range of 12 to 4100 mU/mL. Renal EPO secretion rates were variable and averaged 155+/-105 mU/min when hematocrit was 31.3%+/-1.6% (n=5) and 1124+/-300 mU/min post-exchange transfusion when hematocrit was 15.6%+/-0.8% (n=12). In contrast, umbilical venous and arterial EPO concentrations (range 9-35 mU/mL), although highly correlated (r=0.94), were not different during normoxia (Po(2)=21.6+/-0.5 mm Hg, n=9). Under hypoxic conditions (Po(2)=15.6+/-0.4 mm Hg, n=9), umbilical vein EPO concentration (range 151-1245 mU/mL) was higher than arterial concentration (range 140-951 mU/mL) in eight of nine paired samples by 13.6%+/-3.3% (P<.01). Under these conditions, estimated umbilical EPO secretion rate was 27,900+/-11,500 mU/min. CONCLUSION Under nonanemic, normoxic basal conditions, the kidneys secreted EPO into the fetal circulation, whereas secretion by the placenta was not demonstrated. In the phlebotomy-induced fetal anemia experiments, the kidney demonstrated marked, progressive increases in the rate of EPO production. Similarly, in the fetal hypoxemia experiments, the placenta demonstrated progressive increases--albeit an order of magnitude greater than the kidneys--in EPO production rate. As an extension of these findings, we speculate that the hypoproliferative neonatal anemia that invariably occurs in the early weeks after birth is in part the result of loss of EPO production by the placenta.

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.

Augmentation of coronary conductance in adult sheep made anaemic during fetal life

Maximal coronary conductance with adenosine in anaemic fetal sheep is twice that of non‐anaemic fetuses. To investigate whether this increase in conductance persists into adulthood we studied twin sheep as fetuses and again as adults. Nine anaemic fetuses (118 days gestation) underwent isovolaemic haemorrhage for 18.0 ± 4.6 days (means ±s.d.) during which time the haematocrit was reduced from 39.9 ± 5.2 % to 16.3 ± 3.4 % and oxygen content from 8.6 ± 1.3 to 2.3 ± 0.2 ml dl−1. At 138 days the anaemic fetuses were transfused; at delivery the haematocrit was 29.3 ± 6.8 % compared to nine control fetuses in which the haematocrit was 38.5 ± 4.3 %. The weight at delivery was 3.5 ± 0.36 kg in the anaemic fetuses vs. 4.2 ± 0.83 kg in controls. Twenty‐eight weeks later, we placed an occluder on the descending thoracic aorta and inferior vena cava, a flow probe around the proximal left circumflex coronary artery, and catheters in the left atrial appendage, jugular and carotid vessels. Maximal coronary conductance was determined in the adults by recording coronary blood flow as driving pressure was altered by inflating the occluders while adenosine was infused into the left atrium. Right atrial, left atrial, systolic and mean arterial pressures, systemic vascular resistance and haematocrit were not different between ‘in utero anaemic’ and control adults. The adults that were anaemic in utero weighed less than the controls 39.4 ± 4.6 kg vs. 45.0 ± 5.6 kg. Maximal conductance was greater in the adults that were anaemic in utero: 11.2 ± 4.0 ml min−1 (100 g)−1 mmHg−1 as compared to 6.1 ± 1.8 ml min−1 (100 g)−1 mmHg−1 in the controls. Vascular reactivity of the mesenteric arteries was not different. These data suggest that coronary conductance can be modified in utero by anaemia (high flow and hypoxaemia) and that the remodelled coronary tree persists to adulthood.

Myocardial blood flow and coronary reserve in chronically anemic fetal lambs.

Chronic fetal anemia produces large compensatory increases in coronary blood flow in the near-term fetal lamb. To determine if increased coronary flow in anemic fetuses is associated with decreased coronary flow reserve or, alternatively, an increase in coronary conductance, we measured maximal coronary artery conductance during adenosine infusion before and during anemia. Isovolemic hemorrhage over 7 days reduced hematocrit from 30.6 +/- 2. 7 to 15.8 +/- 2.4% (P < 0.02) and the oxygen content from 7.3 +/- 1. 4 to 2.6 +/- 0.4 ml/dl (P < 0.001). Coronary blood flow increased from control (202 +/- 60) to 664 +/- 208 ml. min(-1). 100 g(-1) with adenosine to 726 +/- 169 ml. min(-1). 100 g(-1) during anemia and to 1,162 +/- 250 ml. min(-1). 100 g(-1) (left ventricle) during anemia with adenosine infusion (all P < 0.001). Coronary conductance, determined during maximal vasodilation, was 18.2 +/- 7.7 before and 32.8 +/- 11.9 ml. min(-1). 100 g(-1). mmHg(-1) during anemia (P < 0. 001). Coronary reserve, the difference between resting and maximal myocardial blood flow interpolated at 40 mmHg, was unchanged in control and anemic fetuses (368 +/- 142 and 372 +/- 201 ml/min). Because hematocrit affects viscosity, anemic fetuses were transfused with blood to acutely increase the hematocrit back to control, and conductance was remeasured. Coronary blood flow decreased 57.3 +/- 18.9% but was still 42.6 +/- 18.9% greater than control. We conclude that in chronically anemic fetal sheep coronary conductance is increased and coronary reserve is maintained, and this is attributed in part to angiogenesis as well as changes in viscosity.Chronic fetal anemia produces large compensatory increases in coronary blood flow in the near-term fetal lamb. To determine if increased coronary flow in anemic fetuses is associated with decreased coronary flow reserve or, alternatively, an increase in coronary conductance, we measured maximal coronary artery conductance during adenosine infusion before and during anemia. Isovolemic hemorrhage over 7 days reduced hematocrit from 30.6 ± 2.7 to 15.8 ± 2.4% ( P < 0.02) and the oxygen content from 7.3 ± 1.4 to 2.6 ± 0.4 ml/dl ( P < 0.001). Coronary blood flow increased from control (202 ± 60) to 664 ± 208 ml ⋅ min-1 ⋅ 100 g-1 with adenosine to 726 ± 169 ml ⋅ min-1 ⋅ 100 g-1 during anemia and to 1,162 ± 250 ml ⋅ min-1 ⋅ 100 g-1 (left ventricle) during anemia with adenosine infusion (all P< 0.001). Coronary conductance, determined during maximal vasodilation, was 18.2 ± 7.7 before and 32.8 ± 11.9 ml ⋅ min-1 ⋅ 100 g-1 ⋅ mmHg-1during anemia ( P < 0.001). Coronary reserve, the difference between resting and maximal myocardial blood flow interpolated at 40 mmHg, was unchanged in control and anemic fetuses (368 ± 142 and 372 ± 201 ml/min). Because hematocrit affects viscosity, anemic fetuses were transfused with blood to acutely increase the hematocrit back to control, and conductance was remeasured. Coronary blood flow decreased 57.3 ± 18.9% but was still 42.6 ± 18.9% greater than control. We conclude that in chronically anemic fetal sheep coronary conductance is increased and coronary reserve is maintained, and this is attributed in part to angiogenesis as well as changes in viscosity.

Responses of amniotic fluid volume and its four major flows to lung liquid diversion and amniotic infusion in the ovine fetus.

We designed experiments to allow direct measurement of amniotic fluid volume and continuous measurement of lung liquid production, swallowing, and urine production in fetal sheep. From these values, the rate of intramembranous absorption was calculated. Using this experimental design, the contribution of lung liquid to the control of amniotic fluid volume was examined. Fetuses were assigned to 1 of 4 protocols, each protocol lasting 3 days: control, isovolemic replacement of lung liquid, supplementation of amniotic fluid inflow by 4 L/day, and supplementation of amniotic inflow during isovolemic replacement of lung liquid. We found no effect of lung liquid replacement on any of the known flows into and out of the amniotic fluid. Although intramembranous absorption increased greatly during supplementation, the amniochorionic function curves were not altered by isovolemic lung liquid replacement. We conclude that lung liquid does not appear to contain a significant regulatory substance for amniotic fluid volume control.

Enlarged cisterna magna in trisomy 18: prenatal ultrasonographic diagnosis.

Ultrasonographic visualization of an enlarged cisterna magna was the initial clue that led to the prenatal diagnosis of trisomy 18 in five patients. Prenatal diagnosis of trisomy 18, a lethal defect, is important for proper patient counseling and management.

Anaemia Stimulates Aquaporin 1 Expression in the Fetal Sheep Heart

Interstitial fluid fluxes are much greater in the fetus than in the adult, and filtration rates are increased over control in most tissues of the anaemic fetus. Increased capillary filtration may lead to cardiac oedema which, in turn, severely impacts cardiac function. Mechanisms that underlie these differences in flux are incompletely understood. One possible mechanism is an increase in capillary water permeability. Therefore, the goal of our study was to determine the level of expression of the water channel aquaporin 1 (AQP1) during cardiac development and in the anaemic fetal sheep heart. Hearts from chronically instrumented anaemic sheep fetuses and hearts from normal early fetal, late fetal, neonatal and adult sheep were used for Northern and Western analyses and immunohistochemistry. We found that AQP1 mRNA levels were lower in the young fetal left ventricle than in the adult left ventricle (P < 0.05). We also found that cardiac AQP1 expression was increased in anaemic fetuses compared to age‐matched controls (P < 0.05). Expression of AQP1 in all groups was greatest in the microvascular endothelium. These data suggest that AQP1 plays an important role in the physiological accommodation to fetal anaemia.

Intramembranous solute and water fluxes during high intramembranous absorption rates in fetal sheep with and without lung liquid diversion

OBJECTIVE To examine mechanisms that mediate increased intramembranous solute and water absorption. STUDY DESIGN Intramembranous solute and water fluxes were measured in fetal sheep under basal conditions and after intraamniotic infusion of lactated Ringers solution of 4 L/d for 3 days with and without lung liquid diversion. RESULTS Intramembranous sodium, potassium, chloride, calcium, glucose, and lactate fluxes increased 2.5- to 7.9-fold, were linearly related to volume fluxes (r = 0.83-0.99), and were unaffected by lung liquid. All clearance rates, except that of lactate, increased to equal the intramembranous volume absorption rate during infusion. CONCLUSION Under basal conditions, passive diffusion makes a minor and bulk flow a major contribution to intramembranous solute absorption. During high absorption rates, the increase in solute absorption above basal levels appears to be due entirely to bulk flow and is unaffected by lung liquid. The increased bulk flow is consistent with vesicular transcytosis.