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Dramatic changes in pulmonary circulation occur shortly after birth with the onset of breath. Fetal adaptation to the extrauterine life consists in a rapid fall in pulmonary vascular resistance and in an increase in pulmonary blood flow (1). It has been demonstrated that several vasoactive agents such as bradykinin, acetylcholine, prostacyclin, and nitric oxide decrease vascular resistance and increase blood flow in fetal lung (2). However, the mechanisms involved in this physiologic adaptation have not been fully elucidated.

Adrenomedullin is a novel peptide first isolated human pheochromocytoma eliciting a long-lasting vasorelaxant activity (3).

Recently De Vroomen et al. (4) have studied the effects of adrenomedullin administration on fetal pulmonary arterial blood flow in fetal sheep, showing that this peptide induces pulmonary vasodilation. Moreover, we have demonstrated the presence of high concentrations of adrenomedullin in cord plasma and amniotic fluid in human term pregnancy (5) and of immunoreactive adrenomedullin in placenta and fetal membranes (6). The aim of this study was to confirm the hypothesis of a physiologic role of adrenomedullin in the regulation of pulmonary circulation at birth (4) determining, in humans, its presence and its localization during fetal lung development and after delivery.

MATERIAL AND METHODS

Population. We collected lung tissues from six abortive fetuses (18-24 wk of gestation), four preterm neonates (27-35 wk), three term infants, and two adults as controls. Characteristics of studied cases are shown in Table 1. The protocol of the study was approved by the University Ethical Committee. Tissues were collected after written permission from relatives.

Table 1 Characteristics of studied cases
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Immunohistochemistry. Tissues were fixed in 4% paraformaldehyde:0.2% gluteraldehyde, washed, and embedded in paraffin as reported previously (7). Sections 5 µm thick were cut and stained by means of the avidin-biotin peroxidase technique (Vector ABC, Vector Laboratories, Burlinghame, CA) using a polyclonal antibody raised in rabbits against purified human adrenomedullin 1-52 (Peninsula Laboratories, Inc., Belmont, CA) at a dilution of 1:600. Negative controls consisted of lung tissue sections incubated with either nonimmune rabbit serum, antibody dilution buffer or with the primary antibody preabsorbed with an excess of human adrenomedullin (1 µM). Sections of human rectus abdominis muscle tissue were stained for adrenomedullin at different dilutions (1:500, 1:750, 1:1000) as negative control. The number of slides examined for each case ranged from 2 to 5, with a median of 3, and both lungs were evaluated.

RESULTS

Adrenomedullin was detected in fetal lungs collected as early as at 18 wk of gestation and was identified in all specimen examined throughout lung development and growth period. The localization of adrenomedullin immunoreactivity was different in specimen collected before and after birth.

Positive immunostaining for adrenomedullin was observed in endothelial cells of specimen collected either from fetuses at different gestational ages or from newborns died after breathing (Fig. 1). Moreover, during fetal development, adrenomedullin was also localized in the cytoplasm of bronchial columnar epithelium cells with an apical distribution. The intensity of immunostaining and the percentage of positive cells increased as gestation progressed, and there was no association with maternal or fetal clinical features. In contrast, bronchial epithelial cells of neonatal lung in specimen obtained after breathing (two cases at term and two preterm) did not stain for adrenomedullin (Fig. 2), independently from the time of surviving. The same pattern of distribution was present in adults, in whom immunoreactive adrenomedullin was localized exclusively in the endothelial cells.

Figure 1
figure 1

Immunohistochemistry for adrenomedullin in endothelial cells lung vessels in specimen collected at: (A) 18 wk of gestation (case 3) and (B) 7 d in term neonate (case 12). Arrows indicate endothelial cells. (Magnification × 160).

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Figure 2
figure 2

Immunohistochemistry for adrenomedullin in bronchial cells (arrows) in specimen collected from fetuses at: (A) 18 wk (case 4), (B) 24 wk (case 5), (C) 30 wk (case 7), (D) 38 wk (case 11) of gestation, and from (E) preterm neonates died after 18 h (case 10), (F) term neonate died after 5 d (case 13). Adrenomedullin immunostaining increases as gestation progresses during lung development and it is absent in bronchial cells after breathing. (Magnification × 100).

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No staining was detected in negative controls.

DISCUSSION

This study indicates that adrenomedullin is present in human fetal lung during ontogenesis and that its localization and distribution changes with the events related to birth.

At birth, pulmonary vascular resistance decreases dramatically, allowing pulmonary blood flood to increase and oxygen exchange to occur. The drop of pulmonary vascular resistance reflects the vasoactive effects of locally acting endothelial-derived agents (i.e. nitric oxide, prostacyclin, endothelin). The presence of adrenomedullin in endothelial cells of pulmonary vessels during fetal life and after birth, together with high circulating adrenomedullin levels in the fetus, suggests that this peptide together with endothelial-derived factors may play a role in decreasing and maintaining a low resistance in pulmonary circulation.

Immunoreactive adrenomedullin in bronchial cells during fetal lung development suggests a role of this peptide also in the regulation of bronchial tone, which is important in determining adequacy of neonatal alveolar ventilation at birth. It has been shown that adrenomedullin is a potent bronchodilator peptide (8) and mRNA for adrenomedullin has been detected in the columnar epithelium and in some glands of normal human lung in adults (9). However, in our study, adrenomedullin immunoreactivity was restricted to endothelial cells and was absent in the epithelium of the lungs. The difference in adrenomedullin immunostaining distribution between fetal and neonatal tissues may suggest that adrenomedullin production from bronchial cells is decreased in functional lung after the process of adaptation to air-breathing. Increased PaO2 may regulate directly, or indirectly through other agents such as nitric oxide (10), adrenomedullin production, and/or release. Recently, it has been found a negative correlation between adrenomedullin concentration and pH values in cord blood at birth (11). Alternatively, lower adrenomedullin immunostaining may also derive from a decreased site of binding at this level, or from an increase in pulmonary adrenomedullin clearance after the normal postnatal adaptation of the lung as demonstrated in piglets (12). We cannot, however, exclude the possibility that the causes of neonatal death may affect adrenomedullin expression in the lung. Thus the absence of the peptide in these tissues could not reflect the physiologic features of normal lung.

Adrenomedullin immunostaining as early as at 18 wk of gestation and its increase during fetal lung development, from the late organogenetic period to term, may indicate a role of this peptide in the mechanisms of fetal lung differentiation and/or maturation. Several studies have already shown that adrenomedullin acts as a true growth factor in normal and malignant tissues (13) and the same adrenomedullin immuno-reactivity pattern that we found in human fetal lungs has been demonstrated in developing mouse and rat epithelial lining bronchial cells with a distribution similar to that of platelet-derived growth factor, transforming growth factor-β, fibroblast growth factors, and insulin-like growth factor I and II (14). Moreover, it has been shown that adrenomedullin increases intracellular cAMP, which affects significantly fetal lung maturity promoting glycogen degradation, essential for the synthesis of surfactant phospholipids and for lung growth and that adrenomedullin secretion is stimulated by thyroid hormones and glucocorticoids (15), both of which are implicated in the stimulation of fetal lung maturity. Therefore, adrenomedullin in columnar epithelial cells of bronchial system may also have an important function in the complex process leading to fetal lung maturation.

In conclusion, this study has shown that adrenomedullin is present in human fetal lung and that its localization and distribution changes during ontogenesis and after birth; however, further studies are needed to clarify the mechanism(s) in which it is involved.