There is a remarkable change in the structure and function of the pulmonary vasculature during the transition from fetal to extrauterine life. In the fetus, the pulmonary vasculature is a high resistance low flow system, and thus right ventricular output is shunted to the placenta for gas exchange. At birth, even with the first breath, pulmonary vascular resistance falls dramatically, and pulmonary blood flow increases rapidly. Failure of this transition can lead to serious neonatal lung disorders characterized by the clinical syndrome of Persistent Pulmonary Hypertension of the Newborn (PPHN) [
The role of vasoactive mediators in achieving successful pulmonary vascular remodeling at birth has begun to emerge [
Current data suggests that disorders associated with PPHN have an overall mortality rate of 10–20%, although ACD/MPV is uniformly lethal despite the use of inhaled nitric oxide therapy often combined with extracorporeal membrane oxygenation [
Thus, we hypothesize that human pulmonary SERT may play a role in the adaptation of fetal pulmonary vasculature to extrauterine life and that abnormalities of SERT activity lead to neonatal pulmonary hypertension. Here we present a study of SERT expression in pre- and postnatal human lungs and in cases of newborn and childhood PHT of varied etiology including ACD/MPV.
Approval for this study was obtained through review by the University of Pittsburgh and Baylor College of Medicine Institutional Review Boards.
Microarray of pulmonary tissue obtained from multiple autopsy cases was employed to establish the specificity and sensitivity of SERT immunostaining of lung endothelium. Additionally, SERT immunostaining of the vascular endothelial of different organs was similarly assessed.
Physiologic temporal-spatial expression of SERT was studied using lung sections from 30 autopsy cases encompassing a range of ages, including 27 cases of 12, 13, 16, 18, 34, 39 and 40 weeks of gestational age (WGA) and 3 postnatal lungs of ages 1, 5, and 10 years. Cases with significant pulmonary pathology were excluded from this group.
SERT expression in a variety pulmonary hypertensive disorders was studied employing lung sections obtained at autopsy from 29 children with PHT due to ACD/MPV (
ACD/MVP patient population with testing for mutation and/or deletions in
Serial number | Gender | Survival in days | Gestation | Weight | Diagnosis | FOXF1 mutation | FOXF1 deletion | Other associated anomalies |
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1 | F | 5 | 39-5/7 | 3850 | Autopsy | NM | NT | Coarctation of aorta; ventricular septal defect; mitral valve malformation; tricuspid valve malformation; malformed atrial septum |
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2 | M | 22 | 39 | 3240 | Autopsy | NM | NT | Bilateral hydronephrosis secondary to posterior urethral valves |
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3 | F | 33 | 36-1/2 | 3120 | Autopsy | NM | NT | None |
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4 | M | 12 | 37 | N/A | Autopsy | NM | NT | Right lung with two lobes |
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5 | M | 4 | 38 | 3295 | Autopsy | NM | NT | Marked hypertrophy of urinary bladder wall; bilaterally dilated ureters and renal pelves |
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6 | F | 9 | 40 | 4180 | Autopsy | NM | NT | Butterfly vertebra; imperforate anus; abnormal pulmonary lobar configuration |
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7 | F | 30 | 41-2/7 | 3595 | Autopsy | NM | NT | Small thymus with adrenal glands; single umbilical artery |
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8 | M | 47 | 40 | 3025 | Autopsy | NM | ND | Bilateral ureteropelvic junction obstruction; hydronephrosis |
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9 | M | 10 | 32 | 2470 | Autopsy | c.225C>A; |
Bilateral bilobed lungs; malrotation of the intestine; bilateral hydronephrosis and hydroureters; annular pancreas and gastroduodenal stenosis | |
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10 | M | 0.5 | 32 | 2435 | Autopsy | c.850dupT; |
Lymphangiectasia; renal cortical cysts; pneumothorax; renal obstructive dysplasia | |
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11 | M | 46 | 40 | 5057 | Autopsy | c.1138T>C; |
Malrotation of the colon with Meckel’s diverticulum | |
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12 | F | 13 | 40 | 3600 | Biopsy | NM | Upstream | None |
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13 | F | 25 | N/A | 3676 | Biopsy | NM | Genic | Abnormal lobation |
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14 | M | 40 | 38 | 2900 | Autopsy | NM | Genic | Ventricular septal defect; duodenal atresia; annular pancreas; imperforate anus; hydronephrosis; tetralogy of Fallot |
NM, no mutation; NT, not tested; ND, tested no deletion; Genic, deletions involving
SERT (mouse monoclonal, clone MAB5618; Millipore, Temecula, CA, USA) and CD31 (mouse monoclonal, clone JC70A; Dako, Carpinteria, CA, USA) antibodies were used as follows: after the routine steps of deparaffinization and dehydration SERT antigen was retrieved by steam-heat treatment in 1 mM EDTA + 10 mM Tris buffer at pH 9.0. After incubation of primary SERT antibody for 30 minutes in room temperature at a dilution of 1/4000, diaminobenzidine (DAB) staining was carried out with utilization of the avidin-biotin-peroxidase system. All sections were counterstained with hematoxylin. Diffuse brown membranous endothelial staining was interpreted as positive while absence of brown staining was deemed negative. For positive controls brain sections including the Raphe nucleus (rich in serotoninergic neurons and recommended by manufacturer) were used; adrenal tissue was used as negative control (Figure
SERT immunohistochemistry was assessed and recorded as positive and negative. In addition, the terms diffuse and patchy were added for the distribution pattern of the stain.
The PCR was carried out in a TC-512 thermocycler (Techne, Minneapolis, MN) utilizing 100 ng template DNA using the following protocol: 5 min at 95°C, followed by 35 cycles of 1 min at 95°C, 1 min at 60°C, 2 min at 72°C, and final extension for 10 min at 72°C. The PCR products were purified using a Sephadex G-50 column and sequenced on a ABI 3130XL DNA Genetic Analyzer (Life Technologies, Grand Island, NY) following the manufacturer’s protocol. The primers used were as follows: forward-GCCAGCACCTAACCCCTAATG and reverse- GAATACTGGTAGGGTGCAAGGAG.
The autopsy tissue microarray showed that in normal lungs SERT is expressed specifically in pulmonary endothelial cells with a diffuse crisp brown staining after birth (Figure
Lungs from the antenatal period included all stages of lung development including pseudoglandular (
SERT expression was absent or only very minimal in the pulmonary microvasculature in every ACD/MPV case (Figures
Several SERT promoter defects have been linked to human disorders [
We found that SERT is specifically expressed in the microvasculature of the developing human lung beginning at about 30 WGA and that SERT expression increases near birth. SERT expression in the pulmonary microvasculature was absent in all 14 ACD/MPV patients tested.
We did not identify any decrease in SERT expression in the lungs of neonates and young children with PHT due to other causes. In addition, SERT promoter length polymorphism in all ACD/MPV patients was without abnormality.
Our study is the first direct evidence of a functional abnormality of the maldeveloped pulmonary microvasculature of ACD/MPV. In this uniformly lethal condition, death is associated with treatment-resistant PHT, suggesting functionally altered pulmonary vasculature [
The finding that the expression of SERT peaks at or near birth points to a regulatory role for SERT in governing normal pulmonary vascular growth and remodeling, particularly at the critical period near birth. SERT expression begins in the late saccular stage (30 WGA) while the morphologic changes of ACD/MPV begin earlier, likely before the canalicular stage (starting at ~20 WGA) [
Due to the rarity of ACD/MPV and because it has largely been defined histologically with most diagnoses made at autopsy examination, it has been very challenging to identify cases in which tissue appropriate for complex molecular studies can be obtained [
The genetics of SERT promoter regulation is complex and several abnormalities have been described that can lead to a decrease in SERT protein expression [
Most pediatric pathologists, radiologists, and neonatologists consider ACD/MPV to be a difficult to diagnose neonatal lung disease. Our study suggests that absence of SERT immunostaining in the pulmonary vasculature in infants and beyond is an excellent surrogate marker for this disorder, currently diagnosed only by microscopic examination of the lung with hematoxylin and eosin staining [
Because diminished SERT expression was unique to ACD/MPV patients with all other patients with neonatal or childhood PH diseases having normal SERT expression, our study provides evidence of a role for SERT-serotonin regulation in the pathogenesis of ACD/MPV. Since the precise mechanism leading to ACD/MPV is unknown, our findings could lead to focused studies to test SERT-relevant pathways [
We conclude that abnormalities in pulmonary SERT protein expression contribute to the pathomechanism of ACD/MPV. We speculate that the absence of SERT activity leading to high levels of serotonin results in the maintenance of high pulmonary vascular resistance and low pulmonary blood flow seen during fetal life and contributes to the characteristic vascular abnormalities and pulmonary hypertension of ACD/MPV.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors would like to thank Dr. Steven H Abman, Department of Pediatrics, University of Colorado, and Dr. Ron Jaffe, Division of Pediatric Pathology, University of Pittsburgh Medical Center, for their advice and help with this manuscript.