Open Access
Pavlovic, Jelena
Graduate Program:
Molecular Medicine
Doctor of Philosophy
Document Type:
Date of Defense:
May 08, 2006
Committee Members:
  • Joanna Floros, Committee Chair/Co-Chair
  • David Spencer Phelps, Committee Chair/Co-Chair
  • Andrew Thomas Henderson, Committee Member
  • Mary Howett, Committee Member
  • Hung Yu Lin, Committee Member
  • Judith Weisz, Committee Member
  • Brian Wigdahl, Committee Member
  • BPD
  • lung development
  • surfactant
  • RDS
  • xenograft
There are approximately 400, 000 infants born prematurely each year in the United States alone. Recent technological advances in perinatal and neonatal medicine have decreased morbidity and increased survival rates of prematurely born infants. Modern therapies, such as surfactant, steroids and gentler ventilation have lead to a drastically decreased age and weight of surviving newborns. As a result, there has been a change in incidence of neonatal pulmonary disease and its pathophysiology. Although current neonatal therapies have many advantages, they can cause long-term changes in lung physiology. Even though ventilatory support of premature neonates is essential, ventilation of the anatomically and biochemically immature lung structure results in alveolar simplification and damage. Neonatal steroid therapy, although beneficial for surfactant production, leads to the arrest of alveolarization that results in decreased number of alveoli. These changes have been proposed to have long term consequences that may extend into adulthood. Furthermore, the underlying genetic background appears to affect the outcome of premature birth and the differential response to therapy, such as steroids. There is a compelling need to study normal lung development, lung pathogenesis due to premature birth, and the effects of current and novel therapeutic treatments on both states. In order to shed light on these processes, a multifaceted but integrated approach is needed. Such approach should incorporate knowledge gained from models of lung development and lung disease, as well as knowledge derived from clinical and translational studies of neonatal pulmonary disease. The work presented in this thesis describes; 1) development of an in vivo model of human fetal lung development and; 2) a study of the genetic background of neonatal pulmonary lung disease. Specifically, this in vivo model allows for the study of human lung developmental processes and could be used to study the effects of current and novel therapeutic modalities on lung development and pathogenesis. In addition, the specific effects of these modalities can be studied by choosing fetal lungs with the genetic background of interest. Clinical and translational studies as the one presented in this thesis provide the starting point for further exploration of these processes. The focus of the studies presented here has been on the role of surfactant proteins in lung development and neonatal lung disease. These proteins are good candidate genes for these studies due to their essential role in lung development, lung maturation, and host defense. Development of a functional alveolar epithelium capable of gas exchange and surfactant secretion is essential for successful adaptation of the fetus to extra-uterine life. Premature birth is commonly characterized by a deficiency and perturbation of the surfactant system. Pulmonary surfactant is a lipoprotein complex produced by type II pneumocytes that acts to reduce surface tension at the air-liquid interface in the alveolus and, thereby, prevents atelectasis. Surfactant proteins (SP)-A, SP-B, SP-C, and SP-D have multiple important roles within the alveolus and are subject to developmentally and hormonally regulated expression. Specific aim I of this thesis describes an in vivo xenograft model of human fetal lung development. In this model, human fetal lung tissues were grafted either beneath the renal capsule or the skin of athymic mice (NCr-nu). Tissues were analyzed from 3 to 42 days post-grafting for morphological alterations by light and electron microscopy (EM), and for mRNA and protein content of surfactant proteins by reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry (ICC), respectively. The changes observed mimic those of human lung development in utero in many respects, including the differentiation of epithelium to the saccular stage. However, each stage of development occurred over approximately one week in the graft in contrast to the eight weeks of in utero development. At all time points examined, all four surfactant proteins (SP-A, SP-B, SP-C, and SP-D) were detected in the epithelium by ICC. Lamellar bodies were first identified by EM in 14 day xenografts. By day 21, significant increase was observed in both the number of lamellar bodies per cell and lamellar body positive cells. Cellular proliferation, as marked by proliferating cell nuclear antigen (PCNA) ICC and elastic fiber deposition resembled those of canalicular and saccular in utero development. Tissues that were grafted longer than 28 days, started to undergo distention of alveoli, presumably due to the accumulation of fluid. These findings indicate that the fetal lung xenograft model can serve as a valuable tool in the study of human fetal lung development. This model can provide the means to study the impact of various pharmacological agents on the development of human fetal lungs in general, and on the surfactant proteins in particular. In order to be able to better study the role of surfactant protein genetic variants in neonatal respiratory disease, rapid and accurate methods of genotyping are necessary. In this respect, Specific aim IIa of this thesis describes the development of a novel pyrosequencing based method for genotyping of single nucleotide polymorphisms of SP-A, SP-B, and SP-D. This primer extension sequencing method has been used to develop the following assays: 1) simplex and multiplex pyrosequencing assays for SP-A1 and SP-A2; 2) molecular haplotype assay for AA19(C/T) and AA50(C/G) SNPs of SP-A1; 3) simplex pyrosequencing assays for SP-B SNP B1580(C/T); 4) simplex and multiplex pyrosequencing assays for SP-D SNPs DA11(C/T) and DA160(A/G); and 5) assays for allele frequency determination in genomic DNA pools for DA11(C/T) and DA160(A/G) SNPs of SP-D. These assays greatly accelerate individual genotype analysis of surfactant proteins thus enabling efficient and reliable genotyping of SPs in samples from individuals with various pulmonary diseases. This high-throughput method of genotyping was applied to the study of surfactant protein genetic variants in Bronchopulmonary dysplasia (BPD) in Specific aim IIb. BPD is a chronic lung disease of light weight prematurely born infants that are mechanically ventilated from birth. It has been suggested that genetic factors contribute to BPD pathogenesis. We hypothesized that genetic variants of surfactant proteins are differentially responsive to disruption of surfactant homeostasis in premature birth and are either protection or susceptibility factors for BPD. In order to determine the role of surfactant protein (SP) genetic variants and microsatellite markers linked to SP-B, a family based association study was conducted using the Transmission Disequilibrium Test (TDT) and Family Based Association Test (FBAT). The study group consisted of 61 families with 71 BPD affected infants. SNP B-18_C of the SP-B gene was identified as susceptibility factor in BPD at 36 weeks (by TDT p=0.001 and by FBAT p=0.018). Allele 6 of SP-B linked microsatellite marker AAGG was found to be a susceptibility factor in BPD (by TDT p=0.004 and by FBAT p= 0.014). Haplotype analysis revealed two SP-A-SP-D susceptibility haplotypes, and eight susceptibility and three protective haplotypes for SP-B. These genetic association data identify variants of SP and SP-linked loci to be linked to BPD. Functional analysis of these variants should be investigated to better understand their role in BPD pathogenesis. Taken together, these studies provide insight into the mechanisms of lung development, dysregulation of lung development and perturbation of the surfactant system seen in premature birth, and the impact of surfactant protein genetic variants on neonatal respiratory disease.