2,4-Dichlorophenyl-p-nitrophenyl ether (nitrofen) is known to induce pulmonary hypoplasia (PH) with or without diaphragmatic hernias (DH) in rats and mice. We determined the timing of administration and dose of nitrofen needed to create left-sided DH and PH in fetal mice. Time-dated pregnant CD-1 mice were gavaged with various doses of nitrofen in the later one-half of gestational days (GD) 8-11. Fetuses were removed by laparotomy at GD 14, fixed, and evaluated histologically. Fetal lung size was inversely related to nitrofen dose. Morphometric analysis of normal and nitrofen-exposed hypoplastic lungs at the pseudoglandular stage revealed significant differences in lung length, surface area, and in the number of airways. Left-sided DH were observed in a "dorsolateral" position accompanied by PH in approximately 30% of GD 14 fetuses exposed to 25 mg nitrofen on GD 8. A minimal portion of liver was present in the hernia. The lungs of fetuses exposed on GD 9, 10, and 11 progressed to near normal size. Murine fetuses exposed to 25 mg nitrofen on GD 8 resulted in PH and DH, whereas other doses created dose-dependent PH alone or none at all on GD 11. Our study established that, to create left-sided DH and PH in murine fetuses, nitrofen dose specificity and time of administration during gestation were crucial.
We hypothesized that exposure of murine fetuses to environmental toxins, such as nitrofen, during early embryogenesis alters vasculogenesis. To address our hypothesis, we assessed protein levels of endothelial cell-selective angiogenic factors: angiopoietin (ANG)-1, vascular endothelial growth factor (VEGF), and mediator of VEGF signaling, VEGF receptor-2 [fetal liver kinase (Flk)-1], a transmembrane receptor tyrosine kinase. VEGF and Flk-1 proteins were lower in hypoplastic lungs from pseudoglandular to alveolar stages than in normal lungs at equivalent developmental time points significant for induction of pulmonary vasculogenesis and angiogenesis. ANG-1 protein was higher in hypoplastic lungs than in normal lungs at all the developmental stages considered in this study, i.e., pseudoglandular, canalicular, saccular, and alveolar stages. We assessed exogenous VEGF-mediated endothelial cell response on extracellular signal-regulated kinase (ERK) 1/2, also referred to as p44/42 mitogen-activated protein kinase. Hypoplastic lungs had more elevated ERK 1/2 protein than normal developing lungs. Exposure to exogenous VEGF activated ERK 1/2 in normal developing lungs but not in hypoplastic lungs. Our results suggest that in hypoplastic lungs: 1) low VEGF signifies negative effects on vasculogenesis/angiogenesis and indicates altered endothelial-mesenchymal interactions; 2) increased ANG-1 protein may be required to maintain vessel integrity and quiescence; and 3) regulation of ERK 1/2 protein is affected in hypoplastic lungs. We speculate that extensive remodeling of blood vessels in hypoplastic lungs may occur to compensate for structurally and functionally defective vasculature.
Although several studies have shown that an induction of insulin-like growth factor (IGF) components occurs during hyperoxia-mediated lung injury, the role of these components in tissue repair is not well known. The present study aimed to elucidate the role of IGF system components in normal tissue remodeling. We used a rat model of lung injury and remodeling by exposing rats to > 95% oxygen for 48 h and allowing them to recover in room air for up to 7 days. The mRNA expression of IGF-I, IGF-II, and IGF-1 receptor (IGF-1R) increased during injury. However, the protein levels of these components remained elevated until day 3 of the recovery and were highly abundant in alveolar type II cells. Among IGF binding proteins (IGFBPs), IGFBP-5 mRNA expression increased during injury and at all the recovery time points. IGFBP-2 and -3 mRNA were also elevated during injury phase. In an in vitro model of cell differentiation, the expression of IGF-I and IGF-II increased during trans-differentiation of alveolar epithelial type II cells into type-I like cells. The addition of anti-IGF-1R and anti-IGF-I antibodies inhibited the cell proliferation and trans-differentiation to some extent, as evident by cell morphology and the expression of type I and type II cell markers. These findings demonstrate that the IGF signaling pathway plays a critical role in proliferation and differentiation of alveolar epithelium during tissue remodeling.
The organogenesis of lung involves several complex mechanisms, including interactions between cells originating from two germ layers--endoderm and mesoderm. Regulation of lung branching morphogenesis with reference to its architecture, growth pattern, differentiation, interactions between epithelium and mesenchyme and / or endothelium, as well as genes regulating these processes have been addressed by the pulmonary biologists through careful molecular biology and genetic experimental approaches. The mammalian lung develops by outpouching from the foregut endoderm as two lung buds into the surrounding splanchnic mesenchyme. Several different regions of the foregut are specified to develop into different thoracic and visceral organs. The lung-buds further elongate and branch, and the foregut longitudinally gets separated into esophagus and trachea. In rodents (mice and rats), this occurs around embryonic day 11, where the right lung bud develops into four different lobes and left lung develops as a single lobe. In humans, these processes occur by 3-4 weeks of embryonic development, where the right lung is a trilobar lung and the left lung is a bilobar lung. Several generations of dichotomous branching occur during embryonic development, followed by secularization and alveolarization pre- and post-natally, which transform a fluid-filled lung into an air-breathing lung able to sustain the newborn. During these different developmental stages from embryonic to newborn stage, the lung architecture undergoes profound changes, which are marked by a series of programmed events regulated by master genes (e.g., homeobox genes), nuclear transcription factors, hormones, growth factors and other factors. These programmed events can be altered by undesirable exposure to overdoses of hormones/vitamins/growth factors, synthetic drugs, environmental toxins, radiation and other agents. In the recent years molecular techniques have opened avenues to study specific functions of genes or their products (proteins) in vivo or in vitro at a cellular or an organelle level, some of these include targeted disruption, knock-in / knock-out genes, in vitro mutagenesis, use of sense and anti-sense oligonucleotides. Some of these aspects with reference to regulation of normal lung development and growth and a specific example of pulmonary hypoplasia as an abnormal lung formation are discussed in this review.
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