Human Fetal Lung Explants Research Articles

Hedgehog Pathway regulates Alveolar Type II Cells in Human Lung Development

Rationale: The Hedgehog (HH) pathway plays an important role in human lung branching morphogenesis, yet its role in human alveologenesis remains understudied. Bronchopulmonary Dysplasia (BPD) patients exhibit simplified alveolar structures, accompanied with increased expression of HH components. Therefore, we hypothesize that HH pathway regulation is crucial for alveologenesis, specifically for proximo-distal patterning and AT2 cell differentiation. Methods: Human fetal lung explants aged 18-22 weeks gestation (WG) were cultured in CKDCI media to induce alveolar marker expression, in the presence or absence of HH activator (SAG). AT2 progenitor cells (HTII280+/CD31-/CD45-) were isolated from fresh human lung tissues (18-22 WG) and plated in Matrigel for 14 days to form organoids. A subset of organoids was treated with SAG for 7 days while in matrigel, while others were lifted to air-liquid interface (ALI) culture and treated for 14 days. RT-qPCR, fluorescent in situ hybridization (FISH), and immunofluorescence staining (IF) were carried out at various steps to assess cell fate. Results: SAG treated human fetal lung explants displayed gross morphological differences compared to the control, e.g. enlarged distal cysting. HH pathway upregulation resulted in a significant decrease of AT2 markers expression ( ABCA3, LAMP3; p<0.05) and increased expression of proximal markers ( TP63, SCGB3A2; p<0.01) as compared to control. FISH for SFTPC (AT2 marker) showed a 3-fold decrease ( p<0.05) following HH pathway activation accompanied by a loss of SOX9+ cells (2.5-fold vs control, p<0.05). The loss of SFTPC+ cells was confirmed by IF ( p<0.05). A decrease in KRT8+, a marker of intermediate AT2 cells, was also observed (1.5-fold vs control, p<0.05). Similarly, in organoid cultures, we found decreased SFTPC+, SOX9+ and KRT8+ cells (1.5-; 2- and 1.5-fold vs control, p<0.05). Furthermore, SAG treatment resulted in the formation of fewer and smaller-sized organoids (1-fold and 1.5-fold vs control, p<0.05). Staining on ALI cultures revealed fewer SFTPC+ cells and more TP63+ cells (1.5-fold vs control, p<0.05). Tight junction proteins (ZO1 and Claudin18) were compromised on ALI membranes demonstrating that HH pathway activation results in larger and fewer tight junctions, as indicated by the TIJOR score (2-fold vs control, p<0.01). Conclusion: In our alveologenesis-like model, HH pathway activation reduces AT2 markers, favoring a proximal fate, ultimately suggesting that the HH pathway regulates late human lung development. Understanding the role of the HH pathway in regulating alveologenesis is essential in pediatric conditions like BPD and could facilitate translational discoveries. CIRM EDUC4-12837 (BR) NIH/NHLBI R01HL141856 (DA). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The effect of VEGF-A isoforms on capillary endothelial cell differentiation in human fetal lung development

Rationale: Single-cell RNA-sequencing has revealed two distinct capillary cell populations in the lung - general capillary cells (gCap) and alveolar capillary cells (aCap). Studies in mouse have demonstrated that VEGF-A188 promotes the formation of aCaps in vitro. Our study investigates the role of VEGF-A in endothelial cell (EC) differentiation into functional pulmonary capillaries during human lung development. We hypothesize that VEGFA-189, the human equivalent of murine VEGFA-188, promotes the formation of aCaps in the developing human lung. Methods: We examined the expression of the different VEGF-A isoforms ( VEGF-A121, 145, 165 and 189) on human fetal lungs aged 10-20 weeks gestation. RT-qPCR was performed to correlate the expression of VEGF-A isoforms to the expression of aCap makers ( CA4, EDNRB, SOSTDC1, HPGD, TBX2 and IL1RL1). In parallel to these analyses, we examined the effects of VEGF-A isoforms by culturing human fetal lung explants (13-20 weeks gestation) for either 2 or 7 days in the presence or absence of either recombinant human protein VEGF-A165 (20 ng/mL) or VEGF-A189 (80 ng/mL) (n=8). To verify this hypothesis of VEGF-A189, we selected VEGF-A165 which has one exon less (exon 6) than VEGF-A189. Explants were collected and analyzed for gCap ( EDN1) and aCap ( TBX2, EDNRB, SOSTDC1, HPGD, and CA4) markers expression by RT-qPCR. Immunofluorescence (IF) staining was also performed on explants to analyze the effect of VEGF-A isoforms on the proliferation of endothelial cell. Results: In native human fetal lung, we demonstrated a simultaneous expression of VEGF-A-121, 145 and 189 isoforms with several aCap markers ( SOSTDC1, EDNRB, HPGD), that peaked at around 18 weeks of gestation. Based on this, we decided to culture human fetal lung explants within this same developmental stage in the presence or absence of either VEGF-A165 or -189. Following 2 days treatment with VEGF-A165, RT-qPCR demonstrated that human fetal lung explants exhibited a significant increase in the expression of EDN1 (p<0.005), accompanied by a significant decrease in expression of CA4 (p<0.005). Interestingly, whereas VEGF-A189 also demonstrated decreased expression of several aCap markers ( CA4, EDNRB, SOSTDC1, and HPGD (p<0.01)) it also showed increased expression of TBX2 and EDN1 (p<0.05). Furthermore, IF staining displayed decreased Ki67/CD31 double positive cells on explants treated with VEGF-A189 with a more elongated and flattened morphology, much like what is observed in aCaps. Prolonging the treatment to 7 days yielded comparable results to the 2 days treatment, with VEGF-A165 and VEGF-A189 downregulating several aCap markers. Conclusion: Our study revealed that the VEGF-A189 isoform does not have the same effect on human lung capillary differentiation as observed in mouse. We noted a contrasting impact of VEGF-A isoforms on the expression of gCap and aCap markers and highlighted the significance of specific VEGF isoforms in capillary EC differentiation. Additional investigations are required to elucidate the underlying mechanisms. California Institute for Regenerative Medicine-CIRM. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Co-exposure to cannabis and nicotine during pregnancy alters fetal lung development

Rationale: The co-use of cigarettes and cannabis is increasing especially in regions where recreational marijuana use has become legal. However, the use of these dependent substances during pregnancy poses a serious risk on fetal development and health (low birth weight, preterm birth, and perinatal death). Nevertheless, the impact of co-exposure to the most prominent cannabinoids found in Cannabis sativa (Trans-Δ-9-tetrahydrocannabinol (THC) and cannabidiol (CBD)) with nicotine on fetal lung development is not well understood. This study aims to determine the effect of nicotine, CBD, and THC, alone or combined, on human lung development through investigating cellular senescence. Methods: In vitro human fetal lung explants from 10–20 weeks gestation were cultured on air-liquid interface and treated with physiological concentrations of CBD, THC, and nicotine, alone or combined, for 72h. Similarly, lung mesenchymal cells (LMCs) were isolated from the same samples and treated for 48h. Both culture types were either fixed for immunofluorescent staining or used for RNA extraction and qRT-PCR. Results: Our results showed that nicotine and CBD induce thickening of the mesenchymal compartment of lung explants. Furthermore, treatment with either CBD or THC, or the two combined, induced an increase in epithelial cysting. Ki-67 staining demonstrated increased proliferation following treatment with nicotine, THC and CBD+THC+nicotine. ACTA2 staining, a marker for smooth muscle cells, decreased following treatment with THC, CBD, and nicotine as well as all 3 combined, while the senescence marker p21 increased following exposure to nicotine, CBD+THC, as well as CBD+THC+nicotine. qRT-PCR data showed decreased expression of ACTA2 in the presence of CBD, THC, CBD+THC, and CBD+THC+nicotine, and increased expression of CDKN1A (encoding p21) and CDKN2A (encoding p16) in response to CBD+THC+nicotine. LMCs treated with nicotine, CBD, and THC demonstrated an increase in cell proliferation and senescence as shown by Ki-67 and p21 staining respectively, and a decrease in ACTA2. These data were confirmed by qRT-PCR that showed a decrease in ACTA2 for the LMCs treated with either nicotine or CBD+THC+nicotine and an increase in senescence genes CDKN1A and CDKN2A. Moreover, the expression of these genes was higher in the LMCs derived from second trimester samples in comparison to those of the first trimester. Conclusion: Our results showed that the co-exposure to nicotine and cannabis induces senescence and alters lung fetal cellular proliferation, especially when used in conjunction. Furthermore, this effect appears to be age dependent. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

FGF18 promotes human lung branching morphogenesis through regulating mesenchymal progenitor cells.

Fibroblast growth factor (FGF) signaling is known to play an important role in lung organogenesis. However, we recently demonstrated that FGF10 fails to induce branching in human fetal lungs as is observed in mouse. Our previous human fetal lung RNA sequencing data exhibited increased FGF18 during the pseudoglandular stage of development, suggestive of its importance in human lung branching morphogenesis. Whereas it has been previously reported that FGF18 is critical during alveologenesis, few studies have described its implication in lung branching, specifically in human. Therefore, we aimed to determine the role of FGF18 in human lung branching morphogenesis. Human fetal lung explants within the pseudoglandular stage of development were treated with recombinant human FGF18 in air-liquid interface culture. Explants were analyzed grossly to assess differences in branching pattern, as well as at the cellular and molecular levels. FGF18 treatment promoted branching in explant cultures and demonstrated increased epithelial proliferation as well as maintenance of the double positive SOX2/SOX9 distal bud progenitor cells, confirming its role in human lung branching morphogenesis. In addition, FGF18 treated explants displayed increased expression of SOX9, FN1, and COL2A1 within the mesenchyme, all factors that are important to chondrocyte differentiation. In humans, cartilaginous airways extend deep into the lung up to the 12th generation of branching whereas in mouse these are restricted to the trachea and main bronchi. Therefore, our data suggest that FGF18 promotes human lung branching morphogenesis through regulating mesenchymal progenitor cells.

Autophagy Is Impaired in Fetal Hypoplastic Lungs and Rescued by Administration of Amniotic Fluid Stem Cell Extracellular Vesicles.

Rationale: Pulmonary hypoplasia secondary to congenital diaphragmatic hernia is characterized by reduced branching morphogenesis, which is responsible for poor clinical outcomes. Administration of amniotic fluid stem cell extracellular vesicles (AFSC-EVs) rescues branching morphogenesis in rodent fetal models of pulmonary hypoplasia. Herein, we hypothesized that AFSC-EVs exert their regenerative potential by affecting autophagy, a process required for normal lung development. Objectives: To evaluate autophagy in hypoplastic lungs throughout gestation and establish whether AFSC-EV administration improves branching morphogenesis through autophagy-mediated mechanisms. Methods: EVs were isolated from c-kit+ AFSC-conditioned medium by ultracentrifugation and characterized for size, morphology, and EV markers. Branching morphogenesis was inhibited in rat fetuses by nitrofen administration to dams and in human fetal lung explants by blocking RAC1 activity with NSC23766. The expression of autophagy activators (BECN1 and ATG5) and adaptor (SQSTM1/p62) was analyzed invitro (rat and human fetal lung explants) and invivo (rat fetal lungs). Mechanistic studies on rat fetal primary lung epithelial cells were conducted using inhibitors for microRNA-17 and -20a contained in the AFSC-EV cargo and known to regulate autophagy. Measurements and Main Results: Rat and human models of fetal pulmonary hypoplasia showed reduced autophagy mainly at pseudoglandular and canalicular stages. AFSC-EV administration restored autophagy in both pulmonary hypoplasia models by transferring miR-17∼92 cluster members contained in the EV cargo. Conclusions: AFSC-EV treatment rescues branching morphogenesis partly by restoring autophagy through microRNA cargo transfer. This study enhances our understanding of pulmonary hypoplasia pathogenesis and creates new opportunities for fetal therapeutic intervention in congenital diaphragmatic hernia babies.

The MicroRNA 29 Family Promotes Type II Cell Differentiation in Developing Lung.

Lung alveolar type II cells uniquely synthesize surfactant, a developmentally regulated lipoprotein that is essential for breathing. Expression of the gene (SFTPA) encoding the major surfactant protein, SP-A, in midgestation human fetal lung (HFL) is dramatically induced by cyclic AMP (cAMP). cAMP induction of SP-A expression is repressed by transforming growth factor β (TGF-β) and by hypoxia. In this study, we found that expression of the microRNA 29 (miR-29) family was significantly upregulated in epithelial cells isolated from mouse fetal lung during late gestation and in epithelial cells isolated from HFL explants during type II cell differentiation in culture. miR-29 expression in cultured HFL epithelial cells was increased by cAMP and inhibited by hypoxia, whereas the miR-29 target, TGF-β2, was coordinately decreased. Knockdown of the miR-29 family in cultured HFL type II cells blocked cAMP-induced SP-A expression and accumulation of surfactant-containing lamellar bodies, suggesting their physiological relevance. This occurred through derepression of TGF-β signaling. Notably, cAMP increased binding of endogenous thyroid transcription factor 1 (TTF-1/Nkx2.1) to the miR-29ab1 promoter in HFL type II cells, and TTF-1 increased miR-29ab1 promoter-driven luciferase activity in cotransfection assays. Together, these findings identify miR-29 family members as TTF-1-driven mediators of SP-A expression and type II cell differentiation through repression of TGF-β signaling.

Effect of Oxygen on the Expression of Hypoxia-Inducible Factors in Human Fetal Lung Explants

Background: Fetal lung development requires proper coordination between lung epithelial and vascular morphogenesis. A major determinant in lung vascular development is vascular endothelial growth factor (VEGF), which is regulated by hypoxia-inducible factors (HIFs). VEGF is expressed in the airway epithelium, while its receptors (VEGFRs) are expressed in the pulmonary mesenchyme. The hypoxic environment in utero is beneficial for fetal organogenesis, especially vascular development. However, little is known about the expression of HIFs and VEGFR-2 in the human fetal lung in vitro. Objectives: The purpose of this study was to investigate the effects of hypoxia on fetal lung morphology and mRNA expression of VEGF, VEGFR-2, HIF-2α, and HIF-3α. Methods: An explant culture technique was used to study the effects of normoxic and hypoxic conditions on human fetal lung. Results: The morphology remained largely unchanged in explants cultured under hypoxic or normoxic conditions. Quantitative RT-PCR showed that the mRNA expression of VEGF-A, but not VEGFR-2 is upregulated in explants cultured at 1.5% compared with 21% oxygen. We observed a nonsignificant increase in HIF-2α and HIF-3α mRNA expression in explants cultured at 1.5% oxygen. These data suggest that the mRNA expression of VEGF, and possibly HIF-2α and HIF-3α, is regulated by hypoxia in the developing human lung. Conclusion: This lung explant culture model appears to be a valuable model to unravel the molecular mechanisms of human lung development.

IS-018 The effect of oxygen concentration on hypoxia-inducible factors expression in human fetal lung explants(International Session V,Better Life for Sick Children, Better Future for Pediatric Surgery,the 45th Annual Meeting of Japanese Society of Pediatric Surgeons)

IS-018 The effect of oxygen concentration on hypoxia-inducible factors expression in human fetal lung explants(International Session V,Better Life for Sick Children, Better Future for Pediatric Surgery,the 45th Annual Meeting of Japanese Society of Pediatric Surgeons)

Differential Response of the Epithelium and Interstitium in Developing Human Fetal Lung Explants to Hyperoxia

Hyperoxia is closely linked with the development of chronic lung disease of prematurity (CLD), but the exact mechanisms whereby hyperoxia alters the lung architecture in the developing lung remain largely unknown. We developed a fetal human lung organ culture model to investigate (a) the morphologic changes induced by hyperoxia and (b) whether hyperoxia resulted in differential cellular responses in the epithelium and interstitium. The effects of hyperoxia on lung morphometry were analyzed using computer-assisted image analysis. The lung architecture remained largely unchanged in normoxia lasting as long as 4 d. In contrast, hyperoxic culture of pseudoglandular fetal lungs resulted in significant dilatation of airways, thinning of the epithelium, and regression of the interstitium including the pulmonary vasculature. Although there were no significant differences in Ki67 between normoxic and hyperoxic lungs, activated caspase-3 was significantly increased in interstitial cells, but not epithelial cells, under hyperoxic conditions. These changes show that exposure of pseudoglandular lungs to hyperoxia modulates the lung architecture to resemble saccular lungs.

Lung Fluid Balance During Development

After completing this article, readers should be able to: 1. Describe the major driving forces for liquid formation in and liquid removal from the lumen of the fetal lung. 2. Delineate the role of a normal volume of liquid within the lumen of the fetal lung for normal lung growth during development. 3. Describe the effects of labor on Na,K-ATPase activity in lung epithelial cells and clearance of lung liquid after birth. 4. Explain the route by which most residual liquid drains from the lung. 5. List the conditions associated with preterm birth that may contribute to delayed removal of fetal lung liquid. Curious students of mammalian development recognized more than a century ago that the lungs are filled with liquid during fetal life, (1) but the origin of that liquid remained obscure until 1948, when an unexpected discovery by two French scientists dispelled the prevailing view that fetal lung liquid derived from intrauterine inhalation of amniotic fluid. In studies designed to explore the ontogeny of the pituitary-adrenal axis, tracheal ligation of fetal rabbits for 9 days resulted in fluid distention of the lungs. (2) This serendipitous finding, subsequently confirmed and embellished by others, (3)(4)(5) established that the fetal lung itself, rather than the amniotic sac, is the source of the liquid that fills the lung during development. This liquid forms a slowly expanding structural template that prevents collapse and promotes growth of the fetal lung. Subsequent studies, conducted mostly with sheep, showed that liquid in the lumen of the fetal lung forms as a result of chloride secretion in the respiratory epithelium, (6)(7) a process that can be inhibited by diuretics that block Na,K-2Cl cotransport. (8)(9) In vitro experiments using cultured explants of lung tissue and monolayers of epithelial cells harvested from human fetal lung have …

Insulin-like growth factor-I receptor-mediated vasculogenesis/angiogenesis in human lung development.

The structural and functional development of the pulmonary system is dependent upon appropriate early vascularization of the embryonic lung. Our previous in vitro studies in a rat model indicated that insulin-like growth factor-I (IGF-I) is a potent angiogenic agent for fetal lung endothelial cells. To assess its role on human vascular lung development, we first examined the expression of IGF-I/II and IGF receptor type I (IGF-IR) in human embryonic and fetal lung tissues at 4-12 wk of gestation. Immunohistochemical and in situ hybridization studies revealed the presence of IGF-I/II-IGF-IR ligands and mRNA transcripts in embryonic lungs as early as 4 wk gestation. Immunotargeting using an anti-IGF-IR neutralizing antibody on human fetal lung explants demonstrated a significant blockade of IGF-IR signaling. Inactivation of IGF-IR resulted in a loss of endothelial cells, accompanied by dramatic changes in fetal lung explant morphology. Terminal transferase dUTP end-labeling assay and TEM studies of anti-IGF-IR-treated lungs demonstrated numerous apoptotic mesenchymal cells. Rat embryonic lung explant studies further validated the importance of the IGF-IGF-IR system for lung vascular development. These data provide the first demonstration of IGF-I/II expression in the human lung in early gestation and indicate that the IGF family of growth factors, acting through the IGF-IR, is required as a survival factor during normal human lung vascularization.

Antisense inhibition of surfactant protein A decreases tubular myelin formation in human fetal lung in vitro.

Surfactant protein A (SP-A) is the most abundant of the surfactant-associated proteins. SP-A is involved in the formation of tubular myelin, the modulation of the surface tension-reducing properties of surfactant phospholipids, the metabolism of surfactant phospholipids, and local pulmonary host defense. We hypothesized that elimination of SP-A would alter the regulation of SP-B gene expression and the formation of tubular myelin. Midtrimester human fetal lung explants were cultured for 3-5 days in the presence or absence of an antisense 18-mer phosphorothioate oligonucleotide (ON) complementary to SP-A mRNA. After 3 days in culture, SP-A mRNA was undetectable in antisense ON-treated explants. After 5 days in culture, levels of SP-A protein were also decreased by antisense treatment. SP-B mRNA levels were not affected by the antisense SP-A ON treatment. However, there was decreased tubular myelin formation in the antisense SP-A ON-treated tissue. We conclude that selective elimination of SP-A mRNA and protein results in a decrease in tubular myelin formation in human fetal lung without affecting SP-B mRNA. We speculate that SP-A is critical to the formation of tubular myelin during human lung development and that the regulation of SP-B gene expression is independent of SP-A gene expression.

VEGF induces airway epithelial cell proliferation in human fetal lung in vitro.

Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen involved in normal and abnormal angiogenesis. VEGF mRNA and protein are abundant in distal epithelium of midtrimester human fetal lung. In the present study, we identified immunoreactivity for KDR, a major VEGF-specific receptor, in distal lung epithelial cells of human fetal lung tissue, suggesting a possible autocrine or paracrine regulatory role for VEGF in pulmonary epithelial cell growth and differentiation. Addition of exogenous VEGF to human fetal lung explants resulted in increased epithelium volume density and lumen volume density in the tissues, both morphometric parameters of tissue differentiation. Cellular proliferation demonstrated by bromodeoxyuridine uptake was prominent in distal airway epithelial cells and increased in the VEGF-treated explants. VEGF-treated explants also demonstrated increased surfactant protein (SP) A mRNA, SP-C mRNA, and SP-A protein levels compared with controls. However, SP-B mRNA levels were unaffected by VEGF treatment. [(3)H]choline incorporation into total phosphatidylcholine was increased by VEGF treatment, but incorporation into disaturated phosphatidylcholine was not affected by exogenous VEGF. Based on these observations, we conclude that VEGF may be an important autocrine growth factor for distal airway epithelial cells in the developing human lung.

Antisense inhibition of epidermal growth factor receptor decreases expression of human surfactant protein A.

Epidermal growth factor (EGF) stimulates surfactant protein A (SP-A) synthesis in fetal lung tissue through ligand binding to the EGF receptor. We hypothesized that inhibition of EGF receptor messenger RNA (mRNA) would block SP-A expression in human fetal lung tissue during alveolar type II cell differentiation in vitro. Midtrimester human fetal lung explants were maintained in serum-free Waymouth's medium for 3 to 5 d in the presence or absence of an antisense 18-mer phosphorothioate oligonucleotide (ON) complementary to the initiation codon region of EGF receptor mRNA. Sense and scrambled ONs similarly modified were used as additional controls. The concentration of EGF receptor mRNA was semiquantitatively determined by reverse transcriptase/polymerase chain reaction (RT-PCR). We found a significant 3-fold decrease in EGF receptor mRNA levels in the antisense-treated groups compared with the control group with no effect in the sense condition. Immunohistochemical staining revealed a decrease in the amount of staining for EGF receptor protein in distal pulmonary epithelial cells in the antisense-treated groups compared with either control or sense conditions. Treatment with antisense EGF receptor ON decreased both SP-A mRNA and protein compared with controls with no effect in the sense condition. The ONs did not affect tissue viability as measured by the release of lactate dehydrogenase. We conclude that selective degradation of EGF receptor mRNA with antisense ON treatment results in a decrease in SP-A expression in human fetal lung. These findings support the critical importance of the EGF receptor for the regulation of SP-A gene expression during human alveolar type II cell differentiation.

Neuregulin-1 and human epidermal growth factor receptors 2 and 3 play a role in human lung development in vitro.

The human epidermal growth factor receptor (HER) family consists of four distinct receptors: HER1 (epidermal growth factor receptor), HER2, HER3, and HER4. Their specific activating ligands are collectively known as neuregulins (NRG). We hypothesized that one member of the NRG family, NRG-1, and the HER family would play a role in fetal lung development. To test this hypothesis, we defined NRG-1 and HER gene expression in mid-trimester human fetal lung tissue. HER2 and HER3 messenger RNA and protein were detected in the fetal lung, but HER4 expression was not detected. Immunohistochemical staining of fetal lung tissue localized HER2 and HER3 protein to the developing lung epithelium. NRG-1 expression was not found in freshly isolated human fetal lung, but it was observed in fetal lung explants after 2 d of explant culture. Immunohistochemistry of cultured human fetal lung explants revealed that NRG-1 protein was also expressed in pulmonary epithelial cells. Exposing human fetal lung to recombinant NRG-1 activated the HER receptor complex as measured by approximately 4-fold increases in receptor phosphotyrosine content. In addition, NRG-1 increased explant epithelial cell volume density approximately 2-fold (P < 0. 03); increased epithelial cell proliferation approximately 2-fold, as determined by bromodeoxyuridine labeling (P = 0.002); and reduced surfactant protein-A (SP-A) levels by 53% (P < 0.05). These data are consistent with an autocrine regulatory process mediated by NRG-1 activation of HER2/HER3 heterodimers expressed on developing human fetal lung epithelial cells. Receptor activation results in increased lung epithelial cell proliferation and volume density, and decreased SP-A production, a marker of type II pneumocyte differentiation.

Vascular endothelial growth factor gene expression in human fetal lung in vitro.

Neonatal respiratory function depends on the development of a well-formed pulmonary capillary bed. Vascular endothelial growth factor (VEGF) is a potent inducer of endothelial cell growth and angiogenesis. High levels of VEGF protein and messenger RNA (mRNA) have been detected in the developing lung, suggesting that VEGF plays a role in the development of the pulmonary capillary bed. To begin to understand the role of VEGF in human lung development, we explored the regulation of VEGF gene expression and the localization of VEGF protein and mRNA in a model of the developing human lung. VEGF protein and mRNA were detected in midtrimester human fetal lung tissue, and their levels increased with time in explant culture. VEGF protein and mRNA were increased by the maintenance of human fetal lung explants in 2% O2 environments compared with 20% O2 environments. VEGF mRNA levels were found to be increased by cyclic adenosine monophosphate (cAMP) in explants that were incubated in 20% O2, but not in those incubated in 2% O2. Immunostaining for VEGF protein demonstrated localization primarily in airway epithelial cells in midtrimester human fetal lung tissue. Immunostaining for VEGF increased with incubation of human fetal lung explants in 2% and 20% O2. Interestingly, VEGF protein was localized primarily in the basement membrane subjacent to airway epithelial cells after 4 d of incubation in 20% O2. Incubation of tissues in the presence of dibutyryl cAMP resulted in an increase in immunostaining for VEGF, primarily in the basement membranes of prealveolar ducts in 20% O2-treated tissues. In situ hybridization studies indicated that VEGF mRNA was present in both mesenchymal cells and airway epithelial cells. These data suggest that VEGF gene expression is regulated by both oxygen and cAMP in the developing human lung. The detection of VEGF mRNA and protein in distal airway epithelial cells and the detection of VEGF protein in the basement membrane subjacent to the airway epithelial cells suggest that translocation of VEGF protein occurs after its synthesis in the epithelium. Localization of VEGF to the basement membrane of airway epithelial cells may be important for directing capillary development in the human lung.

Human surfactant proteins A1 and A2 are differentially regulated during development and by soluble factors.

An RT-PCR method for the relative quantitation of the mRNAs for human surfactant protein (SP) A1 and SP-A2 was developed, verified, and then utilized to determine the relative levels of these mRNAs in fetal and adult lung samples in vivo, as well as in cultured human fetal lung explants and H441 cells. For the cultured tissue and cells, we assessed the effects of a variety of soluble factors known to modulate total SP-A. Comprehensive analysis revealed many significant findings, including the following: both mRNAs were expressed as early as 15 wk of gestation; throughout midgestation, SP-A1 was present at higher levels than SP-A2, with an average ratio of 30:1. In the adult lung, SP-A1 mRNA was present at lower levels than SP-A2, with a ratio of 0.4:1, whereas in H441 cells, the ratio was 0.85:1. In fetal lung cultured for 4 days, both mRNAs increased, with a greater increase in SP-A2 (97-fold) than in SP-A1 (15-fold), resulting in a final ratio of 4:1. Differential regulation was demonstrated for 8-(4-chlorophenylthio)-cAMP, interferon (IFN)-gamma, tumor necrosis factor-alpha, and transforming growth factor (TGF)-beta in the human fetal lung explant system, with SP-A2 being more affected, and for IFN-gamma and TGF-beta in the H441 cells, where SP-A1 showed greater regulation. Of the soluble factors tested, IFN-gamma and TGF-beta had the most potent and consistent effects in both systems.

Insulin inhibits surfactant protein A and B gene expression in the H441 cell line

Fetuses of mothers with uncontrolled gestational diabetes have an increased risk of developing neonatal respiratory distress syndrome and are frequently hyperinsulinemic, thus it has been proposed that high levels of insulin delay fetal lung maturation. We have shown previously that insulin inhibits the accumulation of mRNA for the surfactant-associated proteins A and B (SP-A and SP-B) in human fetal lung explants maintained in vitro. To test the hypothesis that the inhibitory effects of insulin on the surfactant proteins are the result of a direct action of insulin on the lung epithelial cell, we evaluated the effects of insulin in the H441 cell line, a human pulmonary adenocarcinoma cell line that expresses SP-A and SP-B mRNA. We observed that insulin treatment for 48 h decreased SP-A mRNA and protein levels in a concentration-dependent manner when compared to controls. The inhibitory effect of insulin on SP-A mRNA levels was apparent as early as after 4 h of exposure. SP-B mRNA levels were also significantly decreased by insulin in a concentration-dependent manner. Insulin, at 2.5 μg/ml, inhibited SP-A gene transcription by approx. 67%, and inhibited SP-B gene transcription by about 32%. There was no significant effect of insulin on SP-A or SP-B mRNA stability. Thus, we have observed a pattern of insulin inhibition of SP-A and SP-B gene expression in the H441 lung epithelial cell line similar to that previously observed in human fetal lung explants, which are comprised of both epithelial and mesenchymal cells. Our findings provide further evidence that insulin may delay fetal lung maturation by inhibiting SP-A and SP-B gene expression. Furthermore, our findings suggest that the inhibitory effects of insulin are, at least partially, the result of a direct action on the lung epithelial cell.

SP-A2 gene expression in human fetal lung airways.

In the present study, we characterized surfactant protein (SP)-A messenger RNA (mRNA) in mid-trimester human fetal trachea and bronchi. SP-A protein was localized by immunocytochemistry to scattered epithelial cells in the airway surface epithelium and in submucosal glands of the fetal trachea and bronchi. SP-A mRNA (2.2 kb) was detected by Northern blot analysis in human fetal trachea, as well as in primary and more distal bronchi. The levels of detectable SP-A mRNA were highest in the upper airways and were decreased in smaller bronchi in comparison. SP-A mRNA was barely detectable in the distal fetal lung tissue. In contrast, SP-A mRNA was abundant in cultured explants of distal human fetal lung tissue. SP-A1 and SP-A2 mRNA were detected by primer extension analysis in adult human lung tissue and in cultured human fetal lung explants. Only SP-A2 mRNA was detected in RNA isolated from human fetal trachea and bronchi. SP-A mRNA was localized by in situ hybridization in the fetal trachea and bronchi in scattered cells in the surface epithelium and, most prominently, in submucosal glands. Our results suggest that SP-A2, and not SP-A1, is produced in the human fetal tracheal and bronchial epithelium and in submucosal glands.

Surfactant Protein B (SP-B) Promoter Function in Transgenic Mice • 309

SP-B, a hydrophobic protein produced by alveolar type II and bronchiolar Clara cells, helps in the spreading and stability of surfactant film and is required for surfactant function. The SP-B gene is regulated both developmentally and hormonally. To study regulatory elements in the SP-B gene promoter in vivo, we made transgenic (tg) mice using a fragment of the human SP-B promoter (-1039/+431bp) linked to the chloramphenicol acetyl transferase (CAT) reporter gene. This SP-B DNA fragment contains both proximal and distal TTF-1 and HNF-3 transcription factor binding sites which promote lung epithelial cell line specificity in vivo. CAT activity in tg lung tissue was significantly increased compared to wild-type (22.2±3.2 vs 0.4±0.1 cpm/mg prot/min; p<0.01), whereas CAT activity in heart, liver, spleen, kidney, intestine, skeletal muscle and brain was not different. In two established tg lines CAT activity was found both in lungs (150 and 264 cpm/mg/min) and in trachea (50 and 125 cpm/mg/min), sites of type II and/or Clara cells in mice. Immunohistochemistry of lung sections using antibodies to CAT and human SP-B showed immunostaining for both proteins localized to type II and Clara cells. In tg fetal lungs, CAT activity was found at 17-19 days gestation and increased ≥4 fold (n=2) during explant culture. In tg adult lung explants (n=3), levels of CAT activity were maintained for at least 3 days (95±13% (se) of preculture). Transforming growth factor β(TGFβ1, 30 ng/ml), a cytokine which downregulates SP-B in human fetal lung explants, reduced CAT activity in both fetal (52±9% of control at 3 days, n=2) and adult mouse lung explants (60±11% of control at 3 days, n=1; 34±3% at 5 days, n=3) whereas35 S-methionine incorporation into total protein was unaffected(101±7%). In contrast, 12-O-tetradecanoyl phorbol-13 acetate (TPA, 30 nM), which also downregulates SP-B in human fetal lung explants, did not reduce CAT activity in tg adult lung ex vivo (182±54%, n=4). We conclude that the human SP-B promoter fragment (-1039/+431bp) exhibits tissue and lung cell specificity in vivo and confers developmental regulation and TGFβ responsiveness ex vivo. These transgenic animals provide a useful in vivo model to study specific regulatory elements in the SP-B promoter.