Mala R. Chinoy

Lung growth and development.

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.

Fetal lung development: Airway pressure enhances the expression of developmental genes

BACKGROUND/PURPOSE The mechanisms by which static airway pressures in the developing lung affect development are unknown. The in vitro murine fetal lung model with airway ligation reproduces the phenomenon of intraluminal airway pressure in developing lungs. We have applied the technique of differential display of mRNAs to fetal murine lungs that were maintained in organ culture with and without tracheal ligation. The goal of this investigation was to identify genes that are induced or enhanced by airway pressure during lung development. METHODS Fetuses were harvested from CD-1 mice on gestational day (Gd) 14. The lungs were removed and trachea either transected or ligated and organ cultured for 7 days. Total RNA was extracted from cultured unligated controls and ligated lungs. Reverse transcription (RT) of the purified total RNA from each pooled sample was performed with anchor primer H-T11G or C and one of 24 arbitrary primers followed by polymerase chain reaction (PCR) of the RT mixtures. PCR products were electrophoresed on a DNA sequencing gel. Differentially expressed cDNA bands of interest were cut from the dried gel. Each cDNA was then reamplified. Reamplified cDNAs were extracted, PCR amplified, cloned, and sequenced for homology to existing sequences in the GenBank database. RESULTS Sequencing identified 4 differentially expressed genes enhanced by tracheal ligation: hepatoma-derived growth factor (HDGF), ribosomal protein S24, stathmin, and parathyroid hormone (PTH). CONCLUSIONS Genes enhanced by airway pressure or tracheal ligation are mitogenic for fibroblasts, correlate with cell proliferation, regulate cell proliferation and differentiation, and may play a role in growth in distal lung and type II cell differentiation. Further work is necessary to identify the mechanisms by which these genes influence lung maturational processes.

Influence of epidermal growth factor and transforming growth factor beta-1 on patterns of fetal mouse lung branching morphogenesis in organ culture

Transforming growth factor‐β (TGF‐β), a potent inhibitor of epithelial cell proliferation, and epidermal growth factor (EGF), a mitogenic polypeptide that binds to cell surface receptors, are important regulators of cell differentiation; however, their distinct role(s) in lung development and their mechanisms of action are not well understood. We evaluated the effects of these factors on lung morphogenesis in murine fetal lungs at gestational day 14 (time:zero) and again after 7 days in culture. Baseline controls were cultured after tracheal transection in supplemented BGJb medium, and other tracheally transected lungs were cultured following addition of EGF (10 ng/ml BGJb), TGF‐β1 (2 ng/ml BFJb), or with both in combination added to the medium.

Growth factors and dexamethasone regulate Hoxb5 protein in cultured murine fetal lungs

Studies on lung morphogenesis have indicated a role of homeobox( Hox) genes in the regulation of lung development. In the present study, we attempted to modulate the synthesis of Hoxb5 protein in cultured murine fetal lungs after mechanical or chemical stimuli. Murine fetuses at gestational day 14 (GD14) were removed from pregnant CD-1 mice, and lungs were excised and cultured for 7 days in BGJb media. The experimental groups were 1) untreated, unligated; 2) tracheal ligation; 3) supplemented media with either epidermal growth factor (EGF; 10 ng/ml), transforming growth factor (TGF)-β1 (2 ng/ml), dexamethasone (10 nM), EGF+TGF-β1, or EGF+TGF-β1+dexamethasone. After 3 or 7 days, the cultured lungs were compared with in vivo lungs. Immunoblotting signals at 3 days in culture were stronger than those at 7 days. Western blot analyses showed that ligation, EGF, TGF-β1, and EGF+TGF-β1 downregulated Hoxb5 protein to ∼20-70% of Hoxb5 protein levels in unligated, untreated cultured lungs. Furthermore, dexamethasone alone or in combination with EGF and TGF-β1 downregulated Hoxb5 protein by >90% ( P < 0.05) signal strength, similar to that seen in GD19 or in neonatal lungs. Immunostaining showed that Hoxb5 protein was expressed strongly in the lung mesenchyme at early stages in gestation. However, by GD19 and in neonates, it was present only in specific epithelial cells. A persistent level of Hoxb5 protein in the mesenchyme after EGF or TGF-β1 treatments or tracheal ligation was noted. Hoxb5 protein was significantly downregulated by EGF+TGF-β1, and it was least in lungs after dexamethasone or EGF+TGF-β1+dexamethasone treatment. The decrease in Hoxb5 protein was significant only in the groups with dexamethasone added to the media. Thus immunostaining results parallel those of immunoblotting. The degree of Hoxb5 downregulation by dexamethasone or EGF+TGF-β1+dexamethasone was similar to that seen in vivo in very late gestation, which correlated to the advancing structural development of the lung.

Histology-based screen for zebrafish mutants with abnormal cell differentiation

The power of histology to define states of cell differentiation was used as the basis of a mutagenesis screen in zebrafish. In this screen, 7‐day‐old parthenogenetic half‐tetrad larvae from potential carrier females were screened for mutations affecting cell differentiation in hematoxylin and eosin‐stained tissue sections. Seven, noncomplementing, recessive mutations were found. Two mutations affect only the retina: segmented photoreceptors (spr) show a discontinuous photoreceptor cell layer; vestigial outer segments (vos) has fewer photoreceptor cells and degenerated outer segments within this cell layer. Three mutants have gut‐specific defects: the epithelial cells of kirby (kby) are replaced by ballooned cells; the intestines of stuffy (sfy) and stuffed (sfd) contain increased luminal mucus. Two mutations affect multiple organs: disordered neural retina (dnr) has disrupted retinal layering and mild nuclear abnormalities in the gut and liver; and in huli hutu (hht), the retinal cell layers are disorganized and multiple organs have mild to severe nuclear abnormalities that are reminiscent of the atypia of human neoplasia. Each mutation appears to be homozygous lethal. This screen is proof of principle for the feasibility of histologic screens to yield novel mutations, including potential models of human disease. The throughput for this type of screen may be enhanced by automation. Developmental Dynamics, 2003.

Expression profile of IGF system during lung injury and recovery in rats exposed to hyperoxia: a possible role of IGF-1 in alveolar epithelial cell proliferation and differentiation.

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. J. Cell. Biochem. 97: 984–998, 2006.

Effects of dexamethasone, growth factors, and tracheal ligation on the development of nitrofen-exposed hypoplastic murine fetal lungs in organ culture.

BACKGROUND/PURPOSE The addition of growth factors EGF (epidermal growth factor) plus TGFbeta1 (transforming growth factor beta1; E + T) or dexamethasone (DEX) to normal murine fetal lungs in culture enhances lung development. In addition, ligation of the airway in lungs in organ culture, enhances lung development. Nitrofen (2,4-dichlorophenyl-p-nitrophenylether) administration to pregnant mice results in pulmonary hypoplasia in the offspring with many similarities to human hypoplastic lung conditions. This study investigates the effects of growth factors, dexamethasone, and airway ligation on the development of hypoplastic fetal murine lungs in whole-organ culture. We hypothesized that E+T, DEX, or airway ligation will enhance the development and maturation of hypoplastic murine fetal lungs in vitro. METHODS Time-dated pregnant CD-1 mice were given nitrofen, 25 mg, intragastrically at gestational day (Gd) 8. The dams were killed on Gd 14, and the fetuses were removed. The hypoplastic fetal lungs were excised, and the tracheae were transected. The lungs were cultured in serum-free BGJb media in the presence or absence of E+T (10 ng/mL + 2 ng/mL, respectively) or DEX (10 nmol/L). Some lungs were cultured for 7 days with the tracheae ligated. RESULTS Gross morphology under a dissecting stereomicroscope showed that the lungs were larger after E+T, DEX, or tracheal ligation. Histologically, the untreated lungs had progressed from the pseudoglandular stage to a canalicular-like stage with poorly differentiated airways. The E+T-treated lungs had better developed airway branching and small acini; however, thick mesenchyme persisted. The ligated lungs had well-developed airway branching and acinar structures. After DEX treatment the lungs were most developed with very well defined airway branching and expanded acinar structures; however, there was no secondary septation. Ultrastructurally, the hypoplastic lungs at Gd 14 and after 7 days in culture had no glycogen in their epithelial cells, no defined acinar formation, and had damaged mitochondria. The E+T-treated or tracheally ligated lungs had abundant type II cells, secreted lamellar bodies (LBs), and showed infrequent tubular myelin. Mitochondrial damage was noted in these lungs as in the untreated lungs. DEX-treated hypoplastic lungs showed large acini. The acinar walls were thick; however, they had type II cells with abundant LBs and intact mitochondria. The airways were noted to have differentiated cell types. Surfactant secretions in acinar spaces showed tubular myelin structures. CONCLUSIONS E+T, tracheal ligation, or DEX accelerates lung development and maturation of hypoplastic fetal murine lungs compared with untreated controls. DEX had a greater effect with special reference to repair of mitochondrial damage. DEX not only accelerated lung development, but it may have reversed some of the effects nitrofen.

Maintenance of fetal murine pulmonary microvasculature in heart-lung en bloc whole organ culture

The authors have previously shown that murine fetal lungs can be maintained in serum-free whole organ culture and that airway ligation accelerates lung development. In spite of extensive use of lung organ culture systems, the vasculature of the unperfused lung in organ culture has not been studied. The aim of the present study was to compare organ cultures of heart-lung blocks with continuous perfusion of the pulmonary vasculature to those without perfusion, ie, whole lungs cultured without the attached heart. Time-dated pregnant CD-1 mice were killed on gestational day (Gd) 14. The fetuses were removed via laparotomy. Heart-lung blocks and whole lungs without the heart were excised under sterile conditions and cultured in BGJb media. Some of the heart-lungs and whole lungs underwent tracheal ligation whereas others were left with the trachea unligated allowing free egress of airway fluid. After 7 days, the cultured organs were processed for histology, ultrastructural analysis, and immunohistochemistry. (1) Lungs were fixed in 10% formalin, paraffin embedded, and processed for routine H&E staining. (2) Lungs were fixed in 2.5% glutaraldehyde in cacodylate buffer and processed for transmission electron microscopy. (3) Lungs were embedded in CRYOform and flash frozen for immunohistochemical localization of PECAM-1 (CD31) (PECAM-1, Platelet endothelial cell adhesion molecule-1, a selective endothelial cell marker). Our daily observations of the cultured organs showed that the heart maintained synchronized beating for all 7 days in culture. Perfusion of the pulmonary microvasculature was demonstrated. Light microscopically, H&E sections showed that fresh fetal Gd14 pseudoglandular lungs (time-zero) had a defined capillary network, which was more centrally localized and peripherally less developed. The presence of more numerous lung capillaries in the cultured heart-lung blocks was noted when compared with cultured lungs alone. Ultrastructurally, endothelial cells with intact structural integrity were identified only in cultured whole lungs with hearts. Immunohistochemical staining of the whole lungs with rat antimurine PECAM-1 monoclonal antibody performed on cryosections showed the presence of vasculature by specific PECAM-1 localization on endothelial cells. PECAM-1 labeling of capillaries was noted in Gd14 (time-zero) lungs. In addition, the lungs cultured with hearts, ie, perfused lungs, showed more well defined, distinct capillary networks stained with PECAM-1 antibody than unperfused lungs without hearts. Our results showed that microvasculature is present in murine fetal lungs at Gd14. After 7 days in organ culture, the maintenance of lung microvasculature was confirmed histologically, ultrastructurally, and immunohistochemically. The microvasculature in whole lungs cultured as perfused/beating heart-lung blocks was better maintained than the microvasculature of unperfused whole lungs cultured without hearts. A perfused whole lung organ culture model is attractive because the lung architecture is better maintained and may be useful in lung developmental studies as it mimics the in situ heart-lung functional physiological relationship.

Unique spatial and cellular expression patterns of Hoxa5, Hoxb4 and Hoxb6 proteins in normal developing murine lung are modified in pulmonary hypoplasia

BACKGROUND Hox transcription factors modulate signaling pathways controlling organ morphogenesis and maintain cell fate and differentiation in adults. Retinoid signaling, key in regulating Hox expression, is altered in pulmonary hypoplasia. Information on pattern-specific expression of Hox proteins in normal lung development and in pulmonary hypoplasia is minimal. Our objective was to determine how pulmonary hypoplasia alters temporal, spatial, and cellular expression of Hoxa5, Hoxb4, and Hoxb6 proteins compared to normal lung development. METHODS Temporal, spatial, and cellular Hoxa5, Hoxb4, and Hoxb6 expression was studied in normal (untreated) and nitrofen-induced hypoplastic (NT-PH) lungs from gestational day 13.5, 16, and 19 fetuses and neonates using Western blot and immunohistochemistry. RESULTS Modification of protein levels and spatial and cellular Hox expression patterns in NT-PH lungs was consistent with delayed lung development. Distinct protein isoforms were detected for each Hox protein. Expression levels of the Hoxa5 and Hoxb6 protein isoforms changed with development and were altered further in NT-PH lungs. Compared to normal lungs, GD19 and neonatal NT-PH lungs had decreased Hoxb6 and increased Hoxa5 and Hoxb4. Hoxa5 cellular localization changed from mesenchyme to epithelia earlier in normal lungs. Hoxb4 was expressed in mesenchyme and epithelial cells throughout development. Hoxb6 remained mainly in mesenchymal cells around distal airways. CONCLUSIONS Unique spatial and cellular expression of Hoxa5, Hoxb4, and Hoxb6 participates in branching morphogenesis and terminal sac formation. Altered Hox protein temporal and cellular balance of expression either contributes to pulmonary hypoplasia or functions as a compensatory mechanism attempting to correct abnormal lung development and maturation in this condition.

Angiotensin II-induced changes in G-protein expression and resistance of renal microvessels in young genetically hypertensive rats

Altered regulation of cAMP may contribute to enhanced renal reactivity to angiotensin II (Ang II) in spontaneously hypertensive rats (SHR). Such a phenomenon may occur in renal preglomerular arterioles and may involve changes in expression of GTP-binding regulatory proteins. We have examined the effects of Ang II on steady state levels of Gαi-1,2, Gαi-3 Gαs and Gαq in preglomerular arterioles from young marginally hypertensive SHR and on mean arterial pressure (MAP), renal vascular resistance (RVR) and renal cAMP excretion (UcAMP.V). Young (5-6 week old) SHR and Wistar Kyoto (WKY) rats received Ang II (35 ng/kg/min, s.c.) or vehicle for 7 days via osmotic minipumps. Urine was collected over the last 24 h. On day seven, MAP and renal blood flow were measured in anesthetized rats and RVR was determined. Preglomerular arterioles were isolated by perfusing the kidneys with iron oxide and using a series of mechanical steps coupled with the use of a magnet to retain iron-laden vessels. Membranes were prepared and the expressions of Gαi-1,2, Gαi-3, Gαs and Gαq were evaluated by Western immunoblotting. Baseline MAP (124 ± 6 mmHg) was only marginally (p > 0.05) higher in SHR when compared with WKY rats (110 ± 4 mmHg). RBF (3.04 ± 0.16 mL/min) was significantly lower and RVR (41.10 ± 1.37 mmHg.min/mL) was significantly higher in SHR when compared to age-matched WKY rats (4.36 ± 0.30 mL/min and 25.79 ± 1.58 mmHg.min/mL, respectively). Ang II significantly increased MAP in SHR (17 mmHg) but not in WKY rats. These increases in MAP were accompanied by significant increases in RVR in SHR (48% over control) but not in WKY rats. Compared to WKY rats, preglomerular arterioles from SHR exhibited significantly higher basal expression of Gαi-1,2 (11- fold), Gαi-3 (13-fold) and Gαs (3-fold). Chronic infusion of Ang II, however, downregulated the expression of Gαs (by 53%; p < 0.05), Gαi-1,2 ( by 72%; p < 0.05) and Gαi-3 (by 35%; p > 0.05) in SHR preglomerular arterioles but significantly upregulated the expression of these proteins in WKY by 3-, 8- and 15-fold, respectively. Basal levels of Gαq were not different in preglomerular arterioles from the two strains but were downregulated by Ang II in both WKY (74% of basal) and SHR (52% of control). Baseline UcAMP.V was significantly lower in SHR (31.22 ± 6.51 nmol/24 h) compared with WKY rats (65.33 ± 3.60 nmol/24 h). Chronic Ang II infusion significantly increased UcAMP.V in SHR as well as WKY rats. These data clearly demonstrate that expressions of Gi isoforms as well as Gs in renal microvessels are elevated during early stages of hypertension and suggest that the elevated levels of Gi proteins may be directly associated with a blunted adenylyl cyclase-cAMP cascade in the renal microvasculature. Furthermore, Ang II appears to directly downregulate the expression of Gs in young SHR but not in young WKY renal microvessels. Such diversity in its effect on G-protein expression may be important for enhanced renal sensitivity to Ang II in SHR.