Developing Rat Lung Research Articles

Antisense oligodeoxynucleotides targeting PDGF-B mRNA inhibit cell proliferation during embryonic rat lung development

There is increasing evidence to suggest that platelet-derived growth factor (PDGF) or PDGF-like molecules play a role in fetal lung morphogenesis. Our previous studies demonstrated the presence of PDGF-AA and PDGF-BB homodimers in embryonic and fetal rat lung. To explore further the role for PDGF-BB in embryonic lung development, we conducted intervention studies using PDGF-B chain-specific antisense oligodeoxynucleotides in a simple embryonic rat lung explant system. Unmodified antisense PDGF-B oligodeoxynucleotides inhibited, in a concentration-dependent manner, DNA synthesis of embryonic lung. A maximal inhibition of 50% was observed. The inhibitory effect of antisense PDGF-B oligodeoxynucleotides on DNA synthesis was reversed by the addition of exogenous PDGF-BB but not PDGF-AA. Antisense treatment decreased PDGF-BB but not PDGF-AA protein content, as assessed by immunoblot analyses. Incubation of lung explants with PDGF-BB neutralizing antibodies also resulted in an inhibition of DNA synthesis. Morphometric analyses of antisense-treated cultures showed a significant reduction in lung size when compared to control cultures. The epithelial component of the embryonic lungs was specifically reduced, both in mass and DNA labelling index, by antisense treatment. The number of terminal buds of the lung explants was not significantly affected by antisense PDGF-B treatment. Scrambled PDGF-B oligodeoxynucleotides had no effect. These data suggest that PDGF-BB is involved in regulating growth, but not the degree of branching, of embryonic rat lung.

Calcitonin, CGRP and helodermin in endocrine cells of the developing rat lung

Calcitonin gene-related peptide (CGRP), calcitonin and immunoreactive helodermin occur in endocrine cells in the murine airways. In the lungs these cells form clusters, while they occur scattered in the larynx and trachea. In the present study of the developing rat CGRP-immunoreactive cells were more numerous at all stages than the calcitonin- and helodermin-containing ones. A subpopulation of the CGRP-containing cells contained either calcitonin or helodermin. Immunocytochemistry revealed a higher density of CGRP-containing endocrine cells in the lungs of fetal and newborn rats than in the lungs of older rats, while the density of calcitonin-containing cells was largely unchanged. The helodermin-containing cells were few at all developmental stages examined. The concentration and content of CGRP were highest in newborn rats; the levels decreased postnatally. The calcitonin concentration did not change during development while the content increased with age. The concentration and content of helodermin increased with age.

Development of gas-exchange surface area in rat lung. The effect of alveolar shape.

During development, the surface area of the gas-exchange region of the lung can increase by: (1) expansion, (2) subdivision (septation), (3) replication, or (4) change of shape of the basic gas-exchange units (saccules or alveoli). We evaluated the shape of these units in rat lung from birth to adulthood. A shape factor (phi g) was defined in terms of the surface area (Sg) and the volume (Va) of the average unit, using the expression phi g = SgVa-2/3. We studied the lungs of untreated animals and of animals exposed to or treated with hyperoxia (> 95% O2) and/or dexamethasone, each of which is known to inhibit septation in early postnatal life, and with deferoxamine, which protects the lung against the inhibitory effect of hyperoxia. The values found for the shape factor showed no significant difference with regard to age or treatment. This finding suggests that: (1) saccules and alveoli are formed with a certain predetermined shape (close to a hemisphere), (2) any enlargement with time is isotropic, (3) alveolar shape is insensitive to the drastic treatments used, and (4) change of shape is not a mechanism used to increase the gas-exchange surface area of the developing rat lung.

Phospholipid-transfer proteins and their mRNAs in developing rat lung and in alveolar type-II cells

Gene expression of non-specific lipid-transfer protein (nsL-TP; identical with sterol carrier protein 2) and phosphatidylinositol-transfer protein (PI-TP) was investigated in developing rat lung. During the late prenatal period (between days 17 and 22) there is a 7-fold increase in the level of nsL-TP and a 2-fold rise in that of PI-TP. The prenatal increases in the levels of nsL-TP and PI-TP are accompanied by parallel increases in the levels of their mRNAs, indicating pretranslational regulation. Compared with whole lung, isolated alveolar type-II cells are enriched in nsL-TP and its mRNA, but not in PI-TP and its mRNA. The observation that the levels of nsL-TP and its mRNA in rat lung show a pronounced increase in the period of accelerated surfactant formation, together with the observation that the surfactant-producing type-II cells are enriched in nsL-TP and its mRNA, suggest that nsL-TP plays a role in the metabolism of pulmonary surfactant.

Cell differentiation of alveolar epithelium in the developing rat lung: ultrahistochemical studies of glycoconjugates on the epithelial cell surface.

Glycoconjugates on the surface of pulmonary epithelial cells were ultrahistochemically examined in the fetal, neonatal and adult rat lung. Lectin and colloidal iron staining procedures were performed in combination with digestion using carbohydrate-degrading enzymes or methylation. The glycoconjugate composition of columnar cells at 16 days gestation was similar to that of cuboidal cells at 19 days gestation. Glycoconjugate differentiation on the cell surface occurred at 20 days gestation, and especially the loss of soybean agglutinin (SBA) binding sites could be detected on type II cells. The contents of Ricinus communis agglutinin-I (RCA-I) and Concanavalin A (Con A) binding sites on type II cells also began to decrease. On the contrary, the content of sulfated saccharides decreased on the surface of type I cells during development. Glycoconjugate differentiation on both type I and II cells was completed with the disappearance of hyaluronic acid and peanut agglutinin (PNA) binding sites; type I and II cells acquired a similar histochemical composition to that on adult type I and II cells at 5 days after birth. Both type I and II cells share a common early precursor cell, that is, the cuboidal epithelial cell at the canalicular stage.

Protective effect of the 21-aminosteroid drug (U74389F) against hyperoxic inhibition of normal newborn rat lung development

Protective effect of the 21-aminosteroid drug (U74389F) against hyperoxic inhibition of normal newborn rat lung development

Expression of Immunoreactive Cytidine 5′-Triphosphate: Cholinephosphate Cytidylyltransferase in Developing Rat Lung

The principle rate-limiting enzyme required for phosphatidylcholine production is cytidine 5'-triphosphate:cholinephosphate cytidylyltransferase. Two functional forms of cytosolic cytidylyltransferase have been previously identified: an active high-molecular weight multimer (H-form) and a relatively inactive low-molecular-weight species (L-form). In the present study, we examined the maturational changes in enzyme mass in the subcellular fractions of fetal, neonatal, and adult rat lungs. Total enzyme mass, measured by immunoblotting of total cellular lung homogenates, revealed a large amount of immunoreactive enzyme during the fetal and neonatal periods and relatively low levels of enzyme in the adult lung. A similar developmental profile for enzyme mass was noted in the cytosolic and microsomal fractions. Further, in the fetus, the majority of cytosolic enzyme mass was expressed as an inactive form (L-form). Stimulation of fetal cytosol with phosphatidylglycerol converted the enzyme mass from an inactive form (L-form) to an active form (H-form). In the adult, a substantial portion of the cytosolic enzyme mass was expressed as the active species (H-form). These observations suggest that cytidylyltransferase activity early in lung development is accompanied by an increase in enzyme mass, the majority of which exists as an inactive low-molecular-weight species. In contrast, high levels of enzyme activity are maintained in the adult lung, despite relatively low levels of enzyme mass, because a significantly greater portion of the enzyme mass is expressed as an active high-molecular-weight multimer.

Hyperoxic exposure of developing rat lung decreases tropoelastin mRNA levels that rebound postexposure.

These studies were undertaken to determine whether tropoelastin message expression in lung parenchymal tissue is altered in rats reared in an hyperoxic environment during the period of time that alveolar septation normally occurs. Rat pups were exposed to > 95% oxygen from days 4 to 14 and killed during the exposure and recovery periods. Results of in situ hybridizations indicated a delay in peak tropoelastin (TE) message levels in oxygen-exposed rats vs. controls, day 16 vs. day 11, respectively. In addition, lung parenchymal TE mRNA levels in the oxygen-exposed pups remained elevated through day 23, 1 wk after TE mRNA levels had decreased in controls. These observations suggest that the regulatory control of elastin synthesis during lung alveolar septation is altered by hyperoxic exposure. In a separate experiment, rat pups were exposed to > 95% oxygen during the period of alveolarization and followed for 4 wk postexposure. Pulmonary function measurements were conducted to determine whether lung function was altered postexposure and, if so, whether recovery occurred. We also used stereological techniques to quantitate the total length of lung parenchymal elastic fibers to determine whether elastic fiber content in the oxygen-exposed pups was restored to normal levels during the month postexposure. Although the total length of elastic fibers in lung parenchyma was found to be greater in oxygen-exposed than control pups from postnatal days 22 to 41, pressure-volume curves indicated that lungs of the oxygen-exposed pups tended to be more compliant than controls, suggesting that abnormal elastic fiber assembly might have been a contributing factor.

Morphometric analysis of fetal rat lung development.

Applying the zone concept described previously, we quantitatively analyzed fetal rat lung development. The zone concept allowed us to coherently define reference spaces in the developing lung, a prerequisite for morphometric analysis. The peripheral zone I corresponds to a zone of growth of yet undifferentiated tissues; zone II arises from zone I and represents a region of structural and cellular differentiation; zones III and IV comprise the conducting airways and vessels. Lungs of fetal rats aged 17-23 days and 20 hours postnatal were fixed with OsO4 and glutaraldehyde and processed for light and electron microscopic morphometry implemented by point and intersection counting. Fetal lung volume grew in proportion to body weight. Zone II being the largest compartment, its volume changes largely determined lung growth rates. Zone II increased in mass owing to differentiation processes at the interface to zone I where the proximal portions of zone I were continuously shifted into zone II by differentiation. New tissue was generated within zone I. Due to these combined processes zone I changed little in volume until it disappeared at the end of the canalicular stage. The presence in the pseudoglandular stage of half of the parenchymal epithelial mass available around birth indicated that parenchymal development started earlier than assumed so far. While the endothelial surface increased most at birth, the epithelial surface grew by more than 600% at day 21, reflecting the onset of canalisation. The study confirmed the usefulness of the zone concept for morphometry and provided some new insights into lung development.

Expression of the epithelial Na+ channel in the developing rat lung

The adult mature fetal, but not immature fetal, lung is capable of actively transporting Na+ from the alveolar space. The reason for the impaired Na+ transport in the immature lung is not known; however, the apical membrane Na+ channel is the rate-limiting step for epithelial Na+ transport. This study determined whether transcripts coding for the adult rat colonic epithelial Na+ channel (alpha rENaC) were present in the fetal and adult lung and whether they were developmentally regulated. Similarly sized alpha rENaC transcripts were identified in RNA isolated from fetal and adult whole rat lung, primary cultures of fetal and adult alveolar epithelium, and adult rat whole kidneys, suggesting that the lung alpha rENaC is a similar transcript to that found in the salt-deprived rat colonic epithelium. There were low mRNA levels in 17- to 18-day gestational age (GA) fetal lungs and epithelium (term GA = 22 days), but these levels increased markedly during the saccular stage of lung development (20 days GA) and remained high in adult lungs. The combined administration of thyroid-releasing hormone and dexamethasone to pregnant rats between 16 and 18 days GA induced the expression of lung alpha rENaC in their fetuses. We conclude that alpha rENaC is expressed in mature fetal and adult alveolar epithelium and that it is influenced by hormones known to alter maturation of the fetal lung.

Differential Gene Expression of Antioxidant Enzymes in the Perinatal Rat Lung

It has been previously demonstrated that the developing rat lung markedly increases its catalase (CAT) and glutathione peroxidase (GP) activities during the final 10 to 15% of gestation. In the present studies, we tested whether four major antioxidant enzymes (AOE) in perinatal rat lungs might share a similar pattern of developmental AOE gene expression via a pretranslational mechanism. The left lungs of 18-d to term fetuses and early postnatal rat pups were used to measure the concentrations of AOE mRNA by solution hybridization and the right lungs of the same group of animals were assayed for AOE activities. Results revealed differential AOE gene expression in developing rat lungs. Whereas the CAT and GP activities progressively increased prenatally, the superoxide dismutase (SOD) activity either declined [copper-zinc SOD (Cu, ZnSOD)] or remained constant [manganese SOD (MnSOD)] in late gestation. Postnatally, Cu,ZnSOD and CAT activities progressively increased, whereas MnSOD remained constant and GP activity declined slightly. For Cu,ZnSOD, MnSOD, and CAT, the activity changes were generally consistent with the patterns of changes in their mRNA concentrations in both the prenatal and postnatal period, but for GP they were not. At the time of birth, however, the mRNA levels of Cu,ZnSOD and CAT decreased approximately 40%, whereas their enzyme activities increased. For MnSOD, only a slight rise in mRNA level was observed versus approximately 100% increase in its activity at the time of birth. These findings suggest that the AOE are not coordinately regulated, and that developmental regulation of AOE gene expression in the perinatal rat lung is complex and likely exerted at different levels of regulation.

Degradation of Type IV Collagen during the Development of Fetal Rat Lung

Lung structure undergoes rapid remodeling during late gestation as a functional respiratory unit is formed. To determine the role of collagen turnover in this process, particularly the basement membrane component, we studied the degradation of collagen in a series of fetal rats from day 18 of gestation to full term. During the period of rapid cell proliferation to day 20, the collagen level per milligram of lung did not change though the rate of synthesis increased. More than 40% of new collagen was rapidly degraded. At the end of the growth phase, collagen synthesis rose rapidly as the total collagen content increased in the lung. Over this period, little Type I collagenase activity could be detected, but degradation of Type IV collagen was readily measured and was maximal at 18 to 20 days. The enzyme(s) was almost all present in the active form, and evidence for dissolution of the subepithelial basement membrane was also found by electron microscopy. Using isolated fetal epithelial cells and fibroblasts, supernatants of both cell types showed degradative activity for Type IV collagen, particularly at days 18 to 20. The major enzyme involved appears to be a 72 kD collagenase, as shown by zymography and by mRNA expression in both cell types. The results demonstrate that rapid degradation of Type IV collagen occurs during the growth phase of late fetal lung development, and that both epithelial and stromal cells contribute to collagenolytic activity.

The effect of maternal exercise on somatic growth and lung development of fetal rats: Morphologic and morphometric studies

Our study was aimed at elucidating the effect of strenuous maternal exercise (running at a speed of 20 m/min) on fetal somatic growth and lung development. Dams were separated into three groups: (1) exercising during the entire course of pregnancy; (2) exercising from the 16th to the 20th gestational day; and (3) controls treated in the same manner as the exercising animals, but not exercised. There were no differences in the mother's body weight (mother's body weight--total body weight of the fetuses) among the three groups. Overall growth and lung development in fetuses from mothers that exercised during the entire course of pregnancy showed markedly decreased size and weight of body and lung, smaller air spaces and thicker future alveolar walls, and fewer osmiophilic lamellated bodies in alveolar type II cells. The morphometric data of fetuses from mothers that had exercised only during late gestation were similar to those of control animals. The ratio of fetal lung volume to body weight was the same in all three groups. These findings indicate that long-term strenuous maternal exercise induces retardation of fetal lung development, possibly accompanied by a disturbance of overall growth. Maternal exercise represents another means of manipulating lung development.

Protection against acute and chronic hyperoxic inhibition of neonatal rat lung development with the 21-aminosteroid drug U74389F.

Normal lung development involves septation of the large air saccules present at birth to form smaller diameter alveoli with a much increased surface area for respiratory exchange. This process in the newborn animal is markedly inhibited by hyperoxia, and the altered lung morphology that results may be permanent. We tested whether treatment of neonatal rats with the new 21-aminosteroid (21-AS) drug, U-74389F (15 mg/kg/d), could protect against O2-induced inhibition of normal lung development. By morphometric analysis after 10 d in > 95% O2, the lungs of the animals treated with this potent iron chelator and inhibitor of lipid peroxidation showed a substantial protective effect--with reduced mean air space diameter and significantly increased internal surface area compared with O2 control pups. [Air control mean air space diameter = 47.4 microns, internal surface area = 1014 cm2; O2 controls = 61.0 microns (increases 29%), 769 cm2 (decreases 24%); O2 21-AS = 53.4 microns (increases 13%), 919 cm2 (decreases 9%); p < 0.05 between O2 groups.] Similarly, inhibition of lung elastin deposition (involved in septation process) during hyperoxia was significantly ameliorated by 21-AS treatment. In addition, follow-up studies of young adult rats demonstrated permanently enlarged lung alveoli and reduced surface area after neonatal high O2 exposure. These chronic morphologic effects were also significantly reduced by neonatal 21-AS treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical localization of chondroitin sulfate, chondroitin sulfate proteoglycan, heparan sulfate proteoglycan, entactin, and laminin in basement membranes of postnatal developing and adult rat lungs.

Histologic preparations of lungs from 1-, 5-, 10-, 18-, and 25-day-old postnatal and adult rats were examined immunohistochemically with antibodies specific against chondroitin sulfate (CS), basement membrane chondroitin sulfate proteoglycan (BM-CSPG), heparan sulfate proteoglycan (HSPG), entactin, and laminin. A monoclonal antibody specific for the glycosaminoglycan portion (CS) of CSPG and a monoclonal antibody against the core protein of CSPG were used in an immunoperoxidase sequence to stain extracellular matrix (ECM) components of pulmonary basement membranes (BMs). Anti-CS stained airway BM strongly and alveolar BM weakly in the adult rat lung, as well as in vascular and airway adventitia. In developing lungs, immunoreactivity was strong in all ECM sites, including BM, at day 1 postnatal, and progressively diminished thereafter except in vascular and airway adventitia. Anti-CSPG stained alveolar, airway, and vascular BMs, in addition to smooth muscle external laminae (EL), in the adult and developing rat. Immunostaining for CSPG required hyaluronidase digestion, whereas CS staining was lost with the same treatment. A polyclonal antibody to the core protein of HSPG was found to be similarly distributed to CSPG by immunoperoxidase staining in adult and developing rat lungs, with the notable exception that little immunoreactivity for HSPG was found in smooth muscle EL. Commercially obtained polyclonal antibodies to entactin and laminin gave immunostaining comparable to that seen with CSPG, except that entactin showed particular affinity for EL. These results offer a more detailed perspective on previous survey observations of CSPG, HSPG, and entactin in the rat lung, and describe the immunoreactivity of CS for the first time.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural analysis of fetal rat lung development

The primary aim of this morphological investigation was to elaborate a concept allowing us to coherently define reference spaces for morphometric analysis of fetal lung development. Beyond this quantitative goal, morphological analysis of cell types, definition of compartments, and reflection about the prospective fate of their constituents provided per se new insights into the developmental processes. Lungs of rat fetuses aged 17-23 days and newborns aged 20 hours were fixed with an osmium tetroxide and glutaraldehyde mixture and their volume determined. Left lungs were embedded in Epon and investigated by light and electron microscopy. The right lung of one animal per group was embedded in methacrylate and step sections obtained to precisely locate the airways within the mesenchyme. The various cell types, their topographical relationships, and their morphological alterations with ongoing development were analyzed with regard to their prospective potentials of differentiation. The developing lung could be partitioned into four zones further subdivided into defined compartments. Zone I forms a superficial mantle around the lobes and the future acini. Consisting of primitive mesenchymal cells, it represents a zone of growth which disappears with the onset of the saccular stage. Zone II is mainly a zone of differentiation. Its interstitium stains intensely due to a dense population of dark cells. Up to gestational day 19, zone II contains future conductive airways with their vessels. After day 21, it comprises the whole prospective gas exchange region. Zones III and IV contain the elements of the airway tree and vascular system, zone IV corresponding to the most proximal generations with an adventitial layer. For all differentiation processes, a centrifugal directionality is manifested.

Temporal and spatial differences in glycosaminoglycan synthesis by fetal lung fibroblasts

In studies of the ontogeny of flbroblast-epithelial interactions during late fetal lung rat lung development, we have identified two subpopulations of fibroblasts which differed in their ability to promote epithelial cell proliferation or differentiation. As glycosaminoglycans (GAGs) have been implicated in the regulation of these processes we have tested whether the two fibroblast populations synthesize different GAGs and whether the GAG pattern changes with development. Fibroblasts incorporate more [ 3H]glucosamine and Na 2, 35SO 4 into GAGs than epithelial cells. Both cell types deposited a significant amount of newly synthesized GAGs in the cell-matrix layer. GAGs were lost faster from the cell-matrix layer of fibroblasts ( t 1 2 = 12 h ) than from that of epithelial cells ( t 1 2 = 48 h ). Total GAG synthesis by fibroblasts did not change with advancing gestation, but synthesis of sulfated GAGs by epithelial cells declined with advancing gestation. Independent of gestational age epithelial cells synthesized predominantly heparan sulfate. Depending on their proximity to the epithelium, fibroblasts differed in their production of GAGs. Fibroblasts in close proximity to the epithelium mainly produced and secreted hyaluronan. More distant fibroblasts, from the pseudoglandular stage of lung development synthesized primarily heparan sulfate and chondroitin sulfate. This same population of fibroblasts from the canalicular stage of lung development, produced more hyaluronan. As the shift to hyaluronan occurs with the thinning of the alveolar septal wall, this finding suggests that developmentally regulated GAG production by fibroblasts may facilitate epithelial-fibroblast interaction, thus influencing fetal lung growth and differentiation.

Clara cell secretory protein gene expression in bronchiolar epithelium.

To determine the mechanisms of Clara cell secretory protein (CCSP) gene expression, a cDNA clone was isolated and used in RNA blot analysis. A single 600 bp CCSP specific transcript was detected in the developing rat lung on fetal day 18. This transcript increased in abundance during late fetal life such that adult levels were attained within 2 wk postpartum. CCSP gene expression was tissue specific, being confined to lung and trachea at all developmental stages. The abundance of CCSP mRNA in lung tissue was unchanged after the induction of lung injury in adult rats either with lipopolysaccharide or prolonged exposure to hyperoxia. In situ hybridization of lung tissue revealed that CCSP gene expression is localized to the nonciliated epithelial (Clara) cells of the bronchiolar epithelium throughout fetal and postnatal development. Taken together the results indicate that the gene for CCSP is abundantly expressed in a cell-specific fashion in the lung and suggest that analysis of such expression will be useful in elucidating the role of Clara cells in the growth and development of the bronchiolar epithelium.

The regulated expression of mRNA for Clara cell protein in the developing airways of the rat, as revealed by tissue in situ hybridization

Tissue in situ hybridization has been used on sections of developing rat lung to follow the cellular sites of mRNA expression for a protein identified only in bronchiolar Clara cells. The mRNA for this Clara cell protein (CCP) was first detected on gestational day 16 in only one of the two types of tubules existing in the lung at this developmental stage. During the next 2 days CCP mRNA expression increased uniformly only in the epithelium lining the respiratory tubules. By gestational day 19, CCP mRNA expression became limited to secretory epithelial cells lining the bronchi, and terminal bronchioles. By neonatal day 1, an intense hybridization signal was observed along all of the conducting airways, but it was irregular due to the fact that expression of the CCP gene was limited to the secretory epithelial cells. In adult rats, CCP mRNA was expressed not only in secretory cells of the intrapulmonary airways at all anatomical levels, but also in secretory epithelial cells lining the trachea and its glands, as well as in specific alveolar cells thought to be type II pneumocytes. These findings demonstrate that the regulation of the CCP gene during lung development is a complicated process and that the expression of CCP mRNA does not parallel exactly the sequential development of the airways.

Developmental Changes in Tropoelastin mRNA Levels in Rat Lung: Evaluation by In Situ Hybridization

Alveolarization of the immature lung is thought to be influenced by the presence of elastic fibers that could provide structural support for developing septa. Although morphometric studies have established that alveolar septal development occurs from days 4 to 13 in the neonatal rat, the precise time period over which elastin synthesis occurs has proved difficult to determine. We have evaluated the usefulness of in situ hybridization techniques to follow tropoelastin message expression in parenchymal tissue, small vessels, and bronchioles in the developing rat lung from days 4 through 18. This method proved to be sufficiently sensitive to detect differences in rates of tropoelastin message expression from days 4 through 18 (P less than 0.0001). Peak tropoelastin message expression was observed in the small vessels on day 4 and in parenchymal tissue on days 9 through 11. Because the time course of tropoelastin message expression in small vessels differs from that in parenchymal tissue, the use of lung extracts to analyze rates of tropoelastin synthesis in the developing lung may be in error.