Expression of retinoic acid receptor genes in fetal and newborn rat lung.

Lung differentiation and development are affected by vitamin A and its metabolites. One mechanism through which retinoids might exert their effects is through nuclear retinoic acid receptors (RAR). The gene expression profile of the RAR family (alpha, beta, gamma) has previously been determined in both the developing mouse embryo to 14.5 days gestation, and in the adult lung. The purpose of this study was to determine the expression of the RAR genes during the period of gestation that results in the formation of the saccular lung stage. Total RNA was extracted from fetal lungs of Sprague-Dawley rats at gestational days 17, 19, 20, 21, and 22, and from 12-hour-old newborns for Northern hybridization. Two transcripts of RAR alpha mRNA (3.7 and 2.7 kb) were found at each time point. At day 17, the 2.7 kb RAR alpha mRNA was increased two-fold or more than at any other time studied. At days 19-22 the levels of the 3.7 kb RAR alpha species were also lower than day 17 and newborn levels. One RAR beta mRNA transcript (3.4 kb), present at all time points, was significantly higher in the newborn than on days 17-22. Expression of RAR gamma mRNA could only be demonstrated by reverse transcriptase-polymerase chain reaction. We speculate that the higher RAR alpha species at day 17 indicates a role for RAR alpha in the maintenance of the columnar epithelial cells of the glandular phase of lung development.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in tissue fibronectin in elastase induced lung injury.

We studied changes in rat lung fibronectin (FN) content and synthesis after endobronchial administration of elastase. A severe hemorrhagic neutrophilic alveolitis ensued with plasma protein leakage, initial rise in tissue FN content, and sustained rise in FN synthesis. Unlike fibrotic models where initial rises in tissue FN levels are sustained, levels in this model normalized promptly. This, in the setting of increased synthesis is consistent with increased degradation. This degradation of tissue FN may result in the disruption of the lung architecture, interfere with the deposition of newly synthesized matrix and could partly explain the development of emphysema in a model where excess fibronectin synthesis is observed.

Effect of transforming growth factor beta on synthesis of glycosaminoglycans by human lung fibroblasts.

The processes of lung growth, injury, and repair are characterized by alterations in fibroblast synthesis and interstitial distribution of extracellular matrix components. Transforming growth factor beta (TGF-beta), which is postulated to play a role in modulating lung repair, alters the distribution of several matrix components such as collagen and fibronectin. We studied the effect of TGF-beta on the synthesis and distribution of the various glycosaminoglycans (GAGs) and whether these effects may explain its role in lung repair. Human diploid lung fibroblasts (IMR-90) were exposed to various concentrations of TGF-beta (0-5 nM) for variable periods of time (0-18 h). Newly synthesized GAGs were labeled with either [3H]glucosamine or [35S]sulfate. Individual GAGs were separated by size exclusion chromatography after serial enzymatic and chemical digestions and quantitated using scintillation counting. There was a dose-dependent increase in total GAG synthesis with maximal levels detected after 6 h of exposure. This increase was noted in all individual GAG types measured and was observed in both the cell associated GAGs (cell-matrix fraction) as well as the GAGs released into the medium (medium fraction). In the cell-matrix fraction, TGF-beta increased the proportion of heparan sulfate that was membrane bound as well as the proportion of dermatan sulfate in the intracellular compartment. In the medium fraction, TGF-beta increased the proportion of hyaluronic acid, chondroitin sulfate and dermatan sulfate released. We conclude that the role of TGF-beta in lung growth and repair may be related to increased synthesis of GAGs by human lung fibroblasts as well as alterations in the distribution of individual GAGs.

Subcellular changes in capillary endothelial cells during repair of hyperoxic lung injury.

We studied the changes in subcellular ultrastructure associated with the hypertrophy of capillary endothelial cells during repair of hyperoxic (100% O2) lung injury in rats. We used stereologic-morphometric measurements at different magnifications to determine the absolute volume of each subcellular compartment per average capillary endothelial cell. The increases in this value during the first 3 days of postexposure repair were 118% for cytoplasm, 786% for polyribosomes, 310% for rough endoplasmic reticulum, and 79% for mitochondria; the volume of pinocytotic vesicles did not change. By day 7 of repair, only the polyribosomes and rough endoplasmic reticulum were still increased; by day 14 all values were normal. We conclude that the capillary endothelial cell hypertrophy that develops during repair of hyperoxic lung injury is associated with large and heterogeneous increases in subcellular organelles and is not merely due to increases in the cytosol or to cellular edema. These increases seem to be an integral part of the repair process and may be important in the development of tolerance to subsequent oxygen exposure.

Effect of 70% oxygen on postresectional lung growth in rats.

We tested the hypothesis that exposure to hyperoxia could inhibit postresectional compensatory lung growth to the same degree that it inhibits newborn lung growth. We removed the 3 upper lobes of the right lung of rats, allowed them to breathe either air or 70% oxygen after surgery, and performed electron microscopy and morphometry on the left lung at 14 d postresection. Rats that had a thoracotomy without removal of lung were used as controls. Resection of lung tissue resulted in increases of about 100-200% (relative to controls) in the total volume per left lung of alveolar type 1 and type 2 epithelial cells, capillary endothelial cells, interstitial cells, and interstitial matrix; the total capillary and type 1 epithelial surface areas each increased about 40%. Exposure to 70% oxygen did not significantly inhibit postresectional growth, although there was a trend toward a lesser increase in capillary surface area. However, 70% oxygen did result in a 78% greater (relative to the nonexposed resected group) alveolar type 2 cell volume density and a 54% greater interstitial cell volume density; this suggested that increased proliferation of type 2 cells and interstitial cells occurred. Qualitative ultrastructural assessment confirmed that the type 2 cells and fibroblasts appeared increased and that interstitial edema and neutrophil accumulation were also present. We conclude that although 70% oxygen exposure is not entirely innocuous, it does not inhibit postresectional lung growth.

Effect of subcellular matrix on glycosaminoglycan synthesis by human lung fibroblasts.

The influences modulating glycosaminoglycan production by lung cells are not well understood. We examined the effect of three different subcellular matrices, plastic, type I collagen, and reconstituted basement membrane-like material (RBM), on the synthesis of sulfated glycosaminoglycans by cultured IMR-90 human lung fibroblasts. Accumulation of 35SO4-labeled glycosaminoglycans into the cell-matrix layer or medium was measured. Cells on collagen synthesized significantly less total glycosaminoglycans than cells on plastic but had a higher fraction of labeled glycosaminoglycans present in the cell-matrix layer (35 vs. 18%) with the increases being highest for dermatan and chondroitin sulfates. Cells grown on the RBM synthesized significantly more glycosaminoglycans than cells on plastic or collagen and also had 260% more labeled glycosaminoglycans present in the cell-matrix layer than cells on plastic. We conclude that the matrix to which lung fibroblasts are exposed can influence the amount and type of glycosaminoglycans synthesized and the degree of incorporation into the matrix. This may be relevant to fibrotic lungs with increased type I collagen or to severely injured lungs in which intra-alveolar fibroblasts are in contact with denuded basement membranes.

Changes in lung tissue fibronectin content and synthesis during postnatal lung growth.

We studied changes in lung tissue fibronectin content and synthesis during postnatal lung growth in rats. We reasoned that fibronectin, which is important in cell differentiation, migration, and adhesion, and in the organization of the extracellular matrix, might play a role in the rapid cell proliferation and alveolar septal formation that occurs postnatally in mammalian lungs. Newborn rats were sacrificed at 4, 7, 11, 14, and 21 days after birth. The lungs were perfused and lavaged, tissue fibronectin was extracted using urea and heparin (Bray et al, Science 1981; 214:793) and the extracted fibronectin was measured by enzyme-linked immunoassay. Tissue fibronectin synthesis was measured by the in vivo incorporation of 35S-methionine into fibronectin that was extracted from lung tissue and immunoprecipitated. Lavage fibronectin and albumin content and lung tissue collagen (hydroxyproline) content were also determined. Lung tissue fibronectin content per g dry lung almost doubled between days 4 and 7 after birth, was slightly higher at day 14 than at day 7, and decreased sharply between days 14 and 21. Lung tissue fibronectin synthesis per g dry lung increased steadily between days 4 and 14 to reach a peak value of about 2.5 times the 4-day value; it then decreased sharply between days 14 and 21. The period of increased fibronectin content and synthesis (4 to 14 days) coincided with the period during which lung cell proliferation and secondary alveolar septa formation are known to be the most active, and the sharp decrease in fibronectin content and synthesis (between 14 and 21 days) coincided with the period during which lung growth and remodeling markedly decrease.(ABSTRACT TRUNCATED AT 250 WORDS)

Repair of chronic hyperoxic lung injury: changes in lung ultrastructure and matrix.

We studied changes in lung ultrastructure, fibronectin, and collagen during repair of chronic hyperoxic lung injury induced by exposure of rats to 85% oxygen for 14 days. Morphologically, the most persistent changes were in the alveolar interstitium. After 28 days of repair, the extracellular matrix volume was still twofold normal. Total interstitial cell numbers also remained high and interstitial myofibroblast number actually doubled between Days 7 and 14. These changes contrast markedly with repair of acute lung injury induced by 100% oxygen (Thet et al. (1986) Exp. Lung Res. 11, 209-228) in which matrix volume and interstitial myofibroblast number increased initially but then returned to normal. Biochemically, tissue-associated fibronectin was high initially and peaked at 3 days before slowly declining. Tissue collagen content began to increase after the peak in fibronectin content and was over 150% of controls at 28 days; this correlated with an increase in visible collagen fibers. We conclude that changes in lung morphology and matrix after chronic hyperoxic lung injury are more persistent than after acute hyperoxic lung injury and result in a greater degree of chronic interstitial fibrosis.

Changes in lung glycosaminoglycans during postresectional lung growth.

We studied changes in glycosaminoglycan content and concentration during postresectional compensatory lung growth in adult male rats. After right trilobectomy, left lung dry weight was normal at 4 days, increased 74% between 4 and 7 days, and more slowly over the next week. Total glycosaminoglycan content per milligram dry lung weight increased early and rapidly, reaching 189% of the control value at 4 days postresection. The magnitude and temporal pattern of increase was different for different glycosaminoglycan subtypes. Hyaluronate and chondroitin sulfate content were increased by 198 and 113%, respectively, at 4 days, with no further increases subsequently. Heparan sulfate content increased more slowly and steadily, and dermatan sulfate concentrations did not change. At 4 days, the percent of total glycosaminoglycans that was hyaluronate was almost doubled, whereas the percent that was heparan sulfate was decreased; by day 7 the percent compositions had returned to normal. We conclude that changes in glycosaminoglycans occur early in postresectional lung growth and speculate that they may play a facilitatory role.

Changes in lung morphology and cell number in radiation pneumonitis and fibrosis: a quantitative ultrastructural study.

We used stereologic-morphometric techniques to obtain a detailed quantitative picture of the changes in lung ultrastructure of rats at 12 and 26 weeks after unilateral thoracic irradiation with 3000 cGy. At 12 weeks post-radiation, the total number type 1 epithelial cells, type 2 epithelial cells and capillary endothelial cells were decreased 50-70%, total type 1 epithelial and capillary surface areas were decreased 55-60%, and the total volume of intracapillary blood was decreased 75%. The interstitial cells and matrix together accounted for more than 9% of the peripheral lung tissue volume including air, compared to 3% in controls. The numerical density of interstitial cells was increased to 3-fold the control value. The numerical density of interstitial cells was increased to 3-fold the control value. Although fibroblasts still comprised the largest interstitial cell subgroup, the numerical density of mast cells was increased over 150-fold and other inflammatory and immune cells were increased to a lesser extent. At 26 weeks post-radiation, the number, volume, and surface area of the type 1 epithelium and capillary endothelium had further decreased to only 5-10% of control values. The total number of type 2 epithelial cells was reduced by 75% but the volume density was actually increased because of a 4-fold increase in the mean cell volume. The interstitial cells and matrix now comprised over 77% of total peripheral lung tissue volume including air as compared to 6% in controls. Mast cells and plasma cells comprised 11% and 19% of all interstitial cells respectively and the densities of these cells were 540 and 180-fold the control value respectively. The relation of these morphometric findings to the results of previous morphologic studies is discussed.

Unilateral paraquat-induced lung fibrosis: evolution of changes in lung fibronectin and collagen after graded degrees of lung injury.

We describe a model of pulmonary fibrosis in which doses of paraquat ranging from 0.001 mg/kg to 1.0 mg/kg were instilled into the right lung of rats. Lung injury, as measured by right lung lavage albumin content and differential neutrophil count, ranged from undetectable to extremely severe, depending on the dose. Lung fibrosis, as assessed by collagen content and electron microscopy, showed similar dose-response effects. Mortality was minimal. Lavage fibronectin increased after high doses of paraquat, peaked at 2 d postinjury, decreased sharply after 3 d and was normal by 7 d. The temporal pattern was similar to that for albumin. Cultured alveolar macrophages obtained at 4 d postinjury did not have significant increases in fibronectin release. Tissue fibronectin content increased more slowly than lavage fibronectin, peaking at 4 d postinjury, and was still elevated at 7 and 14 d postinjury. Incorporation of [35S]methionine into tissue fibronectin by lung explants obtained at different times postinjury showed a similar time course. Lung collagen content increased steadily between 4 and 14 d postinjury. We conclude that, in our model, graded degrees of lung injury and fibrosis can be produced by varying the dose of unilaterally instilled paraquat and that the increases in lavage fibronectin were related mainly to capillary permeability whereas increases in tissue fibronectin represented parenchymal synthesis. The time course of changes in lung tissue fibronectin and collagen was consistent with the proposed roles of fibronectin in tissue repair and fibrosis. The ability of our model to produce graduated degrees of lung injury and fibrosis should be useful in further studies on the pathogenesis of postinjury lung fibrosis.

Role of ornithine decarboxylase and polyamines in early postnatal lung growth.

We assessed the importance of ornithine decarboxylase (ODC) and polyamines in early postnatal lung growth. Lung-ODC activity in newborn rats rose rapidly after birth and was highest at 4-6 days of age. Lung putrescine and spermidine specific contents also peaked during this period, but spermine specific content remained relatively unchanged. The temporal pattern of these changes differed markedly from that in the heart, brain, and kidney where ODC activity is highest at birth then rapidly declines. The period of peak lung-ODC activity and polyamine specific content correlated with rapid increases in lung DNA content, protein content, and weight. The specific irreversible ODC inhibitor, alpha-difluoromethylornithine, significantly reduced lung-ODC activity and putrescine and spermidine specific content; it also caused significant early reductions in lung DNA and protein content without simultaneously affecting body weight and appearance. Morphometrically, the lungs of alpha-difluoromethylornithine-treated rats had significantly fewer type 2 epithelial cells, interstitial cells, and capillary endothelial cells than the lungs of controls. We conclude that ODC and polyamines play an important role in postnatal lung growth and that alpha-difluoromethylornithine can be used as a probe to disrupt lung growth.

Changes in lung tissue and lavage fibronectin after paraquat injury in rats.

Lung lavage fibronectin is known to be elevated after experimental lung injury; however, changes in lung tissue fibronectin content probably more directly reflect tissue injury and repair. We injured rat lungs with a single injection of paraquat and serially measured the fibronectin content of both lung lavage fluid and lung tissue. Changes in lung ultrastructural morphology and collagen content were also assessed. Although both lavage and tissue content of fibronectin increased, the temporal patterns of increase were very different. Lavage fibronectin peaked at 1 day post-injury, was normal by 7 days, and paralleled changes in lavage albumin. Tissue fibronectin was normal at 1 day post-injury, peaked at 4 days and was still high at 7 and 14 days. The period of increased tissue fibronectin coincided with electron microscopic evidence of cell injury and repair at 7 days. By 14 days post-injury the lungs appeared virtually normal. Lung collagen content remained normal throughout the period of injury and repair. We conclude that increases in lung lavage fibronectin do not necessarily correlate with changes in lung tissue fibronectin and that the latter may more accurately reflect lung tissue repair. In addition, even large increases in both measurements do not necessarily predict the development of post-injury fibrosis.

Sequential changes in lung morphology during the repair of acute oxygen-induced lung injury in adult rats.

We studied changes in lung ultrastructure and collagen content during the repair of acute lung injury in adult rats exposed to 100% O2 for 60 h and recovering in ambient air. In the interstitium, during the first 3 days of repair, the number of neutrophils decreased 16-fold, and monocytes and lymphocytes increased to 7-fold and 4-fold the respective control values. Myofibroblasts increased about 5-fold and the volume of the interstitial matrix remained high. By 7 days, the differential count of inflammatory cells was normal although the number of total interstitial cells and myofibroblasts decreased more slowly. In the capillary endothelium, after 3 days of repair, the cells were hypertrophied and had organelle-rich cytoplasm, and total cell number had increased back to control values; endothelial cell number increased an additional 63% between 3 and 7 days of repair. In the epithelium, type 2 cells increased 150% during the first 3 days of repair before decreasing; type 1 cell number did not change. After 28 days of repair, the lungs appeared qualitatively almost normal; however, interstitial cell number and collagen content were still increased. We conclude that the repair of oxygen-induced lung injury involves a complex pattern of morphologic changes that has important similarities to those occurring during repair on other tissues such as the skin.

A technique for unilateral instillation of agents into the rat lung.

We describe a technique for unilateral instillation of agents into the right lung of rats. We intubated rats perorally with a 16-gauge flexible polyethylene catheter and through it introduced narrow-bore (less than 1 mm diam) polyethylene tubing into the trachea and beyond the carina into the right main-stem bronchus. This maneuver was facilitated by placing the animal supine with the cephalad end tilted up and the right side tilted down. We tested the effectiveness of our technique by instilling Evans blue dye into the right lung of 14 rats and spectrophotometrically quantitating the amount of dye present in homogenates from each lobe of the right and left lungs. Ninety-seven percent of the instilled dye was recovered from the right lung, and distribution of dye per gram of tissue was uniform among the four lobes. The technique described should be useful in producing severe degrees of unilateral lung injury and fibrosis in rodents without the high mortality that can be associated with bilateral lung injury.

Changes in cell number and lung morphology during early postpneumonectomy lung growth.

We studied early postpneumonectomy lung growth in adult rats with the aim of determining which lung cells and tissues were involved and what the magnitude of the changes was. Ultrastructural stereological-morphometric measurements were performed 7 days after left pneumonectomy; lung weight, DNA, and protein were also measured. Relative to sham controls, the number of type 2 epithelial cells per right lung increased 34% and the number of capillary endothelial cells increased 28%. The total type 1 epithelial cell volume and surface area both increased 26%, and the total volume and luminal surface area of the capillary endothelial cells increased 24 and 22%, respectively. The number of interstitial cells did not change significantly. The total number of lung cells in the alveolar region was increased 22%; this correlated with a 26% increase in right lung DNA content. We conclude that the main changes during early postpneumonectomy lung growth are increases in the number of type 2 epithelial cells and capillary endothelial cells and in the total capillary and alveolar epithelial surface area.

Repair of oxygen-induced lung injury in adult rats. The role of ornithine decarboxylase and polyamines.

We studied the repair of lung injury in adult rats exposed to 100% oxygen for 60 h, then placed in ambient air. Lung ornithine decarboxylase (ODC) activity and polyamine (putrescine, spermidine, and spermine) content during repair were correlated with changes in lung ultrastructure. The effect of difluoromethylornithine (DFMO), a selective Irreversible ODC inhibitor, was also studied; ODC activity increased to 25-fold baseline 2 days after injury and returned to normal by 7 days. Polyamine content increased to 3-fold baseline during the first 3 days. During the same period, the number of capillary endothelial cells and the capillary surface area almost doubled, and the number of type 2 epithelial cells increased 2.5-fold. The DFMO treatment lowered ODC activities below baseline, reduced the increase in polyamine content, and also reduced the morphometric parameters described above to only 60 to 70% of the values during normal repair. It also caused a significant decrease in the number of type 1 epithelial cells during repair, suggesting that deficient replacement by differentiating type 2 epithelial cells occurred. We conclude that marked changes in lung ODC activity and polyamine content occur during the repair of oxygen-induced injury to the lung and that selective inhibition of these changes adversely affects repair.

Changes in lung hyaluronidase activity associated with lung growth, injury and repair.

We measured lung hyaluronidase activity in rats during postnatal life and during the repair of oxygen-induced lung injury. Hyaluronidase activity increased rapidly after birth and peaked at 16-fold the initial value at 8 days. The peak preceded decreased cell proliferation and the onset of differentiation; this is consistent with current concepts of the role of hyaluronidase. During the repair of lung injury, hyaluronidase activity increased to 2.5-fold the control value at 1 day post-injury, but had decreased by 3 days. This early peak is probably related to simultaneous cell proliferation and differentiation. We postulate that changes in hyaluronidase can influence lung growth and repair and that the system may be amenable to manipulation.

A simple method of intubating rats under direct vision.

Intubation of rats was performed under direct vision using a fiberoptic light guide for illumination. The technique was atraumatic, easily learned, and the success rate was high.