Kazuhiro Sugahara

Double intratracheal instillation of keratinocyte growth factor prevents bleomycin-induced lung fibrosis in rats.

Alveolar re‐epithelialization is necessary in the repair of damaged alveolar epithelium after lung injury. Keratinocyte growth factor (KGF) has been shown to be a potent proliferation and differentiation factor for rat alveolar type II cells. The present study examined whether KGF would prevent bleomycin‐induced lung fibrosis. Adult rats were anaesthetized and recombinant human KGF (rhKGF) (150 μg/kg) or saline was injected intratracheally at 48 h before and 24 h after bleomycin (Bleo, 5 mg/kg) instillation. Seven and 14 days after the last administration, rat lungs were processed for lung physiology, immunohistochemistry, and in situhybridization. Double instillation of KGF prevented the loss of body weight and reduction in total lung capacity (TLC) due to Bleo, and markedly attenuated the protein accumulation and mRNA expression of collagen types I and III and the decreased expression of surfactant protein mRNAs in the fibrotic lesions of Bleo‐treated rats. KGF may play an important role in maintaining alveolar epithelium and repairing the damaged epithelium after lung injury.

Differential expression of CCAAT enhancer binding protein family in rat alveolar epithelial cell proliferation and in acute lung injury.

Abstractu2002Although alveolar reorganization after acute lung injury depends on regeneration of alveolar epithelial cells, there is little knowledge of regulation of pulmonary healing process. Transcription factors may play key roles in this regulation. To investigate whether the CCAAT enhancer binding protein (C/EBP) family, α, β, and δ, were involved in alveolar reorganization after injury, we examined expression of C/EBP proteins and mRNAs in lung injuries induced by lipopolysaccharide (LPS) or bleomycin (Bleo) and in cell proliferation by keratinocyte growth factor (KGF). By immunohistochemistry, we demonstrated that C/EBPα and C/EBPβ were expressed in alveolar type II cells and alveolar macrophages, but C/EBPδ was expressed restrictedly in some of alveolar type II cells in a spatial pattern in the control lungs. Further, these three C/EBP family members were differentially expressed in alveolar cell proliferation and in acute lung injury, in which, interestingly, C/EBPα and C/EBPδ were reciprocally expressed in alveolar type II cell proliferation and in pulmonary fibrosis. However, expressions of their mRNAs by in situ hybridization were dramatically increased in the affected lesions of the lungs by LPS and Bleo, and Northern blot analysis showed an increased abundance of the mRNA for C/EBPβ in LPS-treated lungs and for C/EBPδ in Bleo-treated lungs, compared with those in the control lungs. Thus, differential expression of the C/EBP family may be required to maintain and reorganize the basic integrity of alveolar structure during pathological states, which suggests an important role for the C/EBP family in maintaining normal alveolar architecture and function and in repairing the damaged epithelium after injury.

Effects of soluble factors and extracellular matrix on DNA synthesis and surfactant gene expression in primary cultures of rat alveolar type II cells

Abstractu2002Proliferation and differentiation of epithelial cells are thought to be regulated by soluble factors in extracellular fluid and insoluble components of the extracellular matrix. We have examined the combined effects of soluble factors and an extracellular matrix (EHS matrix) on DNA synthesis, cell proliferation, and surfactant protein gene expression in primary cultures of alveolar type II epithelial cells. Cells on EHS matrix cultured in DMEM containing insulin, cholera toxin, EGF, aFGF, 5% rat serum, and 15-fold concentrated bronchoalveolar lavage fluid (D-GM) formed larger aggregates than cells cultured on the same substratum in DMEM containing 5% rat serum (D-5). Cells cultured in D-GM on EHS matrix incorporated more [3H]-thymidine than cells on the same substratum in D-5, with an eight-fold increase seen on day 4 of culture. This increase in [3H]-thymidine incorporation was accompanied by a labeling index of greater than 65% of the cells. Cell counts showed that exposure of type II cells on EHS matrix to D-GM resulted in increased cell number on day 4 of culture. [3H]-thymidine autoradiography combined with immunostaining with anti-cytokeratin, anti-SP-A, and anti-vimentin antibodies demonstrated that the proliferating cells were epithelial cells that contained SP-A. Type II cells cultured on plastic in D-GM also showed increased [3H]-thymidine incorporation compared to cells cultured in D-5. The level of [3H]-thymidine incorporation by cells on plastic, however, was significantly less than that seen in cells cultured in the same medium on EHS matrix. Type II cells cultured on EHS matrix in D-GM had a decreased abundance of mRNAs for SP-A and SP-C than cells cultured on EHS matrix in D-5 as determined by Northern analysis. This inhibition was reversed by switching from D-GM to D-5 on day 4 and culturing the cells for an additional 4 days. In contrast, SP-B mRNA was increased in response to D-GM. This increase was not reversed by switching from D-GM to D-5 on day 4. These results suggest that the interaction of soluble factors and extracellular matrix components has a strong influence on type II cell proliferation, which were partially associated with the reversible inhibition of lung tissue-specific protein mRNAs. Their dynamic interplay among the type II cell, the extracellular matrix, and growth factors may determine multicellular functions and play an important role in normal lung development and in the repair of the lung epithelium following injury.

The effect of fibronectin on cytoskeleton structure and transepithelial resistance of alveolar type II cells in primary culture

SummaryThe cytoskeleton of alveolar type II cells on different matrices has been examined, and the bioelectric properties of these cells grown as monolayers in primary culture has be measured using Ussing-type chambers, to determine whether the extracellular matrix affects the cytoskeletal organization of alveolar type II cells and whether any such interactions influence their physiological functions. Alveolar type II cells cultured on a fibronectin substratum spread slowly over a 6-day period to produce cells of extremely large diameter. The cytoskeletal structure of these cells was characterized by a more marked accumulation of large bundles of actin and a finer network of keratin than cells grown on a collagen substratum. The transepithelial resistances of monolayers grown on a fibronectin substratum were much higher than those on a collagen substratum. These results indicate that alveolar type II cells cultured on fibronectin can form tighter, better organized and more polarized monolayers in primary culture, which suggests that fibronectin may have a physiologically important role in the maintenance of the alveolar wall.

Studies of the lung in diabetes mellitus

To evaluate the effects of chemically induced diabetes on lung tissue, we examined the ultrastructure of the lung of alloxan-induced diabetic rats. Fifty male Wistar rats were made diabetic by a single intraperitoneal injection of alloxan (200 mg/kg of body weight): they were sacrificed from one to four weeks later. The alloxan-induced diabetes produced significant morphological alterations in the lung. These include marked dilatation of the cisterna of the granular endoplasmic reticulum, dilation of the Golgi saccules and the appearance of glycogen granules as a cluster in the cytoplasm of the granular pneumocytes and the interstitium. These findings were well correlated with the severity of diabetes mellitus. The altered granular pneumocytes were observed in about 50% of animals and in most (87.5%) of the observed pneumocytes 2 weeks and 4 weeks after alloxan treatment respectively. The average number of lamellar inclusion bodies per granular pneumocyte decreased to about half of that of the control in diabetic rats 4 weeks after alloxan treatment, and minimum thickness of the capillary basement membrane was approximately 35% thicker than that of the control (diabetics; 879±189 Å, controls; 649±100 Å). The ultrastructural alterations of the lung in diabetic rats indicate disorders in the pulmonary capillaries and in the metabolism of pulmonary surfactant, which may cause pulmonary dysfunction in diabetic patients.

Studies of the lungs in diabetes mellitus II. Phospholipid analyses on the surfactant from broncho-alveolar lavage fluid of alloxan-induced diabetic rats

Abstract Present experiments were undertaken to analyze the phospholipid content and composition in the surfactant from broncho-alveolar lavage fluid of alloxan-induced diabetic rats. The diabetic rats 4 weeks after alloxan treatment had significantly less phospholipid contents in the white layer from broncho-alveolar lavage fluid than the control. As to the phospholipid composition, the white layer of the diabetic rats contained significantly less phosphatidyl choline, and more sphingomyelin and lysophosphatidyl choline compared with the control. These results suggest that diabetes mellitus may induce a disturbance in the synthesis of pulmonary surfactant.

Effects of halothane and enflurane on epithelium-dependent contraction and ion transport of canine tracheal epithelium

To gain insight into the cellular mechanisms involved in bronchodilation induced by inhalation anesthetics, we investigated whether halothane and enflurane can modulate functions of airway epithelium, such as epithelium-mediated bronchodilation and transepithelial transport. To measure the isometric tension of airways, paired rings of canine bronchi (4–6 mm OD), with and without the epithelium were mounted in Krebs-Ringer solution, gassed with 95% O2 and 5% CO2, and isometric tension was continuously recorded. To determine transepithelial transport, the posterior membranous portion of the trachea was mounted in Ussing-type chambers and the potential difference (PD), short-circuit current (SCC), and transepithelial resistance (R) were determined.Halothane and enflurane increased the contractile responses of the trachea to acetylcholine (ACh) in strips either with or without epithelium. However, this enhancement of the contractile responses by volatile agents was much larger with the epithelium than without. Furthermore, halothane tended to gradually increase and then decrease SCC of the trachea, but these changes were not statistically significant. These results indicate that halothane may modulate contractile response of the isolated trachea to ACh, but has no effect on ion transport by airway epithelium. The responsiveness of the trachea may be regulated independently of ion transport by airway epithelium.