Auswahl der wissenschaftlichen Literatur zum Thema „Fibroblast-pneumocyte factor“

Lungs exposed to elevated O2 concentrations suffer an initial loss of type I pneumocytes, followed by a reparative type II pneumocyte hyperplasia. We hypothesized that type II pneumocyte hyperplasia after exposure of young adult rats to 85% O2 in vivo would be temporally related to 1) an increased concentration of intrapulmonary basic fibroblast growth factor (bFGF), a potent stimulator of type II pneumocyte DNA synthesis in vitro, and 2) an upregulation of pneumocyte receptors for bFGF (FGF-R). Increased rat lung bFGF mRNA, relative to air-exposed control animals, was observed at 4 days of exposure, with no increase at days 6 and 14 of exposure. Parallel changes were observed with bFGF receptor (flg) mRNA. Nuclear runoff assays confirmed increased transcription of both bFGF and flg genes in response to 85% O2, whereas increased translation at 6 days of exposure was confirmed by protein immunoanalysis. Immunohistochemistry demonstrated a broad distribution of bFGF throughout the lung, including the alveolar epithelium, which increased after 6 and 14 days of exposure to 85% O2. Our findings are compatible with a role for bFGF in O2-mediated pneumocyte hyperplasia.

We hypothesize that compensatory lung growth after unilateral pneumonectomy in a murine model is, in part, angiogenesis dependent and can be altered using angiogenic agents, possibly through regulation of endothelial cell proliferation and apoptosis. Left pneumonectomy was performed in mice. Mice were then treated with proangiogenic factors [vascular endothelial growth factor (VEGF); basic fibroblast growth factor (bFGF)], VEGF receptor antibodies (MF-1, DC101), and VEGF receptor small molecule chemical inhibitors. Lung volume and mass were measured. The lungs were analyzed using immunohistochemistry by CD31 staining, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling, type II pneumocytes staining, and proliferating cell nuclear antigen. Compensatory lung growth was complete by postoperative day 10 and was associated with diffuse apoptosis of endothelial cells and pneumocytes. This process was accelerated by VEGF, such that growth was complete by postoperative day 4 with similar associated apoptosis. bFGF had no effect on lung growth. MF-1 and DC101 had no effect. The VEGF receptor small molecule chemical inhibitors also had no effect. VEGF, but not bFGF, accelerates growth. VEGF receptor inhibitors do not block growth, suggesting that other proangiogenic factors play a role or can compensate for VEGF receptor blockade. Diffuse apoptosis, endothelial cell and pneumocyte, occurs at cessation of both normal compensatory and VEGF-accelerated growth. Angiogenesis modulators may control growth via regulation of endothelial cell proliferation and apoptosis, although the exact relationship between endothelial cells and pneumocytes has yet to be determined. The fact that bFGF did not accelerate growth in our model when it did accelerate regeneration in the liver model suggests that angiogenesis during organ regeneration is regulated in an organ-specific manner.

Glucocorticoids induce lung fibroblasts to produce fibroblast-pneumocyte factor, a peptide that stimulates type II cells to synthesize pulmonary surfactant. This effect is known to be more apparent in cells derived from female fetuses, a characteristic that has been attributed to sex-linked differences in the fibroblasts. In the current study, it has been shown that dexamethasone enhances both β-adrenergic receptor (β-AR) activity (1.3- to 1.6-fold increase) and (−)-isoproterenol-induced secretion of surfactant (1.8- to 1.9-fold increase) in type II cells. However, fibroblast-conditioned media (FCM), prepared in the presence of dexamethasone, generates a much greater response to (−)-isoproterenol (3.1- to 3.8-fold increase). Furthermore, each of these effects is more pronounced if both cell types are female-derived. It is hypothesized that the enhanced response to glucocorticoids is the result of a synergistic effect between the steroid and a component of FCM. Neuregulin-1β (NRG1β), which is elevated in FCM generated in the presence of dexamethasone, influences not only the rate of surfactant secretion and the β-AR activity in type II cells, but also enhances in both sexes the cellular response to (−)-isoproterenol. These results suggest that NRG1β might be more effective than glucocorticoids in treating prematurely born male infants, which are known to respond poorly to glucocorticoids. Given that glucocorticoids are known to induce higher levels of β-AR mRNA, the effect of NRG1β, alone and in combination with dexamethasone, on β-AR gene expression was measured using qRT-PCR. Whereas NRG1β had no effect alone, in combination with dexamethasone it produced up to a 4.2-fold elevation in the level of β-AR mRNA.

Mammalian lung develops as an evagination of ventral gut endoderm into the underlying mesenchyme. Iterative epithelial branching, regulated by the surrounding mesenchyme, generates an elaborate network of airways from the initial lung bud. Fibroblast growth factors (FGFs) often mediate epithelial-mesenchymal interactions and mesenchymal Fgf10 is essential for epithelial branching in the developing lung. However, no FGF has been shown to regulate lung mesenchyme. In embryonic lung, Fgf9 is detected in airway epithelium and visceral pleura at E10.5, but is restricted to the pleura by E12.5. We report that mice homozygous for a targeted disruption of Fgf9 exhibit lung hypoplasia and early postnatal death. Fgf9−/− lungs exhibit reduced mesenchyme and decreased branching of airways, but show significant distal airspace formation and pneumocyte differentiation. Our results suggest that Fgf9 affects lung size by stimulating mesenchymal proliferation. The reduction in the amount of mesenchyme in Fgf9−/− lungs limits expression of mesenchymal Fgf10. We suggest a model whereby FGF9 signaling from the epithelium and reciprocal FGF10 signaling from the mesenchyme coordinately regulate epithelial airway branching and organ size during lung embryogenesis.

The fetal lung undergoes extensive physiological and biochemical maturation prior to birth in preparation for its postnatal function as an organ for gas exchange. Pulmonary surfactant, a substance that reduces surface tension and prevents alveolar collapse, is produced by type II pneumocytes within the lung. Reduced ability to produce surfactant leads to neonatal respiratory distress syndrome. Synthesis of the phospholipid component of surfactant, phosphatidylcholine (PC), is stimulated by fibroblast-pneumocyte factor (FPF), a protein expressed by fibroblast cells within the fetal lung. Although its function is well known, the identity of this important protein has remained a mystery. Recent research has suggested that FPF may be neuregulin-1, a growth factor found in many tissues during development. Enhanced synthesis of PC (and therefore detection of FPF) is measured using a tissue culture-based method. Primary cultures of lung fibroblasts and type II pneumocytes are prepared, and fibroblast-conditioned medium (FCM) is exposed to the type II cells. Resultant PC synthesis is measured using radioisotope-labeled PC-precursor and a chloroform-based lipid extraction method. Initial results using this method were very inconsistent, so a study was undertaken to determine which parts of the method could be contributing to this inconsistency. Cell density of type II cultures (measured in μg DNA.plate-1) was shown to have a significant effect on results. Treatment of fibroblasts with 100 nM dexamethasone and exposure of type II cultures to the resultant FCM caused a mean 9.17% increase in PC synthesis, but when only type II cultures with a cell density below 25 μg DNA.plate-1 were analyzed, this value increased to 17.56%. Type II cultures with cell density above this threshold value showed a mean increase in synthesis of only 3.39%. The consistent application of [3H]-choline chloride also had a significant effect on results. Experiments utilizing phorbol 12-myristate 13-acetate to stimulate fibroblasts were very inconsistent. The mean activity of the initial [3H]-choline chloride solution prepared for these experiments was found to be 2.04 μCi.mL-1, compared to a mean of 4.79 μCi.mL-1 for all other experiments. Observations from this section of the study led to considerable revision of the method used to measure PC synthesis. Quadrupolar ion trap mass spectrometry (MS) was used to analyze FCM and determine if neuregulin-1 (NRG1) could be FPF. A mass spectrum was obtained for recombinant NRG1, with predominant ions of 1068, 1142 and 1246 m/z. All three of these ions were also detected in both control and dexamethasone-treated FCM. Partial fragmentation of 1068 m/z of NRG1 was achieved using MS2, and generated a base peak of 1047 m/z. This fragmentation was also observed in 1068 m/z from FCM. LC/MS was utilized to quantify NRG1 in FCM, using a standard curve generated using recombinant NRG1. Control FCM had a NRG1 concentration of 19.85 μg.mL-1, while the concentration in dexamethasone treated FCM was 41.59 μg.mL-1. FCM which had given no positive response to dexamethasone when tested using the indirect cultured cell system had a control NRG1 concentration of 20.85 μg.mL-1, and a dexamethasone treated concentration of 22.84 μg.mL-1. These values were not significantly different from the control value for FCM in those fibroblast cultures that had generated a positive response to dexamethasone. Results of this section of the study have provided strong evidence that NRG1 is a major component of FPF, and a review of the NRG1 signaling pathway further supports this conclusion. Insulin-like growth factors (IGFs) are functionally related to neuregulins and are known to be important in fetal development. The effect of IGF-II on synthesis of surfactant PC and its subsequent secretion from type II pneumocytes was studied. In terms of PC synthesis, IGF-II was tested at concentrations of 0.4, 0.6 and 0.8 μM. The mean increase in synthesis was found to be 6.00, 6.15 and 6.91%, respectively. These values were not significantly different from control values. Secretion of PC was tested over the concentration range of 0.1 to 1.6 μM, with no significant effect observed. Possible inhibition by IGF-II was also studied, using the known stimulants of secretion, neuromedin C and isoproterenol. No significant effect on the enhanced level of secretion was observed when IGF-II was added with either secretagogue. Lack of an appropriate receptor and/or the possibility that cultured cells may not exactly mimic the situation in vivo are probably the reasons IGF-II has no effect on either synthesis or secretion.

The fetal lung undergoes extensive physiological and biochemical maturation prior to birth in preparation for its postnatal function as an organ for gas exchange. Pulmonary surfactant, a substance that reduces surface tension and prevents alveolar collapse, is produced by type II pneumocytes within the lung. Reduced ability to produce surfactant leads to neonatal respiratory distress syndrome. Synthesis of the phospholipid component of surfactant, phosphatidylcholine (PC), is stimulated by fibroblast-pneumocyte factor (FPF), a protein expressed by fibroblast cells within the fetal lung. Although its function is well known, the identity of this important protein has remained a mystery. Recent research has suggested that FPF may be neuregulin-1, a growth factor found in many tissues during development. Enhanced synthesis of PC (and therefore detection of FPF) is measured using a tissue culture-based method. Primary cultures of lung fibroblasts and type II pneumocytes are prepared, and fibroblast-conditioned medium (FCM) is exposed to the type II cells. Resultant PC synthesis is measured using radioisotope-labeled PC-precursor and a chloroform-based lipid extraction method. Initial results using this method were very inconsistent, so a study was undertaken to determine which parts of the method could be contributing to this inconsistency. Cell density of type II cultures (measured in mcg DNA.plate-1) was shown to have a significant effect on results. Treatment of fibroblasts with 100 nM dexamethasone and exposure of type II cultures to the resultant FCM caused a mean 9.17% increase in PC synthesis, but when only type II cultures with a cell density below 25 mcg DNA.plate-1 were analyzed, this value increased to 17.56%. Type II cultures with cell density above this threshold value showed a mean increase in synthesis of only 3.39%. The consistent application of [3H]-choline chloride also had a significant effect on results. Experiments utilizing phorbol 12-myristate 13-acetate to stimulate fibroblasts were very inconsistent. The mean activity of the initial [3H]-choline chloride solution prepared for these experiments was found to be 2.04 mcg Ci.mL-1, compared to a mean of 4.79 mcgCi.mL-1 for all other experiments. Observations from this section of the study led to considerable revision of the method used to measure PC synthesis. Quadrupolar ion trap mass spectrometry (MS) was used to analyze FCM and determine if neuregulin-1 (NRG1) could be FPF. A mass spectrum was obtained for recombinant NRG1, with predominant ions of 1068, 1142 and 1246 m/z. All three of these ions were also detected in both control and dexamethasone-treated FCM. Partial fragmentation of 1068 m/z of NRG1 was achieved using MS2, and generated a base peak of 1047 m/z. This fragmentation was also observed in 1068 m/z from FCM. LC/MS was utilized to quantify NRG1 in FCM, using a standard curve generated using recombinant NRG1. Control FCM had a NRG1 concentration of 19.85 mcg.mL-1, while the concentration in dexamethasone treated FCM was 41.59 mcg.mL-1. FCM which had given no positive response to dexamethasone when tested using the indirect cultured cell system had a control NRG1 concentration of 20.85 mcg.mL-1, and a dexamethasone treated concentration of 22.84 mcg.mL-1. These values were not significantly different from the control value for FCM in those fibroblast cultures that had generated a positive response to dexamethasone. Results of this section of the study have provided strong evidence that NRG1 is a major component of FPF, and a review of the NRG1 signaling pathway further supports this conclusion. Insulin-like growth factors (IGFs) are functionally related to neuregulins and are known to be important in fetal development. The effect of IGF-II on synthesis of surfactant PC and its subsequent secretion from type II pneumocytes was studied. In terms of PC synthesis, IGF-II was tested at concentrations of 0.4, 0.6 and 0.8 mcM. The mean increase in synthesis was found to be 6.00, 6.15 and 6.91%, respectively. These values were not significantly different from control values. Secretion of PC was tested over the concentration range of 0.1 to 1.6 mcgM, with no significant effect observed. Possible inhibition by IGF-II was also studied, using the known stimulants of secretion, neuromedin C and isoproterenol. No significant effect on the enhanced level of secretion was observed when IGF-II was added with either secretagogue. Lack of an appropriate receptor and/or the possibility that cultured cells may not exactly mimic the situation in vivo are probably the reasons IGF-II has no effect on either synthesis or secretion.