Antifibrotic therapy for the treatment of pulmonary fibrosis.

Fibroproliferative lung disease is prevalent and associated with high mortality. The pathogenesis of fibrotic lung disease involves inflammation, mesenchymal cell proliferation, and deposition of interstitial matrix components, such as collagen and fibronectin. Corticosteroids and other immunosuppressive medications have been routinely employed, but have demonstrated only marginal efficacy. Even though this information has been known for some time, the optimal medical regimen for treating pulmonary fibrosis remains largely undefined. This article addresses the rationale for, and efficacy of, antifibrotic regimens used to treat humans with fibrotic lung diseases.

Complicated persistent patent ductus arteriosus with acute pneumonia in an adult.

Patent ductus arteriosus (PDA) is a malady usually identified during childhood. Prompt surgical correction provides definitive therapy with long-term survival. When not corrected, PDA leads to significant morbidity and mortality, making it a rare condition in the adult population. We report the case of a 44-year-old man with a history of persistent PDA admitted for worsening dyspnea and fever. Radiographic studies are illustrative of this complex syndrome. We review the complications and treatment alternatives in these patients.

PDGF-beta receptor expression and ventilatory acclimatization to hypoxia in the rat.

Activation of platelet-derived growth factor-beta (PDGF-beta) receptors in the nucleus of the solitary tract (nTS) modulates the late phase of the acute hypoxic ventilatory response (HVR) in the rat. We hypothesized that temporal changes in PDGF-beta receptor expression could underlie the ventilatory acclimatization to hypoxia (VAH). Normoxic ventilation was examined in adult Sprague-Dawley rats chronically exposed to 10% O(2), and at 0, 1, 2, 7, and 14 days, Northern and Western blots of the dorsocaudal brain stem were performed for assessment of PDGF-beta receptor expression. Although no significant changes in PDGF-beta receptor mRNA occurred over time, marked attenuation of PDGF-beta receptor protein became apparent after day 7 of hypoxic exposure. Such changes were significantly correlated with concomitant increases in normoxic ventilation, i.e., with VAH (r: -0.56, P < 0.005). In addition, long-term administration of PDGF-BB in the nTS via osmotic pumps loaded with either PDGF-BB (n = 8) or vehicle (Veh; n = 8) showed that although no significant changes in the magnitude of acute HVR occurred in Veh over time, the typical attenuation of HVR by PDGF-BB decreased over time. Furthermore, PDGF-BB microinjections did not attenuate HVR in acclimatized rats at 7 and 14 days of hypoxia (n = 10). We conclude that decreased expression of PDGF-beta receptors in the dorsocaudal brain stem correlates with the magnitude of VAH. We speculate that the decreased expression of PDGF-beta receptors is mediated via internalization and degradation of the receptor rather than by transcriptional regulation.

Interstitial fibrosis and growth factors.

Interstitial pulmonary fibrosis (IPF) is scarring of the lung caused by a variety of inhaled agents including mineral particles, organic dusts, and oxidant gases. The disease afflicts millions of individuals worldwide, and there are no effective therapeutic approaches. A major reason for this lack of useful treatments is that few of the molecular mechanisms of disease have been defined sufficiently to design appropriate targets for therapy. Our laboratory has focused on the molecular mechanisms through which three selected peptide growth factors could play a role in the development of IPF. Hundreds of growth factors and cytokines could be involved in the complex disease process. We are studying platelet-derived growth factor because it is the most potent mesenchymal cell mitogen yet described, transforming growth factor beta because it is a powerful inducer of extracellular matrix (scar tissue) components by mesenchymal cells, and tumor necrosis factor alpha because it is a pleiotropic cytokine that we and others have shown is essential for the development of IPF in animal models. This review describes some of the evidence from studies in humans, in animal models, and in vitro, that supports the growth factor hypothesis. The use of modern molecular and transgenic technologies could elucidate those targets that will allow effective therapeutic approaches.

Brainstem activation of platelet-derived growth factor-beta receptor modulates the late phase of the hypoxic ventilatory response.

The early phase of the biphasic ventilatory response to hypoxia in mammals is critically dependent on NMDA glutamate receptor activation within the nucleus of the solitary tract. However, the mechanisms underlying the subsequent development of the typical ventilatory roll-off are unclear and could underlie important roles in the functional and molecular adaptation to oxygen deprivation. Because the growth factor platelet-derived growth factor (PDGF)-BB can modulate the open channel probability of NMDA receptors by activating PDGF-beta receptors, its contribution to hypoxic ventilatory roll-off was examined. Administration of PDGF-BB, but not PDGF-AA, in the nucleus of the solitary tract was associated with significant attenuations of the early hypoxic ventilatory response in conscious rats. Furthermore, marked reductions in the magnitude of hypoxic ventilatory roll-off occurred in mice heterozygous for a mutation in the PDGF-beta receptor. Administration of a PDGF-beta receptor antagonist to wild-type littermates elicited similar declines in hypoxic ventilatory roll-off. The relative abundance of PDGF-beta receptors was confirmed in the nucleus of the solitary tract and other nuclei implicated in the hypoxic ventilatory response. In nucleus of the solitary tract lysates, PDGF-beta receptor tyrosine phosphorylation was temporally correlated with hypoxic ventilatory roll-off formation. Increased PDGF-B chain mRNA expression was induced by hypoxia in the nucleus of the solitary tract, and PDGF-B chain immunoreactivity colocalized with approximately 40% of nucleus of the solitary tract neurons, demonstrating hypoxia-induced c-Fos enhancements. Thus, PDGF-BB release and PDGF-beta receptor activation in the nucleus of the solitary tract are critical components of hypoxic ventilatory roll-off and may have important functional implications in processes underlying survival and acclimatization to hypoxic environments.

Exacerbation of bleomycin-induced lung injury in mice by amifostine.

Bleomycin (BLM) induces lung injury and fibrosis in the murine lung and enhances tumor necrosis factor (TNF)-alpha and collagen mRNA expression in the murine lung. Amifostine is a cytoprotective agent that protects normal tissues from the cytotoxic effects of chemo- and radiation therapy. We investigated the effect of amifostine in BLM-induced lung injury in mice. Mice received intraperitoneal amifostine (200 mg/kg) 30 min before and/or 1, 3, and 7 days after an intratracheal injection of saline or BLM (4 U/kg). The animals were killed 14 days after BLM exposure, and their lungs were studied for TNF-alpha and collagen mRNA expression, hydroxyproline content, and histopathology. Light microscopy demonstrated that amifostine exacerbated the BLM-induced lung injury in mice. Increased TNF-alpha mRNA expression as a result of BLM exposure was not modulated by amifostine treatment. In contrast, amifostine treatment enhanced the BLM-induced expression of alpha(1)(I) procollagen mRNA in the lung. Similarly, mice treated with amifostine before BLM exposure accumulated significantly higher amounts of hydroxyproline (111 +/- 5 microg/lung) than BLM-treated animals (90 +/- 6 microg/lung). These data suggest that amifostine treatment exacerbates BLM-induced lung injury in mice.

Differential expression of platelet-derived growth factor-alpha receptor by Thy-1(-) and Thy-1(+) lung fibroblasts.

Fibroblasts are heterogeneous with respect to surface markers, morphology, and participation in fibrotic responses. This study was undertaken to determine whether Thy-1(-) and Thy-1(+) rat lung fibroblasts, which have distinct and relevant phenotypes, differ in their proliferative responses to platelet-derived growth factor (PDGF) isoforms. Homogeneous populations of Thy-1(-) and Thy-1(+) fibroblasts were found to proliferate equally in the presence of PDGF-BB, but PDGF-AA-mediated proliferation occurred only in Thy-1(-) cells. This differential activity correlated with significantly higher expression of PDGF-alpha receptor in Thy-1(-) fibroblasts as shown by immunoblotting, immunofluorescence, and Northern blotting. There was a rapid increase in c-myc mRNA in Thy-1(-) but not in Thy-1(+) fibroblasts on stimulation with PDGF-AA and PDGF-BB. The PDGF-alpha receptor, which mediates signaling by all PDGF isoforms, has been implicated in numerous clinical and experimental forms of fibrosis and regulates lung morphogenesis. Differential expression of the PDGF-alpha receptor supports distinct roles for Thy-1(-) and Thy-1(+) fibroblast populations in developmental and fibrotic processes in the lung.

Emphysematous lesions, inflammation, and fibrosis in the lungs of transgenic mice overexpressing platelet-derived growth factor.

Because of its expression pattern and its potent effects on mesenchymal cells, platelet-derived growth factor (PDGF) has been implicated as an important factor in epithelial-mesenchymal cell interactions during normal lung development and in the pathogenesis of fibrotic lung disease. To further explore the role of PDGF in these processes, we have developed transgenic mice that express the PDGF-B gene from the lung-specific surfactant protein C (SPC) promoter. Adult SPC-PDGFB transgenic mice exhibited lung pathology characterized by enlarged airspaces, inflammation, and fibrosis. Emphysematous changes frequently occurred throughout the lung, but inflammation and fibrotic lesions were usually confined to focal areas. The severity of this phenotype varied significantly among individual mice within the same SPC-PDGFB transgenic lineage. A pathology similar to that observed in adult mice was noted in lungs from transgenic mice as young as 1 week of age. Neonatal transgenic mice exhibited enlarged saccules and thickened primary septa. Results of these studies indicated that overexpression of PDGF-B induced distinct abnormalities in the developing and adult lung and led to a complex phenotype that encompassed aspects of both emphysema and fibrotic lung disease.

Connective tissue growth factor mRNA expression is upregulated in bleomycin-induced lung fibrosis.

Connective tissue growth factor (CTGF) is a newly described 38-kDa peptide mitogen for fibroblasts and a promoter of connective tissue deposition in the skin. The CTGF gene promotor contains a transforming growth factor-beta1 (TGF-beta1) response element. Because TGF-beta1 expression is upregulated in several models of fibroproliferative lung disease, we asked whether CTGF is also upregulated in a murine lung fibrosis model and whether CTGF could mediate some of the fibrogenic effects associated with TGF-beta1. A portion of the rat CTGF gene was cloned and used to show that primary isolates of both murine and human lung fibroblasts express CTGF mRNA in vitro. There was a greater than twofold increase in CTGF expression in both human and murine lung fibroblasts 2, 4, and 24 h after the addition of TGF-beta1 in vitro. A bleomycin-sensitive mouse strain (C57BL/6) and a bleomycin-resistant mouse strain (BALB/c) were given bleomycin, a known lung fibrogenic agent. CTGF mRNA expression was upregulated in the sensitive, but not in the resistant, mouse strain after administration of bleomycin. In vivo differences in the CTGF expression between the two mouse strains were not due to an inherent inability of BALB/c lung fibroblasts to respond to TGF-beta1 because fibroblasts from untreated BALB/c mouse lung upregulated their CTGF message when treated with TGF-beta1 in vitro. These data demonstrate that CTGF is expressed in lung fibroblasts and may play a role in the pathogenesis of lung fibrosis.

Upregulation of the PDGF-alpha receptor precedes asbestos-induced lung fibrosis in rats.

Platelet-derived growth factor-AA (PDGF-AA) and its matching alpha receptor (PDGF-R alpha) are upregulated in rat lung fibroblasts (RLFs) after exposure to chrysotile asbestos fibers in vitro, which results in asbestos-induced RLF proliferation. We now report our in vivo observations, which show an increase in the expression of PDGF-R alpha mRNA, but not PDGF-beta receptor mRNA, in asbestos-exposed rat lungs when compared with RNA from air-exposed (sham) and iron-exposed lungs. Western analysis of membrane preparations confirmed the observations on mRNA expression by demonstrating an increase in PDGF-R alpha peptide expression in the asbestos-exposed rat lungs, compared with that in the air-exposed lungs. Immunohistochemistry for the PDGF-R alpha was performed on air- and asbestos-exposed rat lungs and revealed a clear increase in staining within interstitial and subepithelial compartments in the exposed animals. These observations, along with our previous report demonstrating an increase in the PDGF-AA isoform expression immediately after asbestos-exposure, suggest a scenario in which a potent lung mesenchymal cell mitogen, PDGF-AA, and its alpha-receptor are upregulated prior to the development of a fibroproliferative lung lesion, and thus may play a central role in the pathogenesis of asbestos-induced lung fibrosis.

Dexamethasone activates expression of the PDGF-alpha receptor and induces lung fibroblast proliferation.

Corticosteroids (CSs) are commonly used for anti-inflammatory therapy in asthma and in interstitial lung diseases. In attempting to understand the mechanisms through which CSs control cell proliferation, we have carried out experiments to test the effects of dexamethasone (Dex) on the growth of lung fibroblasts. Using mouse 3T3 fibroblasts as well as early-passage rat lung fibroblasts (RLFs), we show that the quiescent cells in 1% serum or in serum-free media proliferate significantly in response to the addition of 10(-7) to 10(-9) M Dex. Increases as high as fourfold in cell numbers were recorded for the RLFs after 48 h in culture. A polyclonal antibody to the AB isoform of human platelet-derived growth factor (PDGF) blocked the proliferative response. As expected, the fibroblasts produced primarily PDGF-A chain, and the RLFs exhibited few PDGF-alpha receptors (PDGF-R alpha), the receptor type necessary for binding the AA isoform. Accordingly, we determined that Dex upregulated PDGF-R alpha mRNA and protein. Therefore, we can postulate that Dex-induced fibroblast proliferation is mediated, at least in part, by PDGF-AA, which binds to the PDGF-R alpha.

Rapid activation of PDGF-A and -B expression at sites of lung injury in asbestos-exposed rats.

The development of interstitial pulmonary fibrosis is associated with a variety of inflammatory mediators, including peptide growth factors and cytokines. In the work presented here, we have asked whether or not platelet-derived growth factor (PDGF)-A and -B genes and proteins are expressed in anatomic and temporal patterns consistent with this factor playing a role in the disease process. Using an established rat model of asbestos-induced fibroproliferative lung disease, we demonstrate elevated levels of PDGF-A and -B mRNAs in total lung RNA immediately after a single 5-h exposure to approximately 1,000 fibers/ml of chrysotile asbestos. In situ hybridization revealed the PDGF-A and -B in RNAs primarily in macrophages and bronchiolar-alveolar epithelial cells at sites of initial fiber deposition and lung injury. There was clear evidence of PDGF-A and -B mRNAs in interstitial cells as well. The pattern of in situ hybridization was entirely consistent with the appearance (established by immunohistochemistry) of PDGF-A and -B proteins by 24 h post-exposure in the same cell types. Both mRNAs and proteins remained detectable at the fiber deposition sites for almost 2 wk post-exposures. These findings are consistent with our previous studies showing increased mesenchymal cell proliferation and fibroproliferative lesions that progress at the sites where PDGF-A and -B are expressed. Although it is clear that multiple growth factors are produced simultaneously at sites of initial injury, we suggest that the PDGF isoforms could be playing a central role in the disease process based upon their potent mitogenic effects upon mesenchymal cells.

Chrysotile asbestos stimulates platelet-derived growth factor-AA production by rat lung fibroblasts in vitro: evidence for an autocrine loop.

We have investigated the mitogenic and chemotactic role of platelet-derived growth factor (PDGF) in pulmonary fibrogenesis induced by chrysotile asbestos. Since fibroblasts phagocytize asbestos in the lung interstitium, we have sought to learn whether the fibers alter the production of PDGF-like molecules by rat lung fibroblasts or induce mitogenesis of these fibroblasts in vitro. Conditioned medium as well as cell lysates from fibroblasts exposed to asbestos contained approximately 4-fold more PDGF than unexposed cells as detected by Western blot. Two distinct molecular weight forms of PDGF (36 and 18 kD) were detected by Western blotting. We postulate that these PDGF-like molecules are homologues of human PDGF-AA since we could not detect any PDGF in a sensitive enzyme immunoassay that recognized only PDGF-BB and PDGF-AB. Furthermore, PDGF-A chain mRNA was readily detected by Northern analysis, whereas PDGF-B chain mRNA was not detected by conventional Northern analysis. However, message amplification using a reverse transcriptase polymerase chain reaction allowed detection of the B-chain message. A significant dose-dependent mitogenic effect of asbestos was found by using both a cell proliferation assay and nuclear labeling with bromodeoxyuridine when fibroblasts were exposed under serum-free conditions. This mitogenesis induced directly by asbestos was blocked almost entirely with an anti-PDGF antibody that neutralized all three PDGF isoforms. Thus, these data support our hypothesis that an autocrine loop for PDGF-AA is operative in vitro following exposure to asbestos in lung fibroblasts, and we suggest that this signaling pathway could be significant in the pathogenesis of pulmonary fibrosis.

Reversible binding of platelet-derived growth factor-AA, -AB, and -BB isoforms to a similar site on the "slow" and "fast" conformations of alpha 2-macroglobulin.

The mechanism by which the platelet-derived growth factor (PDGF)-binding protein, alpha 2-macroglobulin (alpha 2M), modulates PDGF bioactivity is unknown, but could involve reversible PDGF-alpha 2M binding. Herein we report that greater than 70% of 125I-PDGF-BB or -AB complexed to alpha 2M was dissociated by SDS-denaturation followed by SDS-polyacrylamide gel electrophoresis, i.e. most of the binding was noncovalent. Reduction of the PDGF.alpha 2M complex following denaturation dissociated the cytokine from alpha 2M by greater than 90%, suggesting covalent disulfide bond formation. Approximately 50% of the growth factor was dissociated by lowering the pH from 7.5 to 4.0. 125I-PDGF-BB bound alpha 2M in a time-dependent manner (t1/2 = approximately 1 h), reaching equilibrium after 4 h. The 125I-PDGF.BB/alpha 2M complex dissociated more slowly (t1/2 = approximately 2.5 h). "Slow" and "fast" alpha 2M bound nearly equal amounts of PDGF-AB or -BB. Trypsin treatment converted PDGF-BB/alpha 2M complex to the fast conformation but did not release bound 125I-PDGF-BB. All PDGF-isoforms (AA, -AB, and -BB) competed for binding with 125I-PDGF-BB binding to slow alpha 2M and fast alpha 2M-methylamine by 65-80%. Other cytokines that bind alpha 2M (transforming growth factor-beta 1 and -beta 2, tumor necrosis factor-alpha, basic fibroblast growth factor, interleukin -1 beta, and -6) did not compete for 125I-PDGF-BB binding slow alpha 2M, but transforming growth factor-beta 1 and basic fibroblast growth factor inhibited 125I-PDGF-BB binding alpha 2M-methylamine by 30-50%. The reversible nature of the PDGF.alpha 2M complex could allow for targeted PDGF release near mesenchymal cells which possess PDGF receptors.

Rhodococcus equi causing human pulmonary infection: review of 29 cases.

Rhodococcus equi is a gram-positive pleomorphic bacillus that has been identified as a life-threatening pulmonary pathogen in the immunocompromised host. Infection with R equi may go unrecognized by physicians unacquainted with its presentation and unaware of the organism's ability to mimic diphtheroids and to stain weakly positive with an acid-fast stain.