Early phase collagen synthesis in lungs of rats exposed to bleomycin

Abstract

Skin wound healing exhibits type III collagen synthesis occurring transiently as early as 10 h after injury, with subsequent synthesis of type I to form a scar. We hypothesized that similar collagen type switching also occurred in the bleomycin model of lung fibrosis in the rat. We could measure elevated lung collagen synthesis rates as early as 4 days after administration of bleomycin. Collagen type I:III ratios in whole lung remained constant for the first 7 days at the control level of 2:1, then increased to as high as 5:1 at day 21. Procollagen mRNA content, expressed as a ratio of type I:III mRNAs, was consistent with the protein synthesis data and the observed ratio of collagen types being made by the lungs at the various time points evaluated. We conclude that a transient increase in type III relative to type I collagen does not occur in the bleomycin rat lung model. Therefore, the sequence of type-specific collagen expression and deposition in the skin wound healing model is not entirely analogous to this widely used animal model of pulmonary fibrosis.

Introduction

Pulmonary fibrosis, as typified by idiopathic pulmonary fibrosis (cryptogenic fibrosing alveolitis), is an inflammatory condition of the lungs that results in the progressive deposition of collagen and other extracellular matrix proteins within the lung parenchyma. The inflammatory process and subsequent increased collagen deposition can result in severely reduced lung compliance and impaired pulmonary function that limits gas exchange, and may ultimately result in respiratory failure. Although the cause of idiopathic pulmonary fibrosis is unknown, agents such as bleomycin have been shown to produce an acute process in animal lungs that has many similarities to pulmonary fibrosis in humans. This has lead to widespread use of the bleomycin animal lung model to study the mechanisms and pathogenesis of this disorder.

A discrepancy appears to exist, however, between the bleomycin animal model and clinical studies of fibrotic conditions with respect to early and late phase collagen type switching. In previous animal studies, elevated lung collagen synthesis has been demonstrated as early as 7 days after the intratracheal administration of bleomycin to rats (Hesterberg et al., 1981). This shift to preferential synthesis of type I collagen (with respect to type III) typically occurs before histopathologic accumulation of collagen is noted within the lung (Reiser and Last, 1981). Total lung collagen content is increased by day 14, and the increase in whole lung collagen content may persist for up to 8 weeks after administration of the drug (Hesterberg et al., 1981). The increase in lung collagen content by day 14 is largely due to increases in type I collagen accumulation. Type III collagen synthesis apparently also increases after bleomycin administration (Reiser and Last, 1981, Shahzeidi et al., 1993); animal studies that have examined the relative ratios of type I to type III collagen synthesis and deposition in the lung typically demonstrate an increase in this ratio. This indicates that type I collagen is the predominant accumulated collagen during the fibrotic response (Reiser and Last, 1981, Reiser and Last, 1986, Nerlich et al., 1987).

An initial increase in the relative amount of type III collagen in the lung parenchyma has been reported in studies of patients with adult respiratory distress syndrome, idiopathic pulmonary fibrosis, sarcoidosis, and infant respiratory distress syndrome that have used immunohistochemical methods (Bateman et al., 1981, Bateman et al., 1983, Kirk et al., 1984a, Raghu et al., 1985, Nerlich et al., 1987). Additional studies examining the content of N-terminal type III procollagen peptide in serum or in bronchoalveolar lavage fluid from patients have also suggested that type III collagen synthesis is increased in fibrotic lung diseases (Low et al., 1983, Kirk et al., 1984b, Cantin et al., 1988, Bjermer et al., 1989, Low et al., 1992, Pohl et al., 1992, Lammi et al., 1997, Chesnutt et al., 1997). These observations suggest that in the human condition, an initial increase in type III collagen may precede the progressive deposition of type I collagen. However, this initial increase in type III collagen is not routinely observed in the bleomycin animal model by biochemical analyses.

Several reasons for this apparent discrepancy may exist, including the heterogeneity of the clinical populations studied, a lack of a defined initiation time point in clinical studies, differences in the route of bleomycin administration, and differences in the methodology used to quantify collagen types present in the various studies. Also, little is known about the relative proportion of collagen types I and III at time points earlier than one week after bleomycin administration. An alternative model of increased collagen synthesis and deposition, skin wound healing, may provide some insight. Skin wound healing also exhibits early and late phase collagen type switching and has been suggested to share a similar biologic basis with pulmonary fibrosis (Crystal et al., 1991). In one wound healing study, an increase in type III collagen was noted as early as 10 h after injury (Clore et al., 1979). Thus, it is possible that a transient increase in type III collagen also occurs in the bleomycin animal model, but at a very early time point that has yet to be thoroughly evaluated.

The present study was designed to test the hypothesis that an early phase (prior to day 7) increase in type III collagen synthesis would occur prior to the late phase increase in type I collagen in the bleomycin rat lung model of pulmonary fibrosis. This would be detected as an initial decrease in the type I:III collagen ratio. Collagen accumulation is dependent on multiple factors, including rates of gene transcription and mRNA translation, post-translational modification and secretion, and degradation of newly synthesized collagen (Bienkowski, 1984, Laurent, 1987, Adams, 1989, Bornstein and Sage, 1989, Raghow and Thompson, 1989, Crouch, 1990). We thus hypothesized that examination of the relative amounts of type I and type III procollagen mRNA transcripts in prefibrotic lung tissue might be a sensitive index for detecting alterations in the relative ratio of collagen types actually being synthesized by lung tissue at a given time point.