Unbalanced collagenases/TIMP-1 expression and epithelial apoptosis in experimental lung fibrosis

Abstract

In this study, we examined the sequential expression of several matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and growth factors as well as the presence of apoptosis in a model of pulmonary fibrosis induced in rats with paraquat and hyperoxia. Animals showing neither clinical nor morphological changes with this double aggression were classified as "resistant". Rats were killed at 1, 2, 3, and 6 wk, and lungs were used for collagen content, gene expression by real-time PCR, gelatinolytic activity by zymography, apoptosis by in situ DNA fragmentation, and protein localization by immunohistochemistry. Our results showed a significant decrease of collagenases MMP-8 and MMP-13, with an increase of TIMP-1 and transforming growth factor-beta. Immunoreactive TIMP-1 was increased in experimental rats and primarily localized in alveolar macrophages. Expression of gelatinases MMP-2 and MMP-9 mRNAs was not affected, but lung zymography revealed an increase in progelatinase B, progelatinase A, and its active form. Epithelial apoptosis was evident from the first week, whereas at later periods, interstitial cell apoptosis was also noticed. Resistant animals behave as controls. These findings suggest that an imbalance between collagenases and TIMPs, excessive gelatinolytic activity, and epithelial apoptosis participate in the fibrotic response in this experimental model.

Novel and Extensive Aspects of Paraquat-Induced Pulmonary Fibrogenesis: Comparative and Time-Course Microarray Analyses in Fibrogenic and Non-Fibrogenic Rats

“…Resistant rats obtained in that study, which were defined as animals that received six weeks of PQ plus hyperoxia but still gained weight, did not develop signs of respiratory insufficiency, and morphologically showed minimal lung lesions or no lesions at all, which were the same results observed in control animals. Although the resistant rats could be equivalent to animals in Group 2 in our study, there was a discrepancy between our study and Ruiz et al (2003); in our study, the induction of TGF-β3 was observed in both Group 1 and Group 2 and the degree of induction was the same at R-Day 7. Hyperoxia enhances PQ toxicity, however there are some reports that hyperoxia inhibits oxidantinduced apoptosis in lung epithelial cells (Franek et al, 2001) and induces necrosis (Jyonouchi et al, 1998;Kazzaz et al, 1996;Li et al, 1997).…”

“…Two approaches were conducted in the present study: time-course microarray analysis to investigate time-dependent changes of the gene expression pro- files that occur simultaneously with changes in pulmonary collagen content induced by PQ in Group 1; and comparative microarray analysis of Group 1 and Group 2 because there is considerable individual variability in sensitivity to PQ toxicity in rats (Hollinger et al, 1978), and some individuals considered to be PQ-resistant do not develop pulmonary fibrosis (Ruiz et al, 2003). Therefore, comparative analysis of Groups 1 and 2 is useful to identify genes that are directly relevant to the fibrotic process.…”

“…These reports suggest that two distinct programs regulate lung inflammation in the early phase and fibrosis in the later phase. There is, however, no report concerning increased collagen content in the later phase after discontinuation of PQ administration; most studies of PQ toxicity only investigated the immediate time period after PQ treatment, whereas some long-term studies intermittently administer PQ once or twice a week throughout the experimental period (Ishida et al, 2006;Ruiz et al, 2003;Selman et al, 1985aSelman et al, , 1985b. Therefore, the time course of gene expression profiles for Group 1 could be critical for understanding the mechanisms of PQinduced pulmonary fibrosis.…”

“…Programmed cell death or apoptosis is a highly regulated process of cell removal in which tissue architecture is preserved and inflammation is minimized (Allen et al, 1997). To date, a considerable body of research has shown that alveolar apoptosis is a critical determinant in the pathways that initiate fibrogenesis in the lung (Adamson et al, 1988;Hagimoto et al, 1997aHagimoto et al, , 1997bRuiz et al, 2003;Budinger et al, 2006;Uhal et al, 1995 and. There are two pathways for regulating caspase activation: one is wellcharacterized and coupled to cell death receptors, and the other is initiated by events that are less clear and involve the release of mitochondrial cytochrome c. The release of cytochrome c from the mitochondria during apoptosis is regulated by members of the family of Bcl-2 related proteins.…”

“…More information about TGF-β3 and its roles in pulmonary fibrosis is needed. Ruiz et al (2003) reported that TGF-β1 was induced under a condition of PQ plus hyperoxia that resulted in fibrogenesis in rats. Resistant rats obtained in that study, which were defined as animals that received six weeks of PQ plus hyperoxia but still gained weight, did not develop signs of respiratory insufficiency, and morphologically showed minimal lung lesions or no lesions at all, which were the same results observed in control animals.…”