Overexpression of MMP9 in macrophages attenuates pulmonary fibrosis induced by bleomycin☆
Introduction
Pulmonary fibrosis is the final consequence of a heterogeneous group of disorders known as interstitial lung diseases (Pardo & Selman, 2002). Lung fibrotic remodeling is characterized by fibroblast/myofibroblast activation and unbalanced extracellular matrix accumulation resulting in extensive structural disorganization of the lung. The irreversible changes in the lung architecture result in progressive organ dysfunction and usually a fatal outcome.
Several studies have shown that matrix metalloproteases (MMPs) play an important role in the pathogenesis of pulmonary fibrosis, and the presence of several of these proteases even in the advanced stages of fibrosis highlights the dynamic nature of scarring within the lung. However, the precise role of MMPs in the molecular mechanisms that characterize the fibrotic response is not completely understood (Pardo & Selman, 2006).
MMPs are a family of peptidases with multiple substrate affinities. They are not only collectively capable of cleaving all components of ECM but can also process bioactive mediators, such as growth factors, cytokines, chemokines, and cell-surface receptors (Nagase, Visse, & Murphy, 2006).
From the 23 members of the human MMPs family, MMP9 (gelatinase B), is one of the enzymes shown to be elevated in several human and experimental interstitial lung diseases (Fukuda, Ishizaki, Kudoh, Kitaichi, & Yamanaka, 1998; Hayashi et al., 1996, Pardo et al., 2000, Pérez-Ramos et al., 1999, Selman et al., 2000; Yaguchi, Fukuda, Ishizaki, & Yamanaka, 1998). In idiopathic pulmonary fibrosis (IPF) MMP9 is highly expressed in the lungs and BAL fluids and localized mainly to epithelial cells, neutrophils, and alveolar macrophages (Selman et al., 2000, Suga et al., 2000).
Experimental models of lung fibrosis, including those induced by paraquat plus hyperoxia, silica inhalation, or bleomycin instillation, have also exhibited an increase in MMP9 activity which has been associated with the disruption of the alveolar epithelial basement membrane (Cisneros-Lira, Gaxiola, Ramos, Selman, & Pardo, 2003; Pardo et al., 2003, Ruiz et al., 2003).
However, despite the putative profibrotic role of MMP9 in lung injury, MMP9 null mice develop similar fibrosis to wild-type littermates, after bleomycin instillation, although the lungs of the MMP9 deficient mice showed minimal alveolar bronchiolization (Betsuyaku, Fukuda, Parks, Shipley, & Senior, 2000).
In order to further understand the role of MMP9 in lung fibrosis, we have analyzed the effect of MMP9 overexpression in bleomycin driven lung fibrosis, using transgenic mice (TG) expressing human MMP9 in macrophages (hMMP9 TG), and following the model for 16 weeks. Our results indicate that hMMP9 TG mice develop reduced lung fibrosis compared to wild-type mice and suggest a possible mechanism related to insulin-like growth factor binding protein-3 cleavage by MMP9.
Section snippets
Generation of transgenic mice
Transgenic mice expressing human MMP9 were generated as follows. The 2.4 kb cDNA of human MMP9 (a gift from G. Goldberg, Washington University) was cloned into a vector containing the splice site and polyadenylation sequence of the β-globin gene, and placed under the control of the Scavenger Receptor A enhancer-promoter (SREP; a gift from Dr. Glass, UC, San Diego), a promoter that allows targeted expression of genes specifically in differentiated macrophages of transgenic mice (Lemaitre et al.,
Evidence of human MMP9 in transgenic mice
Levels of mouse and human MMP9 gene expression were assessed by quantitative real-time PCR in total RNA isolated from lungs of bleomycin or saline-treated mice. Mouse MMP9 mRNA was significantly increased at 1, 4 and 8 weeks after bleomycin as compared with saline-treated controls in both hMMP9 TG and WT mice (p < 0.05; Fig. 1A). No significant difference in the gene expression level was found between the TG and WT mice at any time point. Confirming the genotype of the hMMP9 TG mice, human MMP9
Discussion
In the present study we analyzed the effect of the overexpression of MMP9 a metalloprotease mediating diverse biological functions on bleomycin induced pulmonary fibrosis. Following the model for 4 months, we demonstrated that overexpression of MMP9 in macrophages induces a retarded and diminished lung fibrotic reaction. This effect was preceded by a decrease in neutrophil infiltration at 1 week, and decreased lymphocytes at 1 month post-bleomycin treatment. Interestingly, a lower level of
Acknowledgements
This work was supported by a grant from CONACYT 41043-M and by Universidad Nacional Autonoma de Mexico SDI.PTID.05.6. S.C. was funded by CONACYT.
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This work was submitted in partial fulfillment of the requirements to obtain the PhD degree for S.C. at Universidad Nacional Autonoma de Mexico.