Abstract
BackgroundPulmonary fibrotic diseases induce significant morbidity and mortality, for which there are limited therapeutic options available. Rac2, a ras-related guanosine triphosphatase expressed mainly in hematopoietic cells, is a crucial molecule regulating a diversity of mast cell, macrophage, and neutrophil functions. All these cell types have been implicated in the development of pulmonary fibrosis in a variety of animal models. For the studies described here we hypothesized that Rac2 deficiency protects mice from bleomycin-induced pulmonary fibrosis.MethodsTo determine the role of Rac2 in pulmonary fibrosis we used a bleomycin-induced mouse model. Anesthetized C57BL/6 wild type and rac2 -/- mice were instilled intratracheally with bleomycin sulphate (1.25 U/Kg) or saline as control. Bronchoalveolar lavage (BAL) samples were collected at days 3 and 7 of treatment and analyzed for matrix metalloproteinases (MMPs). On day 21 after bleomycin treatment, we measured airway resistance and elastance in tracheotomized animals. Lung sections were stained for histological analysis, while homogenates were analyzed for hydroxyproline and total collagen content.ResultsBLM-treated rac2 -/- mice had reduced MMP-9 levels in the BAL on day 3 and reduced neutrophilia and TNF and CCL3/MIP-1α levels in the BAL on day 7 compared to BLM-treated WT mice. We also showed that rac2 -/- mice had significantly lower mortality (30%) than WT mice (70%) at day 21 of bleomycin treatment. Lung function was diminished in bleomycin-treated WT mice, while it was unaffected in bleomycin-treated rac2 -/- mice. Histological analysis of inflammation and fibrosis as well as collagen and hydroxyproline content in the lungs did not show significant differences between BLM-treated rac2 -/- and WT and mice that survived to day 21.ConclusionRac2 plays an important role in bleomycin-induced lung injury. It is an important signaling molecule leading to BLM-induced mortality and it also mediates the physiological changes seen in the airways after BLM-induced injury.
Highlights
Pulmonary fibrotic diseases induce significant morbidity and mortality, for which there are limited therapeutic options available
To understand the role of ras-related C3 botulinum toxin substrate 2 (Rac2) in BLM-induced lung injury, we first examined the accumulation of inflammatory cells and inflammatory mediators in the airways of mice 3 and 7 days after induction of bleomycin-induced lung injury
We detected a significant increase in total lung collagen content and in hydroxyproline content in BLM-treated Wild type (WT) mice compared to salinetreated mice (p < 0.05, n =6), while rac2−/− BLM-treated mice did not show significant increase in lung collagen or hydroxyproline content compared to saline-treated rac2−/− animals (Figure 9C and D, n = 7-8 per group)
Summary
Pulmonary fibrotic diseases induce significant morbidity and mortality, for which there are limited therapeutic options available. A ras-related guanosine triphosphatase expressed mainly in hematopoietic cells, is a crucial molecule regulating a diversity of mast cell, macrophage, and neutrophil functions. All these cell types have been implicated in the development of pulmonary fibrosis in a variety of animal models. One way of understanding the cellular and inflammatory mechanisms associated with lung fibrosis has been the intratracheal application of bleomycin (BLM), an antineoplastic antibiotic, to induce a model of pulmonary fibrosis in mice [8]. Rac is a member of the Rho subfamily of ras-related guanosine triphosphatase expressed primarily in hematopoietic cells, and is a crucial molecule regulating a variety of neutrophil and macrophage functions [9,10,11]. A small molecule inhibitor of Rac and Rac alleviates LPSinduced acute lung injury [14]
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