Although dasatinib does not distinguish DDR from DDR
Although dasatinib does not distinguish DDR2 from DDR1 whose knockout mice were similarly refractory to bleomycin-induced lung fibrosis,, it is unlikely that the therapeutic effects of dasatinib treatment starting from 14 days after bleomycin in our model could equally benefit from its inhibition of the two members because DDR1 was considered to primarily regulate the function of epithelial jsh receptor and macrophages., The expression of DDR1 in lung fibroblasts, although positive, was reported to rely on DDR2 signaling. Whatever, multitarget effect is a common characteristic for most kinase inhibitors, which sometimes is conducive to enhancing their efficacy in inhibiting disease progression. For instance, dasatinib suppression of Src activity may additionally contribute to the action of this drug on TGF-β-induced myofibroblast events because Src kinase was reported to serve as a key mediator of TGF-β–p38 pathway., The results derived by the authors, together with the most recently published data, provide important preclinical evidence for the use of dasatinib in the treatment of IPF.
Materials and Methods The heterozygous slie mutant mice, carrying deletion mutation spanning Ddr2 exon 1–17, were provided by Jackson Lab (Bar Harbor, Maine) and intercrossed to produce homozygotes for use. All animal studies were performed according to the protocols and guidelines of the institutional care and use committee. To induce pulmonary damage, 6- to 8-week-old sex- and age-matched wild-type or slie mice were intranasally dropped with bleomycin (Nippon Kayaku, Tokyo, Japan) at 5 mg/kg body weight (BW) or FITC (Sigma, St Louis, MO) at 6 mg/kg BW. For in vivo silencing of DDR2 in mouse lung, a nonspecific control siRNA or DDR2-specific siRNA was modified with 2′-methoxy (2′-OMe) by Genepharma Company (Shanghai, China) to improve stability. The sense sequences of siRNAs are as following: DDR2, 5′-TTGAGATGAATACTAGCTTAG-3′; control, 5′-TTCTCCGAACGTGTCACGTT-3′. The silencing effect of DDR2 siRNA has been validated by our previous in vitro study. One OD siRNA was dissolved in 40 μl D5W solution (5% D-Glucose) and used to treat one mouse per time through nasal instillation as described earlier. For each group, the siRNA treatment was conducted once a week before the harvest. Dasatinib (Sprycel, Bristol-Myers Squibb, New York, NY) was dissolved in 80 mmol/l citric acid (pH 2.1) to make a stock solution of 10 mg/ml. For animal use, dasatinib were given to the mice orally (10 mg/kg BW) by gavage once a day. At least five animals were used in each treatment group and three individual experiments were performed. The lungs dissected from 4–5-week mice were cut into small pieces and then immersed in Dulbecco\'s modified Eagle\'s medium (Invitrogen, Carlsbad, CA) containing collagenase II (2 mg/ml), trypsin (2.5 mg/ml), Dnase I (2 mg/ml), penicillin (100 U/ml), and streptomycin (100 μg/ml) for 12 hours at 37 °C after phosphate-buffered saline wash. The clumps of tissues were removed through filtering and the cells were collected from the supernatant by centrifuge, followed by their maintenance in Dulbecco\'s modified Eagle\'s medium supplemented with heat-inactivated 10% fetal bovine serum (FBS). The phenotypes of fibroblasts after passage 3 were characterized by immunostaining of the epithelial marker pan-cytokeratin and the fibroblastic marker vimentin.