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Role of Abl in airway hyperresponsiveness and airway remodeling.

Cleary RA, Wang R, Wang T, Tang DD - Respir. Res. (2013)

Bottom Line: Interestingly, conditional knockout of Abl did not affect the levels of IL-13 and CCL2 in bronchoalveolar lavage fluid of animals treated with ovalbumin.However, treatment with imatinib and GNF-5 inhibited the ovalbumin-induced increase in IL-13 and CCL2 as well as airway resistance and smooth muscle growth in animals.These results suggest that the altered expression of Abl in airway smooth muscle may play a critical role in the development of airway hyperresponsiveness and airway remodeling in asthma.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Cardiovascular Sciences, Albany Medical College, 47 New Scotland Avenue MC-8, Albany, NY 12208, USA. tangd@mail.amc.edu.

ABSTRACT

Background: Asthma is a chronic disease that is characterized by airway hyperresponsiveness and airway remodeling. The underlying mechanisms that mediate the pathological processes are not fully understood. Abl is a non-receptor protein tyrosine kinase that has a role in the regulation of smooth muscle contraction and smooth muscle cell proliferation in vitro. The role of Abl in airway hyperresponsiveness and airway remodeling in vivo is largely unknown.

Methods: To evaluate the role of Abl in asthma pathology, we assessed the expression of Abl in airway tissues from the ovalbumin sensitized and challenged mouse model, and human asthmatic airway smooth muscle cells. In addition, we generated conditional knockout mice in which Abl expression in smooth muscle was disrupted, and then evaluated the effects of Abl conditional knockout on airway resistance, smooth muscle mass, cell proliferation, IL-13 and CCL2 in the mouse model of asthma. Furthermore, we determined the effects of the Abl pharmacological inhibitors imatinib and GNF-5 on these processes in the animal model of asthma.

Results: The expression of Abl was upregulated in airway tissues of the animal model of asthma and in airway smooth muscle cells of patients with severe asthma. Conditional knockout of Abl attenuated airway resistance, smooth muscle mass and staining of proliferating cell nuclear antigen in the airway of mice sensitized and challenged with ovalbumin. Interestingly, conditional knockout of Abl did not affect the levels of IL-13 and CCL2 in bronchoalveolar lavage fluid of animals treated with ovalbumin. However, treatment with imatinib and GNF-5 inhibited the ovalbumin-induced increase in IL-13 and CCL2 as well as airway resistance and smooth muscle growth in animals.

Conclusions: These results suggest that the altered expression of Abl in airway smooth muscle may play a critical role in the development of airway hyperresponsiveness and airway remodeling in asthma. Our findings support the concept that Abl may be a novel target for the development of new therapy to treat asthma.

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Related in: MedlinePlus

Targeting of the abl gene in smooth muscle cells. (A) SM22-Cre construct possesses a SM22 promoter (PSM22) and a Cre coding region. The abl locus containing a floxed version of the Exon5 of abl and the predicted product of Cre-mediated recombination are also shown. Numbered arrows represent PCR primers. (B) Ethidium bromide-stained agarose gel of PCR products amplified from mouse tails of indicated mouse strains. PCR with primers 3 and 5 demonstrate selective deletion of DNA between loxP sites in the presence of Cre recombinase in vivo. Each lane represents tail DNA samples isolated from an individual mouse of the indicated genotype. (C) Extracts of airway smooth muscle cells from Abl-lox and Ablsm−/− mice were analyzed by immunoblotting. Knockout of the Abl protein is verified in airway smooth muscle. Representative blots from three separate experiments are shown.
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Figure 1: Targeting of the abl gene in smooth muscle cells. (A) SM22-Cre construct possesses a SM22 promoter (PSM22) and a Cre coding region. The abl locus containing a floxed version of the Exon5 of abl and the predicted product of Cre-mediated recombination are also shown. Numbered arrows represent PCR primers. (B) Ethidium bromide-stained agarose gel of PCR products amplified from mouse tails of indicated mouse strains. PCR with primers 3 and 5 demonstrate selective deletion of DNA between loxP sites in the presence of Cre recombinase in vivo. Each lane represents tail DNA samples isolated from an individual mouse of the indicated genotype. (C) Extracts of airway smooth muscle cells from Abl-lox and Ablsm−/− mice were analyzed by immunoblotting. Knockout of the Abl protein is verified in airway smooth muscle. Representative blots from three separate experiments are shown.

Mentions: Abl-lox mice were a gift of Dr. Koleske of Yale University [16]. SM22cre mice were purchased from The Jackson Laboratory. Abl-lox mice (genetic background, 129/Svj) were crossed with SM22cre mice on C57BL/6 background. These mice express Cre recombinase under control of a smooth muscle-specific SM22 promoter. As a consequence, this loxP flanked exon 5 of the abl gene was excised in smooth muscle cells (Figure 1A). The sequences of primers used to identify the genotype were: Primer 1, 5’-CTGTACGTGTCCTCCGAGAG-3’; Primer 2, 5’-CTTCAAGGTCTTCACGGCCA-3’; Primer 3, 5’-GATGTCTCTACAGGGTTAAGATTAAGAGCA-3’; Primer 4, 5’-TGTGCATAGCAGGAAGTCCTCCAGAG-3’; Primer 5, 5’-AGTTAACACACCTCCAGAGTGAGTGCCCT-3’.


Role of Abl in airway hyperresponsiveness and airway remodeling.

Cleary RA, Wang R, Wang T, Tang DD - Respir. Res. (2013)

Targeting of the abl gene in smooth muscle cells. (A) SM22-Cre construct possesses a SM22 promoter (PSM22) and a Cre coding region. The abl locus containing a floxed version of the Exon5 of abl and the predicted product of Cre-mediated recombination are also shown. Numbered arrows represent PCR primers. (B) Ethidium bromide-stained agarose gel of PCR products amplified from mouse tails of indicated mouse strains. PCR with primers 3 and 5 demonstrate selective deletion of DNA between loxP sites in the presence of Cre recombinase in vivo. Each lane represents tail DNA samples isolated from an individual mouse of the indicated genotype. (C) Extracts of airway smooth muscle cells from Abl-lox and Ablsm−/− mice were analyzed by immunoblotting. Knockout of the Abl protein is verified in airway smooth muscle. Representative blots from three separate experiments are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3852349&req=5

Figure 1: Targeting of the abl gene in smooth muscle cells. (A) SM22-Cre construct possesses a SM22 promoter (PSM22) and a Cre coding region. The abl locus containing a floxed version of the Exon5 of abl and the predicted product of Cre-mediated recombination are also shown. Numbered arrows represent PCR primers. (B) Ethidium bromide-stained agarose gel of PCR products amplified from mouse tails of indicated mouse strains. PCR with primers 3 and 5 demonstrate selective deletion of DNA between loxP sites in the presence of Cre recombinase in vivo. Each lane represents tail DNA samples isolated from an individual mouse of the indicated genotype. (C) Extracts of airway smooth muscle cells from Abl-lox and Ablsm−/− mice were analyzed by immunoblotting. Knockout of the Abl protein is verified in airway smooth muscle. Representative blots from three separate experiments are shown.
Mentions: Abl-lox mice were a gift of Dr. Koleske of Yale University [16]. SM22cre mice were purchased from The Jackson Laboratory. Abl-lox mice (genetic background, 129/Svj) were crossed with SM22cre mice on C57BL/6 background. These mice express Cre recombinase under control of a smooth muscle-specific SM22 promoter. As a consequence, this loxP flanked exon 5 of the abl gene was excised in smooth muscle cells (Figure 1A). The sequences of primers used to identify the genotype were: Primer 1, 5’-CTGTACGTGTCCTCCGAGAG-3’; Primer 2, 5’-CTTCAAGGTCTTCACGGCCA-3’; Primer 3, 5’-GATGTCTCTACAGGGTTAAGATTAAGAGCA-3’; Primer 4, 5’-TGTGCATAGCAGGAAGTCCTCCAGAG-3’; Primer 5, 5’-AGTTAACACACCTCCAGAGTGAGTGCCCT-3’.

Bottom Line: Interestingly, conditional knockout of Abl did not affect the levels of IL-13 and CCL2 in bronchoalveolar lavage fluid of animals treated with ovalbumin.However, treatment with imatinib and GNF-5 inhibited the ovalbumin-induced increase in IL-13 and CCL2 as well as airway resistance and smooth muscle growth in animals.These results suggest that the altered expression of Abl in airway smooth muscle may play a critical role in the development of airway hyperresponsiveness and airway remodeling in asthma.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Cardiovascular Sciences, Albany Medical College, 47 New Scotland Avenue MC-8, Albany, NY 12208, USA. tangd@mail.amc.edu.

ABSTRACT

Background: Asthma is a chronic disease that is characterized by airway hyperresponsiveness and airway remodeling. The underlying mechanisms that mediate the pathological processes are not fully understood. Abl is a non-receptor protein tyrosine kinase that has a role in the regulation of smooth muscle contraction and smooth muscle cell proliferation in vitro. The role of Abl in airway hyperresponsiveness and airway remodeling in vivo is largely unknown.

Methods: To evaluate the role of Abl in asthma pathology, we assessed the expression of Abl in airway tissues from the ovalbumin sensitized and challenged mouse model, and human asthmatic airway smooth muscle cells. In addition, we generated conditional knockout mice in which Abl expression in smooth muscle was disrupted, and then evaluated the effects of Abl conditional knockout on airway resistance, smooth muscle mass, cell proliferation, IL-13 and CCL2 in the mouse model of asthma. Furthermore, we determined the effects of the Abl pharmacological inhibitors imatinib and GNF-5 on these processes in the animal model of asthma.

Results: The expression of Abl was upregulated in airway tissues of the animal model of asthma and in airway smooth muscle cells of patients with severe asthma. Conditional knockout of Abl attenuated airway resistance, smooth muscle mass and staining of proliferating cell nuclear antigen in the airway of mice sensitized and challenged with ovalbumin. Interestingly, conditional knockout of Abl did not affect the levels of IL-13 and CCL2 in bronchoalveolar lavage fluid of animals treated with ovalbumin. However, treatment with imatinib and GNF-5 inhibited the ovalbumin-induced increase in IL-13 and CCL2 as well as airway resistance and smooth muscle growth in animals.

Conclusions: These results suggest that the altered expression of Abl in airway smooth muscle may play a critical role in the development of airway hyperresponsiveness and airway remodeling in asthma. Our findings support the concept that Abl may be a novel target for the development of new therapy to treat asthma.

Show MeSH
Related in: MedlinePlus