The role of smooth muscle contraction in causing acute airflow obstruction in asthma is firmly established. The basic premise underlying this project is that long-term inhibition of smooth muscle contraction in asthma may provide a useful adjunct to antiinflammatory therapy in the treatment of this prevalent and morbid disease. Our major goal is to create a new strategy, based on expression of an artificial gene, to inhibit smooth muscle contraction in the airways. We have cloned and characterized the full-length murine gene encoding SM22alpha, a 22 kd protein whose expression is restricted to smooth muscle in adult animals. Our planned approach is to employ the SM22alpha promoter to direct smooth muscle-specific expression of peptides that suppress force generation by actomyosin. To achieve these overall objectives, we propose three specific aims: (1) Identify the cis-acting sequences that restrict expression of SM22alpha to smooth muscle in vivo. We previously demonstrated that bp - 441 to +41 of the mouse SM22alpha gene are necessary and sufficient to direct high level smooth muscle cell-specific expression of a luciferase reporter gene in vitro. We will now identify in vivo the distribution of transcriptional activity of this minimal SM22alpha promoter or larger SM22alpha promoter fragments, in transgenic mice in which these regulatory elements control expression of the lacZ reporter gene. These studies will disclose a smooth muscle-specific transcriptional regulatory element that will be used (below) to control expression of the proposed therapeutic gene in vivo. We will also determine whether induction of allergic airway inflammation alters the magnitude or cellular distribution of SM22alpha promoter activity within the lung. (2) Establish the ability of the SH-1 peptide MIRICRKK to inhibit force generation by airway smooth muscle cells when delivered exogenously or when synthesized endogenously within smooth muscle cells in vitro. SH-1 peptides bind to skeletal muscle actin, inhibit actin- activated myosin ATPase activity, and inhibit force generation by skinned skeletal muscle fibers. We will test whether SH-1 peptide MIRICRKK inhibits actomyosin force generation in skinned sheep tracheal smooth muscle strips in vitro, then confirm that the octapeptide is synthesized by cultured tracheal smooth muscle cells transfected with an artificial minigene encoding MIRICRKK. We will test whether MIRICRKK expression within individual cultured bovine tracheal myocytes suppresses their contractile response to bradykinin (using atomic force microscopy to measure transverse cell stiffness changes), and whether MIRICRKK expression alters myocyte viability or proliferative capacity. These studies should establish whether endogenously synthesized MIRICRKK inhibits actomyosin force generation, and whether it exhibits toxic effects. (3) Evaluate whether infection of airway smooth muscle with replication-deficient adenovirus carrying an artificial gene, in which the SM22alpha promoter control expression of MIRICRKK, inhibits force generation in response to contractile agonists. We will quantify MIRICRKK expression within sheep, cat, or human airway smooth muscle strips infected in vitro, or cat trachealis infected in vivo, and will compare contractile responses of these tissues with those of control virus- infected or non-infected tissues. We anticipate that these studies will establish a novel strategy for inhibiting airway smooth muscle contraction in asthma. If successful, this approach might hold promise in other diseases characterized by vascular or gastrointestinal smooth muscle spasm.
| Chen, Bohao; Moore, Tamson V; Li, Zhenping et al. (2014) Gata5 deficiency causes airway constrictor hyperresponsiveness in mice. Am J Respir Cell Mol Biol 50:787-95 |
| Ma, Lan; Brown, Melanie; Kogut, Paul et al. (2011) Akt activation induces hypertrophy without contractile phenotypic maturation in airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 300:L701-9 |
| Motika, Caroline A; Papachristou, Charalampos; Abney, Mark et al. (2011) Rising prevalence of asthma is sex-specific in a US farming population. J Allergy Clin Immunol 128:774-9 |
| McConville, John F; Fernandes, Darren J; Churchill, Jason et al. (2011) Nuclear import of serum response factor in airway smooth muscle. Am J Respir Cell Mol Biol 45:453-8 |
| Chen, Bohao; Yates, Elena; Huang, Yong et al. (2009) Alternative promoter and GATA5 transcripts in mouse. Am J Physiol Gastrointest Liver Physiol 297:G1214-22 |
| Ober, Carole; Nord, Alex S; Thompson, Emma E et al. (2009) Genome-wide association study of plasma lipoprotein(a) levels identifies multiple genes on chromosome 6q. J Lipid Res 50:798-806 |
| Bellenguez, Céline; Ober, Carole; Bourgain, Catherine (2009) A multiple splitting approach to linkage analysis in large pedigrees identifies a linkage to asthma on chromosome 12. Genet Epidemiol 33:207-16 |
| Bellenguez, Celine; Ober, Carole; Bourgain, Catherine (2009) Linkage analysis with dense SNP maps in isolated populations. Hum Hered 68:87-97 |
| Pinto, Jayant M; Hayes, M Geoffrey; Schneider, Daniel et al. (2008) A genomewide screen for chronic rhinosinusitis genes identifies a locus on chromosome 7q. Laryngoscope 118:2067-72 |
| Hershenson, Marc B; Brown, Melanie; Camoretti-Mercado, Blanca et al. (2008) Airway smooth muscle in asthma. Annu Rev Pathol 3:523-55 |
Showing the most recent 10 out of 163 publications
