Therapeutic targeting of SRC kinase in myofibroblast differentiation and pulmonary fibrosis.
As several forms of lung injury are associated with alveolar fibrin deposition, and fibrin has been pathogenically implicated in the lung fibrotic response, we sought to develop an in vivo gene transfer model of fibrinolytic protease overexpression. To this end, human tissue-type plasminogen activator (t-PA) possesses a high degree of specificity for proteolytic activation of fibrin-bound plasminogen to its active form, plasmin. To construct an effective vector, the cDNA for human t-PA was inserted downstream of a cytomegalovirus early enhancer-promoter into the E1 position of a replication-deficient adenovirus. The adenovirally expressed t-PA was found to be of the expected size and appropriate functional activity both in vitro and in vivo. A single intratracheal instillation of the adenoviral-t-PA construct resulted in a dose- dependent, tissue-specific expression of increased levels of t-PA antigen (100-fold) and t-PA protease activity (4-fold) for at least 2 wk in whole lung lysates. The expressed protein localized to the bronchiolar epithelium and peribronchiolar alveolar cells and did not result in increases in total lung protein or alveolar cell counts at 3 d after instillation. In conclusion, a single intratracheal instillation of adenoviral-cytomegalovirus-t-PA construct will generate dramatic bronchoalveolar compartment overexpression of functional recombinant human t-PA for at least 2 wk. This vector can now be utilized for the determination of the therapeutic potential of t-PA in a number of in vivo model systems.