Adenovirally Mediated Gene Transfer of Functional Human Tissue–type Plasminogen Activator to Murine Lungs

Abstract

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.

Therapeutic Targeting of Src Kinase in Myofibroblast Differentiation and Pulmonary Fibrosis

“…The lung tissues were harvested at day 21 after bleomycin instillation for lung fibrosis analysis and for histologic and biochemical studies. To collect lung tissue for histochemical studies, the lungs were perfused with cold phosphate-buffered saline, then inflated with 10% formalin, fixed overnight, and embedded in paraffin as described elsewhere (Simmons et al, 1998;Ding et al, 2013).…”

Focal Adhesion Kinase (FAK)-related Non-kinase Inhibits Myofibroblast Differentiation through Differential MAPK Activation in a FAK-dependent Manner

“…The genotype of FRNK knock-out (homozygous) mice and their wild type littermates was confirmed by PCR using genomic DNA extracted from tails as published (44), and the absence of FRNK expression in lung tissue from FRNK knock-out mice was confirmed by Western blot (data not shown). The administration of bleomycin and harvest of lung tissues were described previously (45,46). Briefly, the animals (7-10 weeks) were anesthetized, and bleomycin (1 unit/kg of body weight in 50 l of saline) or saline alone (50 l) was slowly instilled through airway to lungs by using an intratracheal catheter.…”

“…Whole lung homogenate supernatants were used for Western blot analysis. The harvested lungs were homogenized in 1% Nonidet P-40 lysis buffer (with the following inhibitors, 100 M phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, 10 g/ml leupeptin, 100 M sodium vanadate, and 20 g/ml TLCK) with a polytron (Brinkmann Instruments, Westbury, NY), and homogenate supernatants were extracted by centrifugation (14,000 ϫ g for 20 min at 4°C) (46).…”

“…The HA-tagged FRNK cDNA was engineered into a replication-deficient adenovirus vector by using the Adeno-X Expression System 2 according to the manufacturer's manual (Clontech, Mountain View, CA). The Ad-FRNK was rescued and amplified in 293 cells, purified by CsCl gradient centrifugation, and used to infect cells as described previously (46,51). Adenoviral vectors encoding a green fluorescent protein (Ad-GFP) or a luciferase construct (Ad-Luc) were initially used to optimize the infection conditions on subconfluent (70 -80%) cells cultured in serum-containing or serum-free conditions as described previously (46,51).…”

“…The Ad-FRNK was rescued and amplified in 293 cells, purified by CsCl gradient centrifugation, and used to infect cells as described previously (46,51). Adenoviral vectors encoding a green fluorescent protein (Ad-GFP) or a luciferase construct (Ad-Luc) were initially used to optimize the infection conditions on subconfluent (70 -80%) cells cultured in serum-containing or serum-free conditions as described previously (46,51). The cells in serum-free medium (DMEM with 1% BSA) were infected with Ad-FRNK or control vectors (Ad-Luc or Ad-GFP) 24 h prior to TGF-␤1 treatment.…”