In Vivo and in Vitro Vascular Gene Transfer of Timp-1 Using an Adenoviral Vector

Abstract

Extracellular matrix breakdown is essential for the migration and proliferation of vascular smooth muscle cells (VSMC) in balloon angioplasty restenosis. Tissue inhitor of metalloproteinases-1 (TIMP-I) potently inbibits the matrix metalloproteinase enzynes and may m o w this process. The effects of overexpression of TIMP-1 were investigated and the gene delivered to the rat carotid artery. A replication deficient recombinant adenoviral vector AvI.TIMP-I containing the cDNA for human TIMP-I was constructed. Its ability to express TIMP-I in rat primary VSMC was compared with cells infected a PGalactosidase expressing adenovirus Avl.pGal and control cells. Westem blottmg showed a dose dependent protem expression in response to AvI.TIMP-I (n=3). Reverse zymography showed biologically active protein with an increase proportional to the multiiplicky of infection (MOI) of Av1.TIMF' -1 (n=3). TIMP-I is a mitogen in some cell types but no change in H3 thymidine incorporation was seen versus control cells (n=6). Studies of migration of VSMC infected with Av1.TIMP-1, Avl.pGal, and control cells across an 8 CM pore sizs membrane showed inhiition of migration by viral infection alone (68% reduction in migration by Avl.pGal p<0.05, n=9) but a 90% reduction by AvI.TIMP-I indicating a significant additional effect ofthe TIMP-I versus f3 Galactosidase (p<0.05, n=9). In contrast VSMC invasion through an artificial basement membrane bamer was not affected by infection with Avl.~GaI but showed 30% inbibition by infection of the cells with AvI.TIMp-I @<0.05, n=12). Balloon injured rat carotid arteries exposed to Avl.PGal and AvI.TIMP-I in vivo showed transgene expression 2 and 14 days later. Immunohistochemistry and western blotting demonstrated human =-I. Conclusion: We have generated an adenoviral vector overexpressing biologically active TIMP-1. This vector inhibits migration and invasion of VSMC and successll protem expression follows in vivo administration after vascular injury. This vector nlaybe of interest for gene therapy.Glucocorticoids such as cortisol are immunoregulatory and promote reactivation of TB. Cortisol activity is regulated in each tissue by its inactivation to cortisone. It has previously been shown that the mtio of cortisol to cortisone is elevated in patients with active pulmonary TB .This study aimed to establish whether altered cortisol metabolism is a constitutive abnormality in TB. We studied 5 patients with active pulmonary TB (ATB). 7 with cured pulmonary TB (CTB). 7 healthy controls (CON) and 3 disease controls (pneumonia, DC). Ratios of urinary metabolites of cortisol and cortisone. measured by gas chromatography and mass spectrometry in 24 h urine, showed that a high ratio is exclusive to ATB (1.52 +I-0.17; vs 0.93 +/-0.06 in CTB, p4.05: 0.77 +I-0.02 CON, p4.01 and 0.76 DC, ~4 . 0 1 ) . This is attributable, at least in part, to enhanced conversion of cortisone to cortisol, since serum cortisol was higher after an oral dose of cortisone in ATB (peak 992+/-107nmol/l vs582+/-M nmolll in CON; P4.01 and...