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...
The application of local gene transfer to the investigation or therapy of coronary vascular disease will require high efficiency of gene transfer in vivo. This is a function of the technique of local drug delivery, the vector employed and the underlying vascular disease.We employed an adenoviral vector carrying the gene for p Galactosidase driven by the rous sarcoma virus promoter (Avl.PGal) and showed 80% of cultured pig vascular smooth muscle cells expressing the transgene (MOI 200). 4 large white landrace pigs (15-20 kg) underwent angioplasty of a single coronary artery, (left anterior descending (LAD), circumflex (Cx)) with a 3 m m balloon catheter (balloon to artery ratio 1.3). They were allowed to recover for 14 -21 days. Further angioplasty at the same angiographic site (3mm balloon, balloon to artery ration 1.3) and also of the proximal segment of the previously uninjured left coronary branch.Immediately after angioplasty approximately 1.5ml of a suspension of 4 x 108 pfu A v l . p a l was delivered to the site using a 2.75mm double skinned microporous balloon, the MIC2 catheter (Cordis). Three days later the animals were killed and the coronary arteries retrieved and placed in X-Gal prior to processing for histology.Previously injured vessels showed neointimal hyperplasia. Four of 7 vessels examined showed successful gene transfer as evidenced by prussian blue nuclear staining. Three of these four positive vessels had sustained a primary injury prior to gene transfer, the fourth had a pre-existing neointimal lesion. In three of the vessels medial nuclear staining was sparse and seen in relationship to angioplasty-induced dissection. In the pre-injured vesssel sparse neointima cells showed positive staining. In the fourth vessel, a primary injury with extensive dissection, the adventitial fibromyoblasts showed evidence of approximately 25% gene transfer. Vessels without evidence of gene transfer were generally larger, suggesting poor apposition of the delivery catheter. Distal myocyte staining (with myositis) implied that intralumenal delivery had occurred. ARTERY THE EFFECT OF PRE-ESISTING LESIONS.
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