ABSTRACT
Therapeutic and research applications of direct gene transfer in the coronary vasculature are limited by varying transgene activity found in the vessel wall. It remains to be determined whether this is due to difficulties in delivery of recombinant DNA into the coronary arteries or if it relates to the state of recipient cells in the arterial wall. Accordingly, using a clinically applicable protocol, we have examined the time course of transgene (luciferase) expression in porcine coronary vasculature following transcatheter gene transfer (1 day to 4 weeks). Liposome-mediated transfection by means of a transcatheter approach resulted in detectable luciferase activity in all transfected vessels at 1 day after gene delivery. However, the activity of a reporter gene rapidly declined at 7 days thereafter, with only 33% of coronary arteries demonstrating luciferase activity. Comparable levels of transgene sequences were present in transfected vessels, although no correlation with luciferase activity was found. To further determine possible mechanisms responsible for rapidly declining transgene expression in vivo, cell proliferation was induced in the vessel wall by means of either balloon denudation or by intramural injections of platelet derived growth factor BB (PDGF-BB) into the porcine coronary vasculature at the time of gene transfer. Luciferase activity was significantly augmented (23-fold) following mitogenic stimulation in vivo as compared with the control transfected coronary arteries. These data demonstrate that a practical transcatheter approach provides an effective route to deliver recombinant DNA sequences into the coronary arterial wall.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Coronary Circulation , Gene Expression Regulation, Enzymologic , Gene Transfer Techniques , Animals , Base Sequence , Cardiac Catheterization , Cell Division , Cells, Cultured , Coronary Vessels/cytology , Coronary Vessels/metabolism , DNA Primers/genetics , Immunohistochemistry , In Vitro Techniques , Liposomes , Luciferases/genetics , Molecular Sequence Data , Proliferating Cell Nuclear Antigen/metabolism , Swine , Time Factors , TransfectionABSTRACT
BACKGROUND: Smooth muscle cell proliferation and extracellular matrix accumulation are the principal mechanisms leading to vascular restenosis. We have previously demonstrated the growth-inhibitory effect of antisense oligomers targeting the c-myc proto-oncogene in human smooth muscle cells. The goal of this study was to investigate whether c-myc antisense oligomers reduce neointimal formation in balloon-denuded porcine coronary arteries. METHODS AND RESULTS: First, type I collagen synthesis, which reflects synthetic function, was markedly reduced following c-myc antisense oligomers in porcine vascular smooth muscle cells independent of the growth inhibition. These effects in vitro provided the rationale for assessing c-myc antisense oligomers in the prevention of neointima in vivo. Second, the efficiency of single transcatheter delivery of oligomers into denuded porcine coronary arteries was determined. Despite rapid plasma clearance following local delivery, oligomers persisted at the site of injection for at least 3 days, exceeding by severalfold their concentration in peripheral organs. Third, morphometric analyses were carried out in balloon-denuded coronary arteries at 1 month after transcatheter c-myc antisense oligomer administration. Maximal neointimal area was reduced from 0.80 +/- 0.17 mm2 in the control group (n = 12) to 0.24 +/- 0.06 mm2 in the antisense-treated group (n = 13, P < .01). Likewise, a significant reduction in maximal neointimal thickness was observed in the antisense-treated group (P < .01). These changes in vascular remodeling following denuding injury resulted in an increase in residual lumen from 64 +/- 6% in the control group to 81 +/- 5% in the antisense-treated group (P < .05). CONCLUSIONS: (1) Single transcatheter administration allowed for endoluminal delivery of oligomers to the site of coronary arterial injury. (2) C-myc antisense oligomers reduced the formation of neointima in denuded coronary arteries, implying a therapeutic potential of this approach for the prevention of coronary restenosis. (3) It is postulated that the c-myc proto-oncogene is involved in the process of vascular remodeling, regulating smooth muscle cell proliferation and extracellular matrix synthesis.
