Arterial gene transfer represents a novel strategy that is potentially applicable to a variety of cardiovascular disorders. Attempts to perform arterial gene transfer using nonviral vectors have been compromised by a low transfection efficiency. We investigated the hypothesis that cellular proliferation induced by arterial injury could augment gene expression after liposome-mediated gene transfer. Nondenuded and denuded rabbit arterial strips were maintained in culture for up to 21 d, after which transfection was performed with a mixture of the plasmid encoding firefly luciferase and cationic liposomes. In nondenuded arteries, the culture interval before transfection did not affect the gene expression. In contrast, denuded arteries cultured for 3-14 d before transfection yielded 7-13-fold higher expression (vs. day 0; P < 0.005). Transfection was then performed percutaneously to the iliac arteries of live rabbits with or without antecedent angioplasty. Gene expression increased when transfection was performed 3-.7 d postangioplasty (P < 0.05). Proliferative activity of neointimal cells assessed in vitro by 13Hlthymidine incorporation, and in vivo by immunostaining for proliferating cell nuclear antigen, increased and declined in parallel with gene expression. These findings thus indicate that the expression of liposome-mediated arterial gene transfer may be augmented in presence of ongoing cellular proliferation. (J. Clin. Invest. 1994. 93:652461.)

The possibility of using an exclusively percutaneous strategy to deliver foreign DNA to normal and balloon-dilated atherosclerotic arteries was studied by analysis of transfection efficiency in a rabbit model. A total of 22 external iliac arteries from 22 rabbits (10 normal and 12 atherosclerotic) were transfected with a solution of luciferase expression vector plasmid and liposome, using a dual balloon-catheter system. Analysis of the transfected segments revealed luciferase activity in 10 of the 22 arteries (4/10 normal vs 6/12 balloon-injured atherosclerotic, P = NS); no activity could be detected in the contralateral limb arterial segments used as controls. Luciferase activity levels in successfully transfected segments measured 4.10±1.19 (m±SEM) Turner light units (TLU), with 3.03±1.16 TLU found in normals vs 4.81±1.87 TLU in balloon-injured atherosclerotic arteries (P = NS). In situ hybridization of successfully transfected atherosclerotic sections showed expression of the luciferase gene mRNA from rare cells (< 1 / 1,000) limited to the neointimal lesion. Thus, expression of new genetic material may be achieved in both normal and balloon-dilated atherosclerotic arteries following an exclusively percutaneous approach. The low efficiency ofthe current delivery strategy, however, represents a potential limitation that must be improved if this strategy is to be applied as a therapeutic approach to human vascular disease. (J. Clin. Invest. 1992.90:936-944.)

Cell and gene therapy may alter the outcome of renal diseases, such as hereditary nephropathies, acute and chronic glomerulonephritis and allograft nephropathy. However, owing to blockade of many viral and cellular vehicles by the complex glomerular architecture, the exact nature of gene and cell delivery into specific renal compartments remains currently unknown. To study the interaction of viral vectors with a variety of renal cells and mesenchymal stem cells (MSCs), we employed a novel biological three-dimensional (3D) matrix comprised of fibrin microbeads (FMB) in comparison to monolayer cell culture. Our studies showed that renal cells of both established and primary lines can grow efficiently on FMB and differentiate into epithelial structures, as shown by electron microscopy. Gene delivery into renal cells in 3D was observed for several viral vectors and growth in 3D on FMB conferred resistance to renal cancer cells in the context of oncolytic adenoviruses. Finally, MSCs from various rodent species attached to FMB, grew robustly, survived for several weeks and could efficiently be transduced on FMB. Thus, on the basis of growth, differentiation and transduction of renal cells in 3D, FMB emerge as a novel 3D cellular microenvironment that differs substantially from monolayer cell cultures.

These findings document for the first time that light-induced relaxation of vascular smooth muscle, previously documented in vitro, may be reproduced in vivo.