Cyclic stretch is known to alter a number of cellular and subcellular processes, including those involved in nonviral gene delivery. We have previously shown that moderate equibiaxial cyclic stretch (10% change in basement membrane area, 0.5 Hz, 50% duty cycle) of human pulmonary A549 cells enhances gene transfer and expression of reporter plasmid DNA in vitro, and that this phenomena may be due to alterations in cytoplasmic trafficking. Although the path by which plasmid DNA travels through the cytoplasm toward the nucleus is not well understood, the cytoskeleton and the constituents of the cytoplasm are known to significantly hinder macromolecular diffusion. Using biochemical techniques and immunofluorescence microscopy, we show that both the microfilament and microtubule networks are significantly reorganized by equibiaxial cyclic stretch. Prevention of this reorganization through the use of cytoskeletal stabilizing compounds mitigates the stretchinduced increase in gene expression, however, depolymerization in the absence of stretch is not sufficient to increase gene expression. These results suggest that cytoskeletal reorganization plays an important role in stretch-induced gene transfer and expression. Gene Therapy (2006) 13, 725-731.
Cyclic stretch has been shown to alter cell physiology, cytoskeletal structure, signal transduction, and gene expression in a variety of cell types. To determine the effects of stretch on the gene transfer process, we compared the transfection efficiencies of human A549 cells grown either statically or exposed to 10% cyclic stretch (Delta surface area) at 60 cycles/min (1 Hz) for 24 hours prior to, and/or after transfection with pEGFP-N1 and pCMV-lux-DTS using lipoplex or electroporation. Stretching the cells prior to transfection had no effect on gene transfer. By contrast, cyclic, but not continuous, stretch applied immediately after transfection for as little as 30 minutes resulted in a 10-fold increase in gene transfer and expression by either transfection technique. These stretch conditions did not result in rupture of the plasma membrane based on the fact that DNA was unable to enter stretched cells unless either an electric field was applied or the DNA was complexed with liposomes. Taken together with the timing of the stretch response and the known effects of stretch on transcription, these findings suggest that cyclic stretch may be altering the intracellular transport of plasmids to increase gene expression.
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