The effects of cyclic stretch on gene transfer in alveolar epithelial cells

Abstract

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.

Cyclic stretch-induced reorganization of the cytoskeleton and its role in enhanced gene transfer

“…8 As shown in Figure 3a, as little as 30 min of cyclic stretch was also sufficient to decrease the amount of polymeric tubulin as determined by Western blot, but had little to no effect on microfilaments ( Figure 3b). The depolymerization of the microtubules following 30 min of stretch caused a 50% reduction in microtubules that did not recover when the cells were then grown under static conditions for up to 24 h. This suggested that the stretch-induced changes in the microtubule network due to 30 min of cyclic stretch were retained by the cells throughout the course of our experiments.…”

“…After allowing for attachment for 48-72 h, the cells were stretched using 25 mm BioFlex loading stations with a 10% membrane surface area change at 30 cycles per minute and a 50% duty cycle. These parameters are the same as previously reported, 8 and the 25 mm loading stations ensure an equibiaxial stretch in the radial and circumferential directions over the width of the loading post.…”

“…8 As stretch and many cytoskeletal drugs have been shown to increase cellular proliferation, we wanted to make sure that the enhanced expression we measured was not because of enhanced proliferation. When cells plated under similar conditions were either stretched or unstretched, we found no statistical difference in the number of cells between these groups 24 h later ( Figure 5).…”

“…8 When the barriers that plasmid DNA must cross in order to successfully transfect a cell are examined, it becomes apparent that these same barriers are altered by exogenous mechanical forces, namely, alterations in the cytoskeleton and extracellular matrix, [9][10][11] activation of cell signaling pathways 12 and changes in transcription factor activation. 13,14 We concluded that because of the time course of cyclic stretch necessary to induce increased expression, as well as the ability for this enhancement to occur in cells transfected by either electroporation or liposomal transfection, the mechanisms responsible for the stretch-enhanced expression were at the level of either nuclear entry or cytoplasmic trafficking.…”

Non-viral gene delivery: Stretching is the point

“…A more physical approach, cyclic cell stretching, has also been employed as a means of increasing in vitro gene delivery efficiency. 6 Future studies will likely exploit embedded polymer matrices in combination with undifferentiated stem cells to initiate and promote tissue growth in vitro for implantation. Such a system would be widely applicable, as the gene embedded into the matrix could be varied in order to alter the stem cell differentiation pathway.…”