Abstract
Cells in the lung undergo cyclic deformation under both normal breathing processes as well as mechanical ventilation. While large deformations due to disease or mechanical ventilation can cause cellular damage such as that seen in ventilator induced lung injury, our lab has found that smaller, physiologically relevant deformations have a positive effect on gene transfer in an in vitro pulmonary epithelium model, increasing reporter gene expression up to 14-fold in A549 cells, a lung adenocarcinoma cell line frequently used in cellular models of the pulmonary epithelium. This result is not that surprising considering exogenous mechanical forces have been shown by a number of groups to alter the same barriers that gene transfer faces as a vector moves from the outside of the cell to the nucleus; namely alterations in the cytoskeleton and extracellular matrix, activation of cell signaling pathways and changes in transcription factor activation. Previous studies from our lab have shown that this increase in exogenous gene expression is not at the level of cell entry, transcription, or translation, suggesting increased trafficking of the plasmid DNA through the cytosol toward the nucleus. To understand what happens to these cells under stretch conditions, we grew them on deformable silastic membranes to 85% confluency and found that imposing a 10% change in membrane surface area in a biaxial manner at 30 cycles per minute with a duty cycle of 50% caused large cytoskeletal rearrangements within 30 minutes of the applied stretch. Microtubules showed the largest change as assayed by Western Blot, with a 50% reduction in polymerized tubulin after 30 minutes of stretch. Actin showed smaller changes in the amount of F-actin and G-actin in cell lysates, however, immunofluorescence microscopy showed a significant decrease in the length of actin filaments as well as a localization shift toward the periphery of the cells after biaxial cyclic stretch for 24 hours. Stabilization of either the actin cytoskeleton through the addition of jasplakinolide or the microtubule cytoskeleton through the addition of taxol prevented the stretch-induced increase in gene expression. Taken together, these results suggest that the stretch-induced increase in gene expression is due in part to cytoskeletal rearrangements and enhanced trafficking of plasmids through the cytosol.