MK2 is known to regulate the expression of several proinflammatory cytokines, however we focused on the down regulation of TNF-a protein expression as a marker of YARA activity. In previous studies we have noted that cytokine expression is down regulated at 6 hours and continues to be down regulated at 24 hours with the difference between expression in control and treated cells maximized at the later time point. We believe that the prolonged ability of the peptide to suppress cytokine protein expression is due to uptake into and prolonged residence time in caveolae. This is consistent with work by Kim, et al. where they saw prolonged retention of TAT-M13-phage in caveolae, and is subject to further ICI 182780 company investigation. The concentration required to demonstrate efficacy in this cell line was equivalent to that observed in animal models when cells are seeded at low densities on polyacrylamide substrates. The importance of cell density must not be overlooked. Previous studies in our laboratory indicated that confluent mesothelial cells seeded on GW786034 tissue culture plastic exhibited a decrease in cytokine production only when treated with very high concentrations of peptide. However, when cells were seeded at a much lower density on tissue culture plastic, there was evidence of efficacy at 100 mM. Mesothelial cells were initially cultured and evaluated for efficacy at such a high density because it was thought that the cell-cell contact was best for mimicking the in vivo environment where the cells form a monolayer called the mesothelium. 
However, based on the results presented herein, this assumption is clearly incorrect. Cell context is critical to cell behavior, and cells change their genetic profile base on cellcell contacts, how close to the edge of a culture they are, and even cell density. Similar to other mammalian cells, mesothelial cells have the ability to change phenotype, and it is possible that at high seeding concentrations the mesothelial cells are differentiating and are thus not as responsive to YARA. It is also possible that the rate of endocytosis is decreased at high seeding densities, due to phenotypic and metabolic changes at high cell densities. This latter possibility is consistent with the studies of Snijder, et al. where they used both modeling and experiment to show that the rate of endocytosis changed with cell density, and how close the edge of a cell islet the individual cell was. Thus, phenotypic changes due to cell density may explain why such variability is observed in YARA efficacy when cells are seeded at high concentrations, and further explain the high concentration of YARA required to observe efficacy at confluence. Both substrate stiffness and the extracellular matrix have previously been shown to regulate non-viral gene delivery. In these cases, the gene transfer efficiency of plasmidDNA increased with increasing density of RGD peptides, with fibronectin coating and with increasing stiffness. The enhanced efficiency of uptake with higher density of RGD peptides and increased stiffness is attributed to an increase in cellular proliferation, while the enhanced uptake with fibronectin coating is attributed to the promotion of endocytic uptake by clathrin-mediated endocytosis. That stiffness has an opposing role in plasmid-DNA uptake compared to our MK2-inhibitor peptide is not surprising. PlasmidDNA is often cited as being taken up through clathrin-mediated endocytosis, while the MK2-inhibitor in mesothelial cells gets taken up mainly through caveolae-mediated endocytosis. While proliferation can be important for uptake, as seen in the case of plasmid DNA-uptake, it does not appear to have the same effect in the case of YARA uptake.