Matrix stiffness affects endocytic uptake of MK2-inhibitor peptides.

In this study, the role of substrate stiffness on the endocytic uptake of a cell-penetrating peptide was investigated. The cell-penetrating peptide, an inhibitor of mitogen-activated protein kinase activated protein kinase II (MK2), enters a primary mesothelial cell line predominantly through caveolae. Using tissue culture polystyrene and polyacrylamide gels of varying stiffness for cell culture, and flow cytometry quantification and enzyme-linked immunoassays (ELISA) for uptake assays, we showed that the amount of uptake of the peptide is increased on soft substrates. Further, peptide uptake per cell increased at lower cell density. The improved uptake seen on soft substrates in vitro better correlates with in vivo functional studies where 10-100 µM concentrations of the MK2 inhibitor cell penetrating peptide demonstrated functional activity in several disease models. Additional characterization showed actin polymerization did not affect uptake, while microtubule polymerization had a profound effect on uptake. This work demonstrates that cell culture substrate stiffness can play a role in endocytic uptake, and may be an important consideration to improve correlations between in vitro and in vivo drug efficacy

Characterization and intracellular delivery of MK2-inhibitor peptides for inflammatory applications

The inflammatory response is essential for the body\u27s recovery from injury and infection; however, chronic inflammation can lead to tissue destruction. Several different diseases result from chronic inflammation, including rheumatoid arthritis, which is characterized by decreased mobility due to joint erosion and the upregulation of inflammatory cytokines. Although several therapies are available to treat inflammatory diseases, each therapy has disadvantages that limit its use in a portion of the population, including cost, efficacy, and adverse effects. An ideal therapeutic should utilize knowledge of the disease pathology, while showing efficacy and reducing adverse side effects. Our lab has developed a family of peptide inhibitors capable of inhibiting mitogen activated protein kinase activated protein kinase 2 (MAPKAP K2 or MK2) in vitro. MK2 appears to be a promising target for inflammation because it plays a role in regulating several cytokines that perpetuate inflammation. The overall goal of my research is to determine if these peptide inhibitors are applicable as a therapy for inflammation and characterize their mechanism of delivery, thus ensuring that these therapeutics will succeed where other inflammatory therapies have not. Our peptide therapeutics are designed with two different domains: (1) a functional domain that inhibits the kinase MK2 and (2) a cell-penetrating peptide (CPP) domain, which delivers the therapy into the cell. In this dissertation, I address our findings regarding the specificity, toxicity, and efficacy of these peptide inhibitors. We find that choice of cell-penetrating peptide domain dramatically influences the properties of the therapy. In addition, we have characterized the mechanism of intracellular uptake of the MK2 inhibitors in two different cell lines. In investigating the role that substrate stiffness plays in uptake and efficacy, we have improved our in vitro cell culture models to better replicate what is observed in vivo. These results advance our current understanding of the mechanism of action of the MK2-inhibitor peptides and suggest that these peptide inhibitors have the potential to be an effective therapy for inflammatory diseases

Matrix stiffness affects endocytic uptake of MK2-inhibitor peptides.

In this study, the role of substrate stiffness on the endocytic uptake of a cell-penetrating peptide was investigated. The cell-penetrating peptide, an inhibitor of mitogen-activated protein kinase activated protein kinase II (MK2), enters a primary mesothelial cell line predominantly through caveolae. Using tissue culture polystyrene and polyacrylamide gels of varying stiffness for cell culture, and flow cytometry quantification and enzyme-linked immunoassays (ELISA) for uptake assays, we showed that the amount of uptake of the peptide is increased on soft substrates. Further, peptide uptake per cell increased at lower cell density. The improved uptake seen on soft substrates in vitro better correlates with in vivo functional studies where 10-100 µM concentrations of the MK2 inhibitor cell penetrating peptide demonstrated functional activity in several disease models. Additional characterization showed actin polymerization did not affect uptake, while microtubule polymerization had a profound effect on uptake. This work demonstrates that cell culture substrate stiffness can play a role in endocytic uptake, and may be an important consideration to improve correlations between in vitro and in vivo drug efficacy

Effect of actin polymerization on uptake of 3 µM FITC-YARA.

<p>Cells were seeded on soft substrates at 21,000 cells/cm² and treated with YARA and 5 or 25 µg/ml LPA, or seeded on TCPS and treated with YARA and 5 µg/ml CytoD. Uptake was evaluated using flow cytometry.</p

TNF-α cytokine production of mesothelial cells seeded at low density.

<p>Cells were treated with the MK2-inhibitor peptide on polyacrylamide gels of different stiffness compared to tissue culture plastic. Cells were seeded at 21,000 cells/cm². Data is presented as mean ± standard deviation. Asterisks indicate statistically significant difference from IL-1β stimulated control within each substrate group (p<0.05; one-way ANOVA+Tukey post-hoc test).</p

Effect of microtubule polymerization on uptake of 3 µM FITC-YARA.

<p>Cells were seeded on soft substrates at 21,000 cells/cm² and treated with YARA and 1 or 10 10 µg/ml of nocodazole. Uptake was evaluated using flow cytometry.</p

Mesothelial cells stained for F-actin with phalloidin on stiff (A) and soft (B) substrates, and on tissue culture polystyrene (C).

<p>Scale bars are 50 µm.</p

Rheological characterization of polyacrylamide gels that contain 10% acrylamide and various concentrations of bis-acrylamide crosslinker.

<p>Data is displayed as mean ± standard deviation.</p

Inhibition of 3 µM FITC-YARA uptake on soft polyacrylamide gel substrates.

<p>Uptake is inhibited at 4°C (A) and with the pharmacological inhibitor MβCD (B).</p